PC3 cells are an epithelial prostate tumour cell line. A range of Wnts triggers a delayed Ca2+ rise [1] in these and other cancer cell lines. Recorded in patch clamp the cells have low membrane potential [2] but exhibit an outward current activated apparently at (non-physiological) positive potentials [3]. The main candidate channel carrying this K+ current is the BK(Ca) α subunit although a KCNN4 channel is also reported [4]. Without baseline subtraction, a small ‘leak’ current is sometimes apparent in recordings. All of these currents
are modulated by Wnts as described below.

The results demonstrate that QPatch 16 can provide high-quality whole-cell current data from fast voltage-activated Na+ channels, and that protocols can be defined that allow analysis of drug potency (IC₅₀), inactivation kinetic properties, as well as state- and frequency-dependency of drug action.

Changes of the electrical charges across the surface cell membrane are absolutely necessary to maintain cellular homeostasis in physiological as well as in pathological conditions. The opening of ion channels alter the charge distribution across the surface membrane as they allow the diffusion of ions such as K+, Ca++, Cl−, Na+. Traditionally, voltage-gated ion channels (VGIC) are known to play fundamental roles in controlling rapid bioelectrical signaling including action potential and/or contraction. However, several investigations have revealed that these classes of proteins can also contribute significantly to cell mitotic biochemical signaling, cell cycle progression, as well as cell volume regulation. All these functions are critically important for cancer cell proliferation. Interestingly, a variety of distinct VGICs are expressed in different cancer cell types, including metastasis but not in the tissues from which these tumors were generated. Given the increasing evidence suggesting that VGIC play a major role in cancer cell biology, in this review we discuss the role of distinct VGIC in cancer cell proliferation and possible therapeutic potential of VIGC
pharmacological manipulation.

The new adaptive protocol enables increased control of the state that voltage-gated channels are in during an
experiment, leading to reduced variability of data and increased confidence in compound testing results, on a 384-well high throughput automated patch clamp platform.

As revising an ICH regulatory guideline, S7B, for the Non-clinical Evaluation of the Potential for Delayed Ventricular Repolarization (QT Interval Prolongation by Human Pharmaceuticals) was suggested, Comprehensive In
vitro Proarrhythmia Assay (CiPA) activities are being implemented. The suggested S7B revision includes an assay of cardiac ion channels other than hERG, and CiPA-recommended voltage protocols for patch clamping was recently announced. We, Group 5 of the JSPS iSmart (investigation of in silico/in vitro model for arrhythmogenic risk prediction) play a role of wet-data collection. This time we have investigated the applicability of those protocols

This report summarizes the experiments that were done running fast ligand-gated assays on QPatch HTX. QPatch HTX in multi-hole mode patches 10 cells per well instead of just one which has been shown to increase the overall success rate due to the fact that cells with less or no expression of the ion channel of interest will be patched in parallel with other cells that has better expression. The currents from all 10 holes are thus added together and therefore gives rise to increased current amplitudes from each well. The 10 holes obviously takes up more space in the flow channel in the QPlate chip and we were curious to know if these changes had any effect on the measured signal from the ten cells. We set out to test different ligand-gated ion channels that are well known and already validated on QPatch HT to determine if the signal onset was comparable to QPatch HTX in multi-hole mode. Furthermore we investigated the effect of known compounds: agonists, antagonist and modulators to see if any changes in data quality could be detected.

Automated patch clamp (APC) technology was first developed at the turn of the millennium. The increased throughput it afforded promised a new paradigm in ion channel recordings, offering the potential to overcome the time-consuming, low-throughput bottleneck, arising from manual patch clamp investigations. This has relevance to the fast-paced development of novel therapies for chronic pain. This review highlights the advances in technology, using select examples that have facilitated APC usage in both industry and academia. It covers both first generation and the latest developments in second-generation platforms. In addition, it also provides an overview of the pain research field and how APC platforms have furthered our understanding of ion channel research and the development of pharmacological tools and therapeutics. APC platforms have much to offer to the ion channel research community, and this review highlights areas of best practice for both academia and industry. The impact of APC platforms and the prospects of ion channel research and improved therapeutics for chronic pain will be evaluated.

In ion channel safety assessment and drug discovery it is often important to determine the mode of action of a drug candidate, which requires high-quality recordings. We successfully use the QPatch X in multi-hole mode in a screening scenario on Na_v1.5. This correlates with data from Langendorff perfused rabbit hearts, and an in vivo ECG assay using anesthetized rabbit, and effectively discriminates different modes of action of compounds on NaV1.5.
The protocol for QPatch X both tests the decay of the sodium current (30 Hz pulsetrain), and the recovery of the current. The QPatch assay allows combination of these elements into a single protocol, thereby shortening experiment time and costs.
The results show that: 1) Flecainide is a use-dependent blocker of open channels, as demonstrated by a slow decay of I_Na (30 Hz pulsetrain), and delayed recovery from inactivation in the QPatch experiments. Flecainide produces ECG changes (marked PR and QRS prolongation, and slight QTc prolongation), and lethal arrhythmia (VT ~Vf) in both Langendorff hearts and anesthetized rabbits at low doses. 2) Lidocaine is a state-dependent blocker of inactivated channels, inducing fast decay of I_Na (30 Hz pulsetrain) and fast recovery from inactivation. Lidocaine produces slight ECG changes (PR and QRS prolongation), but no arrhythmia in Langendorff-hearts or anesthetized rabbits. 3) Quinidine demonstrates a slow decay of I_Na (30 Hz pulsetrain), and delayed recovery from inactivation. Quinidine shows moderate PR and QRS prolongation, and severe morphological changes in ECGs.
The present results suggest that: 1) The QPatch X offers a time- and cost-effective screening scenario to select safe compounds with Na_v1.5 blocking activity and no proarrhythmic activity, and 2) the pro-arrhythmic activity of the Na_v1.5 blockers flecainide and quinidine might be attributable to their marked delay of recovery from inactivation.

There is no doubt that automated patch clamp (APC) technology has revolutionized research in biomedical science. High throughput ion channel screening is now an integral part of the development and safety profiling of the majority of new chemical entities currently developed to address unmet medical needs. The increased throughput it provides has significantly improved the ability to overcome the time-consuming, low throughput bottlenecks resulting from the more conventional manual patch-clamp method, considered the ‘gold standard’, for studying ion channel function and pharmacology. While systems offering the luxury of automation have only been commercially available for two decades, the road leading to this new technology is long and rich in seminal, hands-on, studies dating back as far as the 18th century. So where does this technology currently stand, and what will it look like in the future? In the current article, we review the scientific history leading to the development of APC systems, examine key drivers in the rapid development of this technology (such as failed ion channel programmes and the issue of drug-induced hERG inhibition and QT interval prolongation), highlight key capabilities and finally provide some perspective on the current and future impact of the technology on cardiac safety assessment and biomedical science.

Effective screening of large compound libraries in ion channel drug discovery requires the development of new electrophysiological techniques with substantially increased throughputs compared to the conventional patch clamp technique. Sophion Bioscience is aiming to meet this challenge by developing two lines of automated patch clamp products, a traditional pipette-based system called Apatchi-1, and a silicon chip-based system QPatch. The degree of automation spans from semi-automation (Apatchi-1) where a trained technician interacts with the system in a limited way, to a complete automation (QPatch 96) where the system works continuously and unattended until screening of a full compound library is completed. The performance of the systems range from medium to high throughputs.

CHO cells expressing the cold-sensitive ion channel TRPM8 were tested on the QPatch, and HEK cells expressing TRPM8 were tested on QPatch HTX. Two agonists of TRPM8 were used for EC₅₀ determinations: menthol and icilin. All data presented was obtained with QPatch and analyzed with QPatch Assay Software

True giga seal patch clamping can be performed in parallel with QPatch multi-hole technology. This new multi-hole functionality has been tested on three different transient receptor potential ion channels (TRPA1, TRPV1 & TRPM8). All three targets were tested in both single-hole mode and in multi-hole mode with different agonists and antagonists. The advantages of testing compounds in multihole mode will be a minimization of biological variance and an increase in current amplitude, since the current response from several cells are summarized. The disadvantage is lack of serial resistance compensation and in some cases large leak currents

Oseltamivir can exert a powerful anti-AF action through its ideal multi-channel blocking effects on I_K, A_Ch, I_Kr and I_Na.

Although developing a novel formulation that can reduce the effective dose against AF may be mandatory, oseltamivir would be a promising seed compound for developing efficacious and safe anti-AF drugs.

Chemical transfection is broadly used to transiently transfect mammalian cells, although often associated with cellular stress and membrane instability, which imposes challenges for most cellular assays, including high-throughput (HT) assays. In the current study, we compared the effectiveness of calcium phosphate, FuGENE and Lipofectamine 3000 to transiently express two key voltage-gated ion channels critical in pain pathways, Ca_V2.2 and Na_V1.7. The expression and function of these channels were validated using two HT platforms, the Fluorescence Imaging Plate Reader FLIPR^Tetra and the automated patch clamp QPatch 16X. We found that all transfection methods tested demonstrated similar effectiveness when applied to FLIPR^Tetra assays. Lipofectamine 3000-mediated transfection produced the largest peak currents for automated patch clamp QPatch assays. However, the FuGENE-mediated transfection was the most effective for QPatch assays as indicated by the superior number of cells displaying GΩ seal formation in whole-cell patch clamp configuration, medium to large peak currents, and higher rates of accomplished assays for both Ca_V2.2 and Na_V1.7 channels. Our findings can facilitate the development of HT automated patch clamp assays for the discovery and characterization of novel analgesics and modulators of pain pathways, as well as assisting studies examining the pharmacology of mutated channels.

TMEM16A (ANO1) is a Ca²⁺-activated Cl- channel (CaCC) that is involved in a plethora of physiological and pathophysiological conditions. The channel was suggested as a target for treatment of asthma, secretory diarrheas, and hypertension. TMEM16A is unique as its gating synergistically depends on voltage and cytosolic Ca²⁺ (Scudieri et al. 2012).
A robust and high-throughput electrophysiology assay for TMEM16A was long awaited, but progress was hampered by the fact that many automated patch clamp devices rely on the use of fluoride (F^–)in the internal solution to promote seal formation. However CaF₂ has very low solubility and the resulting precipitation limits the ability to control precisely the concentration of Ca⁺ in the internal solution.
We recently developed a novel approach to test the pharmacological inhibition of TMEM16A on Qube with following characteristics:

• Consistently high success rates (>80%)
• Low run down
• High degree of pharmacological reproducibility (consistent IC50 values of multiple runs)

TMEM16A (ANO1) is a Ca2+-activated Cl-channel (CaCC) that is involved in a plethora of physiological and pathophysiological conditions. The channel was suggested as target for treatment of asthma, secretory diarrheas, and hypertension. TMEM16A is unique as its gating ynergistically depends on voltage and cytosolic Ca2+ (Scudieri et al. 2012). A robust TMEM16A assay was long awaited but progress was hampered as many automated patch clamp devices rely on the use of fluoride in the internal solution. Fluoride exhibits a very low solubility with calcium which means that calcium that is added to the internal solution to activate the channel has a strong tendency to precipitate, making it impossible to correctly adjust the calcium concentration.

Mental disorders such as schizophrenia are associated with impaired firing properties of fast spiking inhibitory interneurons (FSINs) causing reduced task-evoked gamma-oscillation in prefrontal cortex. The voltage-gated sodium channel Na_V1.1 is highly expressed in PV-positive interneurons, but only at low levels in principal cells. Positive modulators of Na_V1.1 channels are for this reason considered potential candidates for the treatment of cognitive disorders. Here we examined the effect of the novel positive modulator of voltage-gated sodium channels Lu AE98134. We found that Lu AE98134 facilitated the sodium current mediated by Na_V1.1 expressed in HEK cells by shifting its activation to more negative values, decreasing its inactivation kinetics and promoting a persistent inward current. In a slice preparation from the brain of adult mice, Lu AE98134 promoted the excitability of fast spiking interneurons by decreasing the threshold for action potentials. We then tested if Lu AE98134 could normalize the altered firing properties of FSINs in Dlx5/6^+/− mutant mice. FSINs of this model for schizophrenia are characterized by broader action potentials and higher spike threshold. We found that in the presence of Lu AE98134, the firing frequency was increased while the spike duration and the threshold were decreased. Compounds with similar mode of action appear as promising candidates for restoring cognitive deficits present in schizophrenia.

Millions of years of evolution have fine-tuned the ability of venom peptides to rapidly incapacitate both prey and potential predators. Toxicofera reptiles are characterized by serous-secreting mandibular or maxillary glands with heightened levels of protein expression. These glands are the core anatomical components of the toxicoferan venom system, which exists in myriad points along an evolutionary continuum. Neofunctionalisation of toxins is facilitated by positive selection at functional hotspots on the ancestral protein and venom proteins have undergone dynamic diversification in helodermatid and varanid lizards as well as advanced snakes. A spectacular point on the venom system continuum is the long-glanded blue coral snake (Calliophis bivirgatus), a specialist feeder that preys on fast moving, venomous snakes which have both a high likelihood of prey escape but also represent significant danger to the predator itself. The maxillary venom glands of C. bivirgatus extend one quarter of the snake’s body length and nestle within the rib cavity. Despite the snake’s notoriety its venom has remained largely unstudied. Here we show that the venom uniquely produces spastic paralysis, in contrast to the flaccid paralysis typically produced by neurotoxic snake venoms. The toxin responsible, which we have called calliotoxin (δ-elapitoxin-Cb1a), is a three-finger toxin (3FTx). Calliotoxin shifts the voltage-dependence of NaV1.4 activation to more hyperpolarised potentials, inhibits inactivation, and produces large ramp currents, consistent with its profound effects on contractile force in an isolated skeletal muscle preparation. Voltage-gated sodium channels (NaV) are a particularly attractive pharmacological target as they are involved in almost all physiological processes including action potential generation and conduction. Accordingly, venom peptides that interfere with NaV function provide a key defensive and predatory advantage to a range of invertebrate venomous species including cone snails, scorpions, spiders, and anemones. Enhanced activation or delayed inactivation of sodium channels by toxins is associated with the extremely rapid onset of tetanic/excitatory paralysis in envenomed prey animals. A strong selection pressure exists for the evolution of such toxins where there is a high chance of prey escape. However, despite their prevalence in other venomous species, toxins causing delay of sodium channel inhibition have never previously been described in vertebrate venoms. Here we show that NaV modulators, convergent with those of invertebrates, have evolved in the venom of the long-glanded coral snake. Calliotoxin represents a functionally novel class of 3FTx and a structurally novel class of NaV toxins that will provide significant insights into the pharmacology and physiology of NaV. The toxin represents a remarkable case of functional convergence between invertebrate and vertebrate venom systems in response to similar selection pressures. These results underscore the dynamic evolution of the Toxicofera reptile system and reinforces the value of using evolution as a roadmap for biodiscovery.

Ca2+-activated K+ channels (KCa) are integral players in setting the membrane potential and regulating Ca2+ homeostasis. The human genome consists of a total of 8 KCa channels that differ in their unitary conductance, genetic relation to each other and mechanism of Ca2+ binding. The channels are subdivided in two groups according to the aforementioned characteristics. The first group was initially characterized by their structural similarity and comprises of KCa1.1 (BK), KCa4.1, KCa4.2 and KCa5.1. Later studies revealed that these channels have in fact a different activation mode and only KCa1.1 (BK) is a truly Ca2+-activated channel aside from its voltage-dependence. The second group is characterized by their unitary conductance and encompasses three small conductance (SK) channels KCa2.1 (SK1), KCa2.2 (SK2) and KCa2.3
(SK3), and an intermediate-conductance channel KCa3.1 that is also known as SK4, IKCa1, IK-1 or KCa4 encoded by KCNN4 (Wei et al., 2005). KCa3.1 was cloned independently by three groups in 1997, therefore, the channels biophysical and pharmacological properties are already well-established (Wulff & Castle, 2010).

VCC234718, a molecule with growth inhibitory activity against Mycobacterium tuberculosis (Mtb), was identified by phenotypic screening of a 15344-compound library. Sequencing of a VCC234718-resistant mutant identified a Y487C substitution in the inosine monophosphate dehydrogenase, GuaB2, which was subsequently validated to be the primary molecular target of VCC234718 in Mtb. VCC234718 inhibits Mtb GuaB2 with a Ki of 100 nM and is uncompetitive with respect to IMP and NAD+. This compound binds at the NAD+ site, after IMP has bound, and makes direct interactions with IMP; therefore, the inhibitor is by definition uncompetitive. VCC234718 forms strong pi interactions with the Y487 residue side chain from the adjacent protomer in the tetramer, explaining the resistance-conferring mutation. In addition to sensitizing Mtb to VCC234718, depletion of GuaB2 was bactericidal in Mtb in vitro and in macrophages. When supplied at a high concentration (≥125 μM), guanine alleviated the toxicity of VCC234718 treatment or GuaB2 depletion via purine salvage. However, transcriptional silencing of guaB2 prevented Mtb from establishing an infection in mice, confirming that Mtb has limited access to guanine in this animal model. Together, these data provide compelling validation of GuaB2 as a new tuberculosis drug target.

Background Evidence has emerged that small-conductance Ca²⁺-activated K⁺ (SK) channels constitute a new target for treatment of atrial fibrillation (AF). SK channels are predominantly expressed in the atria as compared with the ventricles. Various marketed antiarrhythmic drugs are limited by ventricular adverse effects and efficacy loss as AF progresses.

Methods and Results A total of 43 pigs were used for the studies. AF reversion in conscious long-term tachypaced pigs: Pigs were subjected to atrial tachypacing (7 Hz) until they developed sustained AF that could not be reverted by vernakalant 4 mg/kg (18.8±3.3 days of atrial tachypacing). When the SK channel inhibitor AP14145 was tested in these animals, vernakalant-resistant AF was reverted to sinus rhythm, and reinduction of AF by burst pacing (50 Hz) was prevented in 8 of 8 pigs. Effects on refractory period and AF duration in open chest pigs: The effects of AP14145 and vernakalant on the effective refractory periods and acute burst pacing-induced AF were examined in anaesthetized open chest pigs. Both vernakalant and AP14145 significantly prolonged atrial refractoriness and reduced AF duration without affecting the ventricular refractoriness or blood pressure in pigs subjected to 7 days atrial tachypacing, as well as in sham-operated control pigs.

Conclusions SK currents play a role in porcine atrial repolarization, and pharmacological inhibition of these with AP14145 demonstrates antiarrhythmic effects in a vernakalant-resistant porcine model of AF. These results suggest SK channel blockers as potentially interesting anti-AF drugs.

The human ether-à-go-go related gene (hERG) function is important for cardiac repolarization and inhibition of the channel can prolong the cardiac action potential, which gives increased risk for ventricular arrhythmias including torsade des points (TdP). Therefore, in vitro evaluations of the compound effects is performed on the hERG channel routinely in drug development projects to detect potential arrhythmic side-effects.
Usually these compound measurements are carried out at ambient temperatures. Previously it has been shown that the potency for many compounds has been underestimated when compared to near physiological temperature tests. Therefore, a temperature controlled measuring environment is beneficial when testing compounds for the aims as mentioned here.
Until recently, the only possibility to test compound potency under voltage control conditions has been the manual patch clamp technique. Now automated patch clamp instruments with temperature control have become available making it possible to perform up to 384 parallel recordings at controlled temperatures ranging from 8°C and above.
Here we used an automated patch clamp system, Qube, to study the effect of temperature on concentration response relationships on a panel of compounds known to block the hERG channel. Qube has a temperature controlled test environment and in these studies, we show that temperature merits being taken into consideration when evaluating for hERG pharmacology.

To better understand how compounds function in warm-blooded animals such as humans, it is key to be able to test compound potency at physiological temperature. Our automated patch clamp systems provide real time measurement of temperature, and thus the effect of temperature change. Temperature regulation is done directly at the measurement site, and not by thermal control of the whole system. Four different compounds were tested in a dose-response format on Qube 384 at differing temperatures. All four compounds showed increased effect at elevated temperature compared to room temperature. Qube is suitable for screening compounds at temperatures from 8° C to 40° C (or 8° C above dew point).

T-type calcium channel (Cav3.x) blockers are receiving increasing attention as potential therapeutics for the treatment of pathophysiological disorders and diseases, including absence epilepsy, Parkinson’s disease (PD), hypertension, cardiovascular diseases, cancers, and pain. However, few clinically approved Cav3.x blockers are available, and selective pharmacological tools are needed to further unravel the roles of individual Cav3.x subtypes. In this work, through an efficient synthetic route to the marine fungal product pseudellone C, we obtained bisindole alkaloid analogs of pseudellone C with a modified tryptophan moiety and identified two Cav3.2 (2, IC50 = 18.24 µM; 3, IC50 = 6.59 µM) and Cav3.3 (2, IC50 = 7.71 µM; 3, IC50 = 3.81 µM) selective blockers using a FLIPR cell-based assay measuring Cav3.x window currents. Further characterization by whole-cell patch-clamp revealed a preferential block of Cav3.1 activated current (2, IC50 = 5.60 µM; 3, IC50 = 9.91 µM), suggesting their state-dependent block is subtype specific.

γ-Lactam is an important structural motif in a large number of biologically active natural products and synthetic small pharmaceutical molecules. However, there is currently no effective approach to construct γ-lactam ring directly from natural rigid polycyclic amides. Herein, we report a facile methodology for synthesis of a new group of olean-28,13β-lactams (10a–j) from their corresponding amides, promoted by an easily available reagent 2,3-dichloro-5,6-dicyanobenzoquinone (DDQ), through an intramolecular dehydrogenative C–N coupling reaction via a radical ion mechanism. Biological evaluation indicated that the most active lactam 10h displayed potent antiproliferative activity against human cancer cells but 13.84- to 16.92-fold less inhibitory activity on noncancer cells in vitro. In addition, 10h significantly inhibited the growth of implanted prostate cancer in vivo. Furthermore, 10h induced cell cycle arrest and apoptosis and down-regulated the AKT/mTOR signaling in DU-145 cells. Finally, 10h was more stable in rat plasma and human liver microsomes than CDDO-Me and had little hERG channel inhibitory activity. Collectively, 10h may be a potential antiprostate cancer agent for further investigation.

Over expression of c-Met tyrosine kinase is known to promote tumorigenesis and metastasis, as well as to cause therapeutic resistance. Herein a series of novel 6,11-dihydro-5H-benzo[e]pyrimido[5,4-b][1,4]diazepine derivatives were designed, synthesised and evaluated for their c-Met kinase inhibition. Compounds 17e, 17f, 18a, and 18b were further examined for their anti-proliferative activities against four typical cancer cell lines (PC-3, Panc-1, HepG2, and Caki-1). The promising compound 17f was identified as a multi-target receptor tyrosine kinase inhibitor, which also displayed favourable pharmacokinetic properties in rats, had an acceptable safety profile in preclinical studies, and significant anti-tumour activity in the Caki-1 tumour xenograft model.

Ion channels are responsible for the permeation of ions across the membrane and their central role in cellular physiology is well established. Historically, the direct study of ion channels has been considered technically challenging. As such, a significant barrier to drug discovery for ion channels has been the low throughput of high quality electrophysiological data. The emergence of automated high throughput platforms for studying ion channel kinetics and pharmacology has lowered this barrier. Ion channels are now recognized as increasingly important drug targets and a diverse range of ion channels are implicated in a variety of drug discovery and cardiac safety assessment programs. The TRP (Transient Receptor Potential) superfamily of ion channels play a crucial role in a broad range of sensory functions including vision, taste, olfaction, hearing, touch, pain and thermosensation. Many of the TRP channels are polymodal in their activation and deactivation mechanisms and even with conventional patch clamp electrophysiology, the TRP channels are considered to be a very complex target class. Here we present an update on the significant progress made on the TRP receptor assays with the available automated patch clamp systems.

Several types of drug-resistant epilepsies of infancy have been associated with mutations in the KCNT1 gene, which encodes the sodium-activated potassium channel subunit KNa1.1. Approximately forty different amino acid substitutions have been identified in patients, involving both transmembrane and intracellular domains of the channel subunit, and with one exception all cause gain-of-function in channel gating. Channel inhibition, therefore, is potentially a stratified approach to treat the disorder. To date, quinidine therapy has been trialled with several patients, but mostly with unsuccessful outcomes, which have been linked to its low potency and lack of specificity; effects on cardiac ion channels being a major concern. Here we describe the use of high-resolution structures of KNa1.1 channels, generated through cryo-electron microscopy, to identify novel inhibitors using computational methods. From an initial analysis of seventeen compounds identified using this approach, we found six that inhibited KNa1.1 channels. Their IC50 values ranged between 0.6 and 7.4 μM, compared to 100 μM with quinidine, and likely inhibited through pore-blocking mechanisms. The compounds delivered varying results when evaluating state-dependence and in preliminary toxicity assays, with some demonstrating negligible cellular toxicity and low levels of hERG block. These compounds may provide starting points for the development of novel pharmacophores for KNa1.1 inhibitors, with the view to treating KCNT1-associated epilepsies. Furthermore, this illustrates the potential for utilising ion channel structures generated through cryo-electron microscopy in drug discovery.

Using structure- and ligand-based design principles, a novel series of piperidyl chromane arylsulfonamide Na_v1.7 inhibitors was discovered. Early optimization focused on improvement of potency through refinement of the low energy ligand conformation and mitigation of high in vivo clearance. An in vitro hepatotoxicity hazard was identified and resolved through optimization of lipophilicity and lipophilic ligand efficiency to arrive at GNE-616 (24), a highly potent, metabolically stable, subtype selective inhibitor of Na_v1.7. Compound 24 showed a robust PK/PD response in a Na_v1.7-dependent mouse model, and site-directed mutagenesis was used to identify residues critical for the isoform selectivity profile of 24.

High information-content screenings based on whole-cell current patch-clamp recordings have become available for ion channel pharmacological research with the development of the QPatch automated patch-clamp technology. A significant increase in system throughput was recently achieved by a tripling of the number of parallel operating patch-clamp sites (from 16 to 48) by the introduction of QPatch HT. In a series of hERG screening studies we have subsequently aimed at increasing the throughput further by reducing the average experimental time consumption associated with each IC₅₀ determination, and by employing alternative calculational algorithms. We have investigated the effect of the following three procedures on effective QPatch throughput:

1. Application of multiple drugs per cell
2. Reduction of the duration of the experimental
   protocol
3. Estimation of drug potency (IC₅₀) based on a
   single inhibitor concentration

The new adaptive protocol enables increased control of the state that voltage-gated channels are in during an experiment, leading to reduced variability of data and increased confidence in compound testing results, on a 384-well high throughput automated patch clamp platform.

The aim of this study is to observe the consistency of a current signal (elicited by an agonist or a voltage change) before and after antagonist or blocker application. This report presents data monitored on the automated QPatch 16 based on whole-cell current recordings from ligand-gated ion channels (LGIC); the glutamate-activated ligand-gated ion channels Glu5R expressed in HEK293 cells from Neurosearch and GluR6 (GRIK2) from Millipore as well as acid-sensing ion channel 3 (ASIC3) expressed in HEK293 cells. Similar experiments were performed on voltage-gated ion channels (VGIC); HEK293-Na_v1.2a and CHO-hERG.

With the emergence of multi-drug-resistant strains of Mycobacterium tuberculosis, there is a pressing need for new oral drugs with novel mechanisms of action. A number of scaffolds with potent anti-tubercular in vitro activity have been identified from phenotypic screening that appear to target MmpL3. However, the scaffolds are typically lipophilic, which facilitates partitioning into hydrophobic membranes, and several contain basic amine groups. Highly lipophilic basic amines are typically cytotoxic against mammalian cell lines and have associated off-target risks, such as inhibition of human ether-à-go-go related gene (hERG) and IKr potassium current modulation. The spirocycle compound 3 was reported to target MmpL3 and displayed promising efficacy in a murine model of acute tuberculosis (TB) infection. However, this highly lipophilic monobasic amine was cytotoxic and inhibited the hERG ion channel. Herein, the related spirocycles (1–2) are described, which were identified following phenotypic screening of the Eli Lilly corporate library against M. tuberculosis. The novel N-alkylated pyrazole portion offered improved physicochemical properties, and optimization led to identification of a zwitterion series, exemplified by lead 29, with decreased HepG2 cytotoxicity as well as limited hERG ion channel inhibition. Strains with mutations in MmpL3 were resistant to 29, and under replicating conditions, 29 demonstrated bactericidal activity against M. tuberculosis. Unfortunately, compound 29 had no efficacy in an acute model of TB infection; this was most likely due to the in vivo exposure remaining above the minimal inhibitory concentration for only a limited time.

Drug discovery on ion channels is a slow and complicated process and demands a high throughput system with high data quality, but also with a flexible design and easy-to-analyze data. Sophion Qube is a next-generation giga-seal automated patch clamp (APC) screening instrument, capable of testing thousands of compounds with a single click on a button. Data analysis is as important as data acquisition. In HTS the need for powerful analysis with efficient quality filtering is evident in order to handle the vast amount of data generated on an electrophysiological platform.

Qube is capable of testing up to 16 different cell lines or cell clones in one experiment. This can be utilized to test a different panel of cell lines or for selecting the best suited cell clone before embarking on a HTS campaign. The integrated analysis software, Sophion Analyzer, ensures analysis to keep track of all the results and whenever another QChip is assayed the analysis is done with the same set of user defined criteria.
Here we demonstrate the power of automated analysis by exploring three types of experiments executed on eight different Nav channel subtypes; 1) TTX sensitivity, 2) IV-relationship for activation and inactivation and 3) pulse train suitable for screening for use dependent sodium channels blockers. For every run Nav1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7 and 1.8 were tested in parallel on a QChip. Recorded ion channel whole-cell currents were automatically analyzed for IV-relationships for activation and inactivation (V½ and Boltzmann fit and time constants) and concentration-dependent drug effects (Hill fit and IC50) were analyzed using the Sophion Analyzer. If preferred, data can by a click be exported and calculated by other programs as Spotfire, Genedata screener or implemented in in-house software.
For each subtype, the experiments identified the expected pharmacology for use- and state-dependent drugs as well as biophysical properties. The findings determined the differences between the different subtypes as expected and also that post experiment analysis can be performed with minimum of effort when using integrated, automated analysis software.

High throughput in vitro profile of the cardiac Na_V1.5 peak sodium current (I_Na) is widely used in cardiac safety screening. However, there is no standardized high throughput method to measure late I_Na. A variety of differing protocols used across industry and academia may contribute to variation in the data.
The objectives of this study were to (1) assess pharmacology and biophysical properties of veratridine- and ATX-II-induced late INa together with that induced by a mutation in the channel (ΔKPQ-Na_V1.5), (2) develop a protocol to allow simultaneous measurement of both peak and late INa under a single protocol using automated QPatch system. The planar patch clamp technique (QPatch) was applied to record the peak and late I_Na from the Na_V1.5 channel (human SCN5A gene) or ΔKPQ-Na_V1.5 mutant channel expressed in mammalian cells.
When measured at the maximal response during the ramp of the voltage protocol, the ΔKPQ-Na_V1.5 mutant produced a small late I_Na (41.9 ± 5.4 pA). Veratridine and ATX-II-induced concentration-dependent increases in the late INa. The amplitude of late I_Na were 1162.2 ± 258.5 pA and 392.4 ± 71.3 pA in the presences of 100 μM veratridine and 100 nM ATX-II, respectively. Veratridine inhibited the peak I_Na
(IC50 = 84.1 ± 10.5 μM) and altered the biophysical properties of the INa. ATX-II showed minimal effects on the peak I_Na and preserved the biophysical properties of the I_Na. In the presence of 100 nM ATX-II, potencies of 25 clinical I_Na blockers on peak and late INa were characterized. In addition, the IC₅₀ values of these clinical INa blockers on peak I_Na correlated well with and without ATX-II. The results also demonstrated that the potency of a compound blocking late I_Na could be either overestimated or underestimated if the late I_Na was measured at the end of the depolarizing pulse versus during the ramp.
In conclusion, in the presence of ATX-II, both peak and late I_Na could be assessed simultaneously under a single protocol. Our results suggest that late I_Na may be best assessed using the maximum response obtained during the ramp after 200 ms depolarizing pulse at 40 mV.

With the ever-increasing emphasis on ion channel profiling in safety screening, there has been a drive towards increased throughput with respect to patch clamp profiling. The advent of automated 384 well plate based planar patch clamp instruments such as the Qube^® have significantly aided in this effort, allowing for a significant increase in the number of compounds that can be profiled in a time and cost-effective manner.

To further explore the possibility of increasing efficiencies in these systems we examined whether it would be possible to measure multiple currents from separate cell lines in a single well of a 384 well plate using the Qube^® instrument. Theoretically, the instrument lends itself to this possibility due to the virtue of being able to select plates that contain multiple holes per well. Hence by adding two independent cell lines, each independently stably expressing a recombinant ion channel, it should be possible to patch two or more cell lines per well.

We have compared a variety of conditions in the study in order to generate a robust assay that allows for the simultaneous profiling of both the Na_V1.5 peak and hERG currents. Na_V1.5 and hERG currents were separated temporally by virtue of the design of the voltage protocol. Our results show that the success rates of simultaneously recording both Na_V1.5 and hERG currents in each well were 34.4%, 87.5%, 93.8%, 93.8% and 93.8% in 2, 6, 10, 16 and 36 hole/well QChips, respectively. The potencies of 28 CiPA compounds on Na_V1.5
peak and hERG currents were determined individually in a single cell line format or in a mixed cell format using a 10 hole/well QChip. The correlation of the IC50 for the compounds was excellent for both hERG (R2=0.96) and Na_V1.5 (R2=0.86) when comparing the single cell line format with the mixed cell format.

Our data demonstrate that is possible to generate a robust and reproducible assay using a single voltage protocol and buffer combination that allows for the measurement of the effects of compounds on both hERG and Na_V1.5 currents simultaneously. Such a format can both significantly increase assay throughput and reduce assay costs while maintaining data quality.

High throughput in vitro profiling of the cardiac Na_V1.5 peak sodium current (I_Na) is widely used in cardiac safety screening. However, there is no standardized high throughput method to measure late I_Na. This study assessed the pharmacological and biophysical properties of veratridine and ATX-II, as well as the channel mutation (Na_V1.5-∆KPQ) on the late I_Na. We describe a method for simultaneous measurement of both peak and late I_Na.

Kv1.5

Selective block of Na_V1.7 promises to produce non-narcotic analgesic activity without motor or cognitive impairment. Several Na_V1.7-selective blockers have been reported, but efficacy in animal pain models required high multiples of the IC₅₀for channel block. Here, we report a target engagement assay using transgenic mice that have enabled the development of a second generation of selective Nav1.7 inhibitors that show robust analgesic activity in inflammatory and neuropathic pain models at low multiples of the IC₅₀. Like earlier arylsulfonamides, these newer acylsulfonamides target a binding site on the surface of voltage sensor domain 4 to achieve high selectivity among sodium channel isoforms and steeply state-dependent block. The improved efficacy correlates with very slow dissociation from the target channel. Chronic dosing increases compound potency about 10-fold, possibly due to reversal of sensitization arising during chronic injury, and provides efficacy that persists long after the compound has cleared from plasma.

To save time and cost for drug discovery, a paradigm shift in cardiotoxicity testing is required. We introduce a novel screening system for drug-induced arrhythmogenic risk that combines in vitro pharmacological assays and a multiscale heart simulator. For 12 drugs reported to have varying cardiotoxicity risks, dose-inhibition curves were determined for six ion channels using automated patch clamp systems. By manipulating the channel models implemented in a heart simulator consisting of more than 20 million myocyte models, we simulated a standard electrocardiogram (ECG) under various doses of drugs. When the drug concentrations were increased from therapeutic levels, each drug induced a concentration-dependent characteristic type of ventricular arrhythmia, whereas no arrhythmias were observed at any dose with drugs known to be safe. We have shown that our system combining in vitro and in silico technologies can predict drug-induced arrhythmogenic risk reliably and efficiently.

Cysteine-knot miniproteins (knottins) have potential as therapeutic agents to block ion channels involved in cancer, autoimmunity and pain but suffer from manufacturing difficulties, short half-lives and a lack of specificity. IONTAS have invented a novel molecular format wherein a peripheral CDR loop (e.g. VL CDR2) of an antibody has been removed and replaced by a naturally occurring knottin. In this novel format (termed a KnotBodyTM), the knottin enjoys the extended half-life of an antibody molecule and the peripheral CDRs gain additional diversity within a scaffold which is pre-disposed to blockade of ion channels. This example of successful fusion of one structural domain within another was initially achieved by inserting a trypsin binding knottin (EETI-II) flanked by diverse repertoire of short linker sequences into the CDR2 position of naïve antibody light chain sequences. Functional KnotBodies were selected from this library using phage display technology on the basis of retained trypsin binding and the correct folding of both domains were confirmed using X-ray crystallography. To further demonstrate the merits of this novel format, the modular nature of the KnotBody binding surface was exploited to: (i) improve existing knottin binding by introducing additional VHcontacts; (ii) create a bispecific molecule by introducing a VH chain that binds to a different target; (iii) engineer novel binding specificity on the knottin scaffold by loop diversification; (iv) substitute the selected (EETI-II trypsin binding) knottin with ion channel blocking knottins.

A scaffold-hopping strategy toward Agomelatine based on in silico screening and knowledge analysis was employed to design novel antidepressant agents. A series of 3, 4-dihydroisoquinoline compounds were selected for chemical synthesis and biological assessment. Three compounds (6a-1, 6a-2, 6a-9) demonstrated protective effects on corticosterone-induced lesion of PC12 cells. Compound 6a-1 also displayed low inhibitory effects on the growth of HEK293 and L02 normal cells and it was further evaluated for its potential antidepressant effects in vivo. The forced swim test (FST) results revealed that compound 6a-1 remarkably reduced the immobility time of rats and the open field test (OFT) results indicated a better general locomotor activity of the rats treated with compound 6a-1 than those with Agomelatine or Fluoxetine. Mechanism studies implied that compound 6a-1 can significantly reduce PC12 cell apoptosis by up-regulation of GSH and down-regulation of ROS in corticosterone-induced lesion of PC12 cells. Meanwhile, the down-regulation of calcium ion concentration and up-regulation of BDNF level in PC12 cells may account for the neuroprotective effects. Furthermore, compound 6a-1 can increase cell survival and cell proliferation, promote cell maturation in the rat hippocampus after chronic treatment. The acute toxicity data in vivo indicated compound 6a-1 exhibited less hepatotoxicity than Agomelatine.

Alterations in the ratio of excitatory to inhibitory transmission are emerging as a common component of many nervous system disorders, including autism spectrum disorders (ASDs). Tonic γ-aminobutyric acidergic (GABAergic) transmission provided by peri- and extrasynaptic GABA type A (GABAA) receptors powerfully controls neuronal excitability and plasticity and, therefore, provides a rational therapeutic target for normalizing hyperexcitable networks across a variety of disorders, including ASDs. Our previous studies revealed tonic GABAergic deficits in principal excitatory neurons in the basolateral amygdala (BLA) in the Fmr1–/y knockout (KO) mouse model fragile X syndrome. To correct amygdala deficits in tonic GABAergic neurotransmission in Fmr1–/y KO mice, we developed a novel positive allosteric modulator of GABAA receptors, SGE-872, based on endogenously active neurosteroids. This study shows that SGE-872 is nearly as potent and twice as efficacious for positively modulating GABAA receptors as its parent molecule, allopregnanolone. Furthermore, at submicromolar concentrations (≤1 μM), SGE-872 is selective for tonic, extrasynaptic α4β3δ-containing GABAA receptors over typical synaptic α1β2γ2 receptors. We further find that SGE-872 strikingly rescues the tonic GABAergic transmission deficit in principal excitatory neurons in the Fmr1–/y KO BLA, a structure heavily implicated in the neuropathology of ASDs. Therefore, the potent and selective action of SGE-872 on tonic GABAA receptors containing α4 subunits may represent a novel and highly useful therapeutic avenue for ASDs and related disorders involving hyperexcitability of neuronal networks.

The voltage-gated potassium channel K_V2.1 is the main contributor to the delayed rectifier potassium current in neurons and in the cortex (Du et al. 1998, Murakoshi et al. 1999). It has been suggested that K_V2.1 activation confers neuroprotection in a state of neuronal hyperexcitability via hyperpolarizing the membrane potential (Du et al. 2000, Misonou et al. 2005, Misonou et al. 2005a). Therefore, K_V2.1 may be an attractive target in epilepsy research. Also, the ion channel has previously been shown to play a central role in neurodegeneration, ageing and pancreatic β cell signaling.

It has been shown that the human muscle cell line TE671 has an endogenous nicotinic acetylcholine alpha 1 receptor (nAChRα1) (Morris et al,. 1991). TE671 cells have therefore been used for experiments with nAChRα1 as a target. This brief report describes the experimental set-up of TE671 and nAChRα1 on the QPatch.

Many spider venom-peptides are known to modulate the activity of the voltage-gated sodium channel Na_V1.7, which has emerged as a promising analgesic target. A class of spider venom-peptides (NaSpTx1) in particular have been found to potently inhibit Na_V1.7 (nanomolar IC50), and shown to produce analgesic effects in animals. However, one member of this family, μ TRTX Hhn2b (Hhn2b), does not inhibit mammalian Na_V channels expressed in dorsal root ganglia at concentrations up to 100 μM. This peptide is classified as a NaSpTx1 member by virtue of its cysteine spacing and sequence conservation over functionally important residues. Here, we have performed detailed structural and functional analyses of Hhn2b leading us to identify two non-pharmacophore residues that contribute to hNa_V1.7 inhibition by non-overlapping mechanisms. These findings allowed us to produce a double mutant of Hhn2b that shows nanomolar inhibition of hNa_V1.7. Traditional structure/function analysis did not provide sufficient resolution to identify the mechanism underlying the observed gain of function. However, by solving the high-resolution structure of both the wild type and mutant peptides using advanced multidimensional NMR experiments, we were able to uncover a previously unknown network of interactions that stabilize the pharmacophore region of this class of venom-peptides. We further monitored the lipid binding properties of the peptides and identified that one of the key amino acid substitutions also selectively modulates the binding of the peptide to anionic lipids. These results will further aid the development of peptide-based analgesics for the treatment of chronic pain.

The MaxCyte® STX™ Scalable Transfection System uses a proprietary flow electroporation technology that can transfect up to 1E10 cells with target, reporter and protein expression plasmids, as well as other molecules, in less than 30 minutes. Transfected cells can be assayed immediately or cryopreserved for future use. Here we demonstrate the use of the MaxCyte STX system in coordination with Corning’s ® HYPERFlask® Cell Culture Vessel to provide an efficient and economical solution for culturing large numbers of adherent CHO cells before and after transfection. The HYPERFlask Cell Culture Vessel features Corning’s HYPER (High Yield PERformance) technology, which utilizes a gas permeable film to provide gas exchange between the internal culture environment and the external atmospheric environment. The unique, space-saving, 10-layer film design results in 1720 cm2 cell growth surface area, which is approximately 10 times that of a normal T-175 flask. We also show that cells transfected with a plasmid encoding a Kv1.3-GFP fusion protein exhibited good seal formation and strong potassium currents following large scale electroporation and cryopreservation. Ion channel activity was assayed on the Sophion QPatch automated patch clamp system. High viability and consistent levels of expression were obtained in three independent large scale electroporations, illustrating robustness and reproducibility of the transfection process. These results demonstrate that the MaxCyte STX system offers a time and labor saving alternative to stable cell line generation for ion channel assays.

The Comprehensive in vitro Proarrhythmia Assay (CiPA) proposal provides an attractive perspective for increasing the efficacy of the drug development process. A detailed electrophysiological analysis of Na_v1.5 (peak and late currents), K_v4.3 (I_to), hERG (I_Kr), KvLQT1/minK (I_Ks), Ca_v1.2 and Kir2.1 (IK1) upon addition of a potential drug is a major part of assessing the drug’s proarrhythmic risk. Gaining a better understanding of the complex relationship between QT-elongation and the occurence of Torsades des Pointes (TdP), potentially enables compounds with properties that today are considered as problematic to be further developed. High throughput screening (HTS) supports this quest and is furthermore of great importance for discovering pharmacologically active substances and understanding ion channels.

Qube is a giga-seal automated patch clamp (APC) instrument, providing 384 amplifiers for the consumable QChip 384 with its integrated electrodes. The QChip has built-in microfluidic flow channels that ensure a fast and complete exchange of liquid for reliable measurements on ligand-gated ion channels and sequential additions to the same site. Here, four different cell lines expressing the cardiac ion channels Na_v1.5, Ca_v1.2, K_v1.5 and hERG, were transferred from a cell clone cell transfer plate (ccCTP) onto the same QChip. A range of pharmacological substances and voltage step protocols were applied to address the suitability of Qube for measuring different cell populations in parallel.

The Qube is the new gigaseal-based 384-channel planar patch clamp system developed by Sophion Bioscience A/S. The system is equipped with 384 individual amplifiers providing continuous recordings at a sampling rate up to 50 kHz from all wells simultaneously. The consumable for the Qube is built on Sophion’s tried and tested silicon technology for optimal giga-seal formation and has been designed to have an effi cient liquid fl ow system which is necessary for testing fast ligand-gated ion channels (LGIC) such as the nicotine acetylcholine receptors (nAChR). In this work we present recordings obtained using the Qube system from two LGICs: ASIC1a and nAChRα1.
The acid-sensitive channel (ASIC) belongs to the ENaC/DEG family. ASIC1a is expressed in the brain and in the peripheral nervous system where it is involved in modulating response to pain (1). The nicotinic acetylcholine receptor alpha 1 (nAChRα1) belongs to the Cys-loop family and is expressed both centrally and in the peripheral nervous system. In the periphery nAChRα1 modulate e.g. the synaptic transmission at the neuromuscular junction (2). Here we show activation of these LGICs by their respective ligand (H+ and acethylcholine (ACh), respectively) and pharmacological block by known reference compounds (amiloride and tetracaine, respectively). These data demonstrate: 1) the design of the Qube consumable allows fast liquid exchange for recordings of fast LGICs and 2) the design of the Qube consumable allows multiple liquid additions to the same recording unit.

Drug discovery on ion channel targets has been an under-explored territory because suitable screening tools for patch clamp were missing. This changed during the 2000’s with the introduction of automated patch clamp (APC) devices. Still, the throughput and running cost of APC devices did not allow for the employment of the patch clamp assays in primary screening. Because of this bottleneck, APC has remained a secondary screening tool. We are now presenting data from the first true gigaseal-based 384-channel planar patch clamp system, the Qube, capable of providing the throughput needed for primary screening. The Qube is a collection of years of experience with planar patch clamp devices: High quality, reliability and easy to use concepts are the core in both the tried-and-tested silicon technology of the consumable, and in the technologies used in the instrument. The Qube offers GΩ seals and efficient integrated liquid flow in 384-well format.
We show here that the Qube provides exceptional data quality for electrophysiological assays with fast desensitizing ligand-gated ion channels. The fast liquid exchange permits accurate XC50 estimations. We have tested the acid sensing ion channel (ASIC1) and the nicotinic acetylcholine receptor (nAchRα1) on the Qube in both agonist and antagonist configurations, showcasing the instrument’s broad flexibility in terms of assay design. The results prove that: 1) Repetitive agonist applications result in reproducible current responses. 2) The agonist can be applied and washed off an unlimited number of times as long as the cell is stable. 3) XC50 values correspond to expected values (QPatch and literature). Collectively, our results show that the design of the flow channels on the Qube consumable enables recordings on ligand-gated ion channels with the highest throughput capacity seen so far, without compromising data quality, reproducibility or reliability.

• QPatchを用いて３型QT延長症候群の変異チャネルから振幅の小さなNaV1.5 (late)電流を高精度で記録した．
• 薬理的な活性化剤を用いずにNaV1.5 (late)電流を安定して記録することで薬物の安全性評価がより高い信頼性で実施可能となった．
• Nav1.5 (late)電流に作用するNaチャネル阻害剤性を用いて新たに開発した試験法の薬理学的バリデーションを実施した．

心毒性における副作用は，未だに創薬プロセスにおいて新規化合物の開発が中止される要因となっており（Valentin and Redfern, 2017），ヒトの臨床リスク予測のためには前臨床においてより厳格なin vitro, ex vivoおよびin vivoアッセイやモデルが必要とされている．現在製薬業界では従来のヒト遅延整流性カリウムイオンチャネル（hERG, K_V11.1） アッセイおよびQT延長検出アッセイに対する過度の依存から脱却する方向にあり，特に心不整脈による患者のリスク予測についてよりバランスの取れたアッセイを提供する新たな取り組みが進められている．FDAが主導するComprehensive in vitro Proarrhythmia Assay（CiPA）と日本の規制当局（JiCSA，CSAHi）は，パッチクランプによる電気生理学スクリーニングパネルに他のヒト心臓イオンチャネルを追加することでより正確にモデル化し，不整脈リスクを予測することを目指している．この広範なデータを最新式のヒト心室活動電位のin silicoモデルで解析することにより不整脈の予測が行われる．英国，EU，米国，日本のワーキンググループの5年間にわたる広範な共同作業の結果，心筋イオンチャネルアッセイの‘ビッグ6’パネル（hERG，Na_V1.5，Ca_V1.2， K_VLQT1，K_V4.3，およびK_ir2.1）から得られたデータを，標準的なin silico活動電位モデルに組み入れることで，十分にリスクプロファイルが明らかになったほとんどの臨床薬（ただしすべてではない）について，催不整脈リスクを正確に予測できることが明らかとなった．

心毒性における副作用は，未だに創薬プロセスにおいて新規 化合物の開発が中止される要因となっており（Valentin and Redfern, 2017），ヒトの臨床リスク予測のためには前臨床に おいてより厳格なin vitro, ex vivoおよびin vivoアッセイやモ デルが必要とされている．現在製薬業界では従来のヒト遅延整 流性カリウムイオンチャネル（hERG, KV11.1） アッセイおよ びQT延長検出アッセイに対する過度の依存から脱却する方向 にあり，特に心不整脈による患者のリスク予測についてよりバ ランスの取れたアッセイを提供する新たな取り組みが進められ ている．FDAが主導するComprehensive in vitro Proarrhythmia Assay（CiPA）と日本の規制当局（JiCSA，CSAHi）は，パッチクランプによる電気生理学スクリーニングパネルに他
のヒト心臓イオンチャネルを追加することでより正確にモデ ル化し，不整脈リスクを予測することを目指している．この 広範なデータを最新式のヒト心室活動電位のin silicoモデルで 解析することにより不整脈の予測が行われる．英国，EU，米 国，日本のワーキンググループの5年間にわたる広範な共同 作業の結果，心筋イオンチャネルアッセイの‘ビッグ6’パネル （hERG，NaV1.5，CaV1.2， KVLQT1，KV4.3，およびKir2.1 ）から得られたデータを，標準的なin silico活動電位モデルに 組み入れることで，十分にリスクプロファイルが明らかになったほとんどの臨床薬（ただしすべてではない）について，催不 整脈リスクを正確に予測できることが明らかとなった．FDAおよびCiPAワーキンググループによる最近の研究では，いわゆる ‘Milnes’電位プロトコール（Milnes et al., 2010; 図 1）で得られたhERG-薬物動態データを，改良型の‘ダイナミッ ク’O’Hara-Rudy in silicoモデルに適用することで，キニジン やシサプリドなどの化合物の心臓への負荷を正確に予測できる ことが示された（Li et al., 2017）．薬物のhERGチャネルへ の結合と解離の動態は化合物の効力を決定する基本的要素で あるが，チャネルのポアに捕捉される化合物がより高い臨床 でのリスクを有するという証拠が示された（Pearlstein et al.,2016）．薬物の作用，効力および心臓への負荷の重要性を鑑 み，hERGチャネルへの結合動態特性および薬物捕捉性を確実 に測定するために，これまでは高忠実度が保証されたマニュア パッチクランプ記録のみが採用されてきた．本レポートでは，自動パッチクランプ（APC）プラットフォー ム上で，ダイナミックな薬物-hERG動態を正確に記録すること ができる‘Milnes’電位プロトコールの適用例を報告する.

The QPatch 16 significantly increases throughput for gigaseal patch clamp experiments, making direct measurements in ion channel drug discovery and safety testing feasible. Released to the market in the Autumn of 2004 by Sophion Bioscience, the QPatch originated from work done at NeuroSearch (Denmark) in the early days of automated patch clamp. Today, the QPatch provides many unique features. For example, only the QPatch includes an automated cell preparation station making several hours of unattended operation possible. The 16-channel electrode array, called the QPlate, includes glass-coated microfluidic channels for less compound absorption and, hence, more accurate IC50 values. The microfluidic pathways also allow for very small amounts of compound used for each experiment (∼ 5 µl per addition). Only the QPatch has four independent pipetting heads for more efficient liquid handling (especially for ligand-gated ion channel experiments). Patch clamp recordings with the QPatch match the high quality of conventional patch clamp and in some cases the results are even better. For example, only the QPatch includes 100% series resistance compensation for the elimination of false positives due to voltage errors. Finally, the modular QPatch 16 was designed with more channels in mind. The upgrade pathway to 48-channels (the QPatch HT) will be discussed.

The conventional patch clamp has long been considered the best approach for studying ion channel function and pharmacology. However, its low throughput has been a major hurdle to overcome for ion channel drug discovery. The recent emergence of higher throughput, automated patch clamp technology begins to break this bottleneck by providing medicinal chemists with high-quality, information-rich data in a more timely fashion. As such, these technologies have the potential to bridge a critical missing link between high-throughput primary screening and meaningful ion channel drug discovery programs. One of these technologies, the QPatch automated patch clamp system developed by Sophion Bioscience, records whole-cell ion channel currents from 16 or 48 individual cells in a parallel fashion. Here, we review the general applicability of the QPatch to studying a wide variety of ion channel types (voltage-/ligand-gated cationic/anionic channels) in various expression systems. The success rate of gigaseals, formation of the whole-cell configuration and usable cells ranged from 40-80%, depending on a number of factors including the cell line used, ion channel expressed, assay development or optimization time and expression level in these studies. We present detailed analyses of the QPatch features and results in case studies in which secondary screening assays were successfully developed for a voltage-gated calcium channel and a ligand-gated TRP channel. The increase in throughput compared to conventional patch clamp with the same cells was approximately 10-fold. We conclude that the QPatch, combining high data quality and speed with user friendliness and suitability for a wide array of ion channels, resides on the cutting edge of automated patch clamp technology and plays a pivotal role in expediting ion channel drug discovery.

Native cells represent a promising alternative to cell lines due to more physiological behaviour.

Primary brain neurons and astrocytes were tested on Qube 384

• Neuronal Na+ and K+ currents were measured in voltage clamp
• Neuronal action potentials were measured in current clamp
• Astrocyte K+-currents were elicited with both ramp- and step-protocols

A series of dihydropyrazole derivatives was developed as potent, selective, and brain-penetrating T-type calcium channel blockers. An optimized derivative, compound 6c, was advanced to in vivo studies, where it demonstrated efficacy in the WAG/Rij rat model of generalized nonconvulsive, absence-like epilepsy. Compound 6c was not efficacious in the basolateral amygdala kindling rat model of temporal lobe epilepsy, and it led to prolongation of the PR interval in ECG recordings in rodents.

Recent work by FDA and HESI CiPA working groups indicate that in vitro hERG, Nav1.5 and Cav1.2 potency data in addition to dynamic hERG kinetic data is required to accurately predict proarrhythmic risk. Below we explore two key challenges in exclusively using automated patch clamp for risk prediction:

1. Metrion has previously shown the ability to implement the difficult Milnes protocol on QPatch but the challenges of producing full concentration-response formats required for in silico models are unknown
2. Cav1.2 inhibition values for compounds such as verapamil have been
   variable, with literature IC50 values >10 µM compared to the 202 nM IC50 obtained by manual patch clamp in the original CiPA study. The similar potency of verapamil against Cav1.2 and hERG make it a Multiple Ion Channel Effect (MICE) compound and “rescues” it from being classed as a high-risk proarrhythmia compound as shown by Li et al.
3. QPatch dynamic hERG and Cav1.2 data from multiple voltage protocols
   were used to assess proarrhythmic risk using the FDA’s in silico model.

The activity of positive allosteric modulators (PAMs) of α7 nicotinic acetylcholine receptors (AChRs), including 3-furan-2-yl-N-p-tolyl-acrylamide (PAM-2), 3-furan-2-yl-N-o-tolylacrylamide (PAM-3), and 3-furan-2-yl-N-phenylacrylamide (PAM-4), was tested on a variety of ligand- [i.e., human (h) α7, rat (r) α9α10, hα3-containing AChRs, mouse (m) 5-HT3AR, and several glutamate receptors (GluRs)] and voltage-gated (i.e., sodium and potassium) ion channels, as well as on acetylcholinesterase (AChE) and β-amyloid (Aβ) content. The functional results indicate that PAM-2 inhibits hα3-containing AChRs (IC50 = 26 ± 6 μM) with higher potency than that for NR1aNR2B and NR1aNR2A, two NMDA-sensitive GluRs. PAM-2 affects neither the activity of m5-HT3ARs, GluR5/KA2 (a kainate-sensitive GluR), nor AChE, and PAM-4 does not affect agonist-activated rα9α10 AChRs. Relevant clinical concentrations of PAM-2–4 do not inhibit Nav1.2 and Kv3.1 ion channels. These PAMs slightly enhance the activity of GluR1 and GluR2, two AMPA-sensitive GluRs. PAM-2 does not change the levels of Aβ42 in an Alzheimer’s disease mouse model (i.e., 5XFAD). The molecular docking and dynamics results using the hα7 model suggest that the active sites for PAM-2 include the intrasubunit (i.e., PNU-120596 locus) and intersubunit sites. These results support our previous study showing that these PAMs are selective for the α7 AChR, and clarify that the procognitive/promnesic/antidepressant activity of PAM-2 is not mediated by other targets.

Selective inhibitors of voltage-activated K⁺ channels are needed for the treatment of multiple sclerosis. In this work it was discovered that porphyrins bearing 2–4 carbon alkyl ammonium side chains predominantly blocked the K_v1.1 current whilst K_v1.2 was susceptible to a porphyrin bearing polyamine side chains.

Many rapidly firing neurons, including those in the Medial Nucleus of the Trapezoid Body (MNTB) in the auditory brainstem, express “high threshold” voltage-gated K_v3.1 potassium channels that activate only at positive potentials and are required for stimuli to generate rapid trains of actions potentials. We now describe the actions of two imidazolidinedione derivatives, AUT1 and AUT2, which modulate K_v3.1 channels. Using CHO cells stably expressing rat K_v3.1 channels, we found that lower concentrations of these compounds shift the voltage of activation of K_v3.1 currents towards negative potentials, increasing currents evoked by depolarization from typical neuronal resting potentials. Single channel recordings also showed that AUT1 shifted the open probability of K_v3.1 to more negative potentials. Higher concentrations of AUT2 also shifted inactivation to negative potentials. The effects of lower and higher concentrations could be mimicked in numerical simulations by increasing rates of activation and inactivation respectively, with no change in intrinsic voltage-dependence. In brain slice recordings of mouse MNTB neurons, both AUT1 and AUT2 modulated firing rate at high rates of stimulation, a result predicted by numerical simulations. Our results suggest that pharmaceutical modulation of K_v3.1 currents represents a novel avenue for manipulation of neuronal excitability, and has the potential for therapeutic benefit in the treatment of hearing disorders.

Venomous animals occupy one of the most successful evolutionary niches and occur on nearly every continent. They deliver venoms via biting and stinging apparatuses with the aim to rapidly incapacitate prey and deter predators. This has led to the evolution of venom components that act at a number of biological targets – including ion channels, G-protein coupled receptors, transporters and enzymes – with exquisite selectivity and potency, making venom-derived components attractive pharmacological tool compounds and drug leads. In recent years, plate-based pharmacological screening approaches have been introduced to accelerate venom-derived drug discovery. A range of assays are amenable to this purpose, including high-throughput electrophysiology, fluorescence-based functional and binding assays. However, despite these technological advances, the traditional activity-guided fractionation approach is time-consuming and resource-intensive. The combination of screening techniques suitable for miniaturization with sequence-based discovery approaches – supported by advanced proteomics, mass spectrometry, chromatography as well as synthesis and expression techniques – promises to further improve venom peptide discovery. Here, we discuss practical aspects of establishing a pipeline for venom peptide drug discovery with a particular emphasis on pharmacology and pharmacological screening approaches.

Studies of GABA_A ion channels using the automated patch clamp platform Qube 384 with focus on:

• Short ligand exposure with repetitive stimulations with EC₅₀
  concentrations of GABA
• Effects of agonists, antagonists and modulators
• Cumulative and non-cumulative concentration-response relationships
• Characterizing the pharmacological properties of four cell lines expressing different GABA_A subtypes

We here report pharmacological data (EC₅₀ and IC₅₀ values, tests for state-dependency of blockers and steady-state inactivation characteristics) from eight different types of voltage- and ligand-gated ion channels using the automated QPatch 16 system. The data obtained were in accordance with published literature values (not shown). We conclude that the QPatch technology enables fast and reliable characterization of electrophysiological ion channel properties.

Human genetic studies have implicated the voltage-gated sodium channel Na_V1.7 as a therapeutic target for the treatment of pain. A novel peptide, μ-theraphotoxin-Pn3a, isolated from venom of the tarantula Pamphobeteus nigricolor, potently inhibits Na_V1.7 (IC₅₀ 0.9 nM) with at least 40–1000-fold selectivity over all other Na_Vsubtypes. Despite on-target activity in small-diameter dorsal root ganglia, spinal slices, and in a mouse model of pain induced by Na_V1.7 activation, Pn3a alone displayed no analgesic activity in formalin-, carrageenan- or FCA-induced pain in rodents when administered systemically. A broad lack of analgesic activity was also found for the selective Na_V1.7 inhibitors PF-04856264 and phlotoxin 1. However, when administered with subtherapeutic doses of opioids or the enkephalinase inhibitor thiorphan, these subtype-selective Na_V1.7 inhibitors produced profound analgesia. Our results suggest that in these inflammatory models, acute administration of peripherally restricted Na_V1.7 inhibitors can only produce analgesia when administered in combination with an opioid.

In isolated human atrial cardiomyocytes, inhibition of K_2P3.1 K⁺ channels results in action potential (action potential duration (APD)) prolongation. It has therefore been postulated that K_2P3.1 (KCNK3), together with K_2P9.1 (KCNK9), could represent novel drug targets for the treatment of atrial fibrillation (AF). However, it is unknown whether these findings in isolated cells translate to the whole heart. The purposes of this study were to investigate the expression levels of KCNK3 and KCNK9 in human hearts and two relevant rodent models and determine the antiarrhythmic potential of K_2P3.1 inhibition in isolated whole-heart preparations. By quantitative PCR, we found that KCNK3 is predominantly expressed in human atria whereas KCNK9 was not detectable in heart human tissue. No differences were found between patients in AF or sinus rhythm. The expression in guinea pig heart resembled humans whereas rats displayed a more uniform expression of KCNK3 between atria and ventricle. In voltage-clamp experiments, ML365 and A293 were found to be potent and selective inhibitors of K_2P3.1, but at pH 7.4, they failed to prolong atrial APD and refractory period (effective refractory period (ERP)) in isolated perfused rat and guinea pig hearts. At pH 7.8, which augments K_2P3.1 currents, pharmacological channel inhibition produced a significant prolongation of atrial ERP (11.6 %, p = 0.004) without prolonging ventricular APD but did not display a significant antiarrhythmic effect in our guinea pig AF model (3/8 hearts converted on A293 vs 0/7 hearts in time-matched controls). These results suggest that when K_2P3.1 current is augmented, K_2P3.1 inhibition leads to atrial-specific prolongation of ERP; however, this ERP prolongation did not translate into significant antiarrhythmic effects in our AF model.

In patch clamp recordings cell dialysis of intracellular constituents can be minimized by employing perforated whole-cell formation.
This method allows electrical access to the cell while maintaining the integrity of the majority of cytoplasmic components. Here we report how to perform perforated patch clamp recordings on Qube 384 with or without the use of intracellular (IC) solution exchange (IC exchange).
• Recommendations for perforated patch clamp experiments with or without using IC exchange
• Comparison of four perforating agents (seal resistance, current level and success rate)
The first part of this report consists of a fast guide for using perforated patch on Qube 384, followed by an in-depth run-through of the experiments underlying our recommendations

Many ion channels are recognized as amenable targets for a range of disease states and conditions. However, the process of discovering drugs is highly influenced by the chemical doability, the biological confidence in rationale of the approach and the ‘screenability’. To date, the absence of informative high throughput technologies for ion channel screening has resulted in ion channels remaining a largely unexplored class of drug targets. This, however, is about to change – a large increase in the number of data points per day should be achieved by the introduction of automated ‘high throughput’ patch clamp machines.

The advent of higher throughput patch clamp electrophysiology systems has begun to change the face of ion channel drug discovery. Systems such as IonWorksHT, PatchXpress and QPatch should allow electrophysiology to be re-positioned from an occasional reagent and compound ‘spot-check’ method, to a frontline gene expression analysis tool and drug screening workhorse. This article chronicles the evolution of these systems, their technical capabilities and impact on drug discovery.

The human α7 nicotinic acetylcholine receptor (α7-nAChR) is a neuronal ligand-gated, fast desensitizing, non-selective cation channel. It is pentahomomeric, consisting of five 50 kD α7 subunits, each composed of 502 amino acids. The α7-nAChR is involved in memory and cognition, and it is widely distributed throughout the nervous system, especially in cholinergic neurons projecting to hippocampus and cortex. The α7-nAChR has also been found to be associated with pathophysiological states. Importantly, it is involved in widespread human neuro-degenerative and psychotic disorders, including Alzheimer’s disease and schizophrenia. Therefore the therapeutic potential of α7-nAChR is substantial, and electrophysiological and pharmacological characterization of the receptor has become an increasingly important issue. The fast kinetics (milliseconds) makes precise patch clamp measurements a challenging task. The present study addresses the possibility of efficient and precise characterization of α7-nAChR function and pharmacology by means of the QPatch automated patch clamp system. Compared to conventional whole-cell patch clamp the system enables a highly increased throughput by simultaneous and asynchronous operation of 16 parallel patch clamp experiments.

Cultured cells from mammalian cell lines incubated at 37 °C can be used for automated patch clamp (APC), including compound screening, for only a relatively brief period of time. Usually this time period extends from the time of ~50 % confluence to full confluence. For CHO cells, the preferred cell type for APC, expressing hERG potassium channels the duration of the usable period is 1-2 days. We here report that incubating CHO-hERG cells at a reduced temperature, 30 °C, for 1-5 days prior to APC experiments is advantageous in several ways: (1) it increases the percentage of cells with acceptable hERG currents (> 50 pA), (2) it increases the average whole-cell current significantly, and (3) it
slows cell proliferation and extends the usable period up to at least 5 days. In addition, we have tested whether the altered conditions affect the IC50 obtained for known hERG inhibitors. For all experiments the automated patch clamp system QPatch 16 was used.

Drug-induced QT interval prolongation, ventricular arrhythmia and sudden death are one of the leading causes for drug withdrawal from the market or denied regulatory approval. Preclinical profiling for off-target cardiac ion channel interactions using The Comprehensive in vitro Proarrhythmia
Assay (CiPA) paradigm has been initiated to overcome drug attrition due to hERG inhibition only. The aim is to improve the prediction of a drug’s proarrhythmic liability for the pharmaceutical industry. This new paradigm includes a panel of in vitro assays that integrates effects of the test compounds on several cardiac ion channels. Voltage-gated ion channels in this panel, particularly hCa_V1.2 and hKCNQ1/hminK, exhibit rapid
activities decrease when contact between membrane and cytosol is disrupted upon patch excision (run-down). The loss of channel activity without pharmacological interaction poses a challenge for developing accurate, precise and robust assay yielding high success rate, minimal current run-down and reliable pharmacological results. In the present study, hCa_V1.2 and hKCNQ1/hminK cardiac ionic currents were validated on QPatch HT patch clamp system with the specific focus on preventing current run-down. The results demonstrate suitability of these assays for screening and profiling of drug effects by significantly reducing run-down (<20%) by optimizing protocol parameters. Reference pharmacology was
assessed for Ca_V1.2 (Nifedipine IC₅₀ =144nM) and hKCNQ1/hminK (Chromanol 293B IC₅₀ =12.3μM). The optimized assays for hCa_V1.2 and hKCNQ1/hminK on the QPatch are able to provide data that is comparable to manual patch clamp, which allows the assessment of a novel compounds proarrhythmic risk.

Optical modulation of ion channels is traditionally studied using a manual patch clamp system combined with a light source. This approach, however, is limited by very low throughput. In the present work, we show data recorded using a 384-well based automated patch clamp system equipped with 384 integrated light sources (Qube Opto 384).

Optogenetics
In this study, we evaluated Channelrhodopsin 2 (ChR2), a light-sensitive non-selective cation channel permeable to Na+, K+ and Ca2+ opened upon illumination (Berndt et. al., 2012). Furthermore, we employed the chloride-conducting channelrhodopsin (iC++, Govorunova et Al. 2015), which was developed from a non-selective cation conducting channelrhodopsin through a mutational approach. Finally, we used photoactivatable adenylyl cyclase, bPAC, to modulate cellular cAMP levels and thereby alter biophysical properties of the HCN2 channel (Stierl et Al., 2011).

Optopharmacology
Compound activation by light enables the pharmacological manipulation of receptors, ion channels and other proteins with a high degree of temporal control. We used caged GABA (Rubi-GABA, Zayat et. al., 2003) to study the light activation of ligands, in combination with the microfluidic flow channel of the QChip 384, to give a higher degree of experimental control.

Large-conductance Ca(2+)-activated K(+) channels (BK, KCa1.1, MaxiK) are important regulators of urinary bladder function and may be an attractive therapeutic target in bladder disorders. In this study, we established a high-throughput fluorometric imaging plate reader-based screening assay for BK channel activators and identified a small-molecule positive modulator, NS19504 (5-[(4-bromophenyl)methyl]-1,3-thiazol-2-amine), which activated the BK channel with an EC50 value of 11.0 ± 1.4 µM. Hit validation was performed using high-throughput electrophysiology (QPatch), and further characterization was achieved in manual whole-cell and inside-out patch-clamp studies in human embryonic kidney 293 cells expressing hBK channels: NS19504 caused distinct activation from a concentration of 0.3 and 10 µM NS19504 left-shifted the voltage activation curve by 60 mV. Furthermore, whole-cell recording showed that NS19504 activated BK channels in native smooth muscle cells from guinea pig urinary bladder. In guinea pig urinary bladder strips, NS19504 (1 µM) reduced spontaneous phasic contractions, an effect that was significantly inhibited by the specific BK channel blocker iberiotoxin. In contrast, NS19504 (1 µM) only modestly inhibited nerve-evoked contractions and had no effect on contractions induced by a high K(+) concentration consistent with a K(+) channel-mediated action. Collectively, these results show that NS19504 is a positive modulator of BK channels and provide support for the role of BK channels in urinary bladder function. The pharmacologic profile of NS19504 indicates that this compound may have the potential to reduce nonvoiding contractions associated with spontaneous bladder overactivity while having a minimal effect on normal voiding.

In Gram-negative bacterial sepsis, production of excess pro-inflammatory cytokines results in hyperinflammation and tissue injury. Anti-inflammatory cytokines such as IL-10 inhibit inflammation and enhance tissue healing. Here, we report a novel approach to treat septicemia associated with intra-abdominal infection in a murine model by delicately balancing pro- and anti-inflammatory cytokines. A novel oligosaccharide compound AVR-25 selectively binds to the TLR4 protein (IC50 = 0.15 µM) in human peripheral blood monocytes and stimulates IL-10 production. Following the cecal ligation and puncture (CLP) procedure, intravenous dosing of AVR-25 (10 mg/kg, 6–12 h post-CLP) alone and in combination with antibiotic imipenem protected both young adult (10–12 week old) and aged (16–18 month old) mice against polymicrobial infection, organ dysfunction, and death. Proinflammatory cytokines (TNF-α, MIP-1, i-NOS) were decreased significantly and restoration of tissue damage was observed in all organs. A decrease in serum C-reactive protein (CRP) and bacterial colony forming unit (CFU) confirmed improved bacterial clearance. Together, these findings demonstrate the therapeutic ability of AVR-25 to mitigate the storm of inflammation and minimize tissue injury with high potential for adjunctive therapy in intra-abdominal sepsis.

NIH/3T3 is a mouse embryonic fibroblast cell line. NIH/3T3 cells are established from NIH Swiss mouse embryos. These cells are highly contact inhibited and are sensitive to sarcoma virus focus formation and leukaemia virus propagation. These cells have now lost their contact inhibition. Contact inhibition is the natural process of arresting cell growth when two or more cells come into contact with each other. The established NIH/3T3 line was subjected to more than 5 serial cycles of subcloning in order to develop a subclone with morphologic characteristics best suited for transformation assays. It is therefore used for DNA transfection studies.
Cystic fibrosis transmembrane conductance regulator (CFTR) functions as a chloride channel and controls the regulation of other transport pathways. Mutations in the CFTR gene have been found to cause cystic fibrosis (CF) and congenital bilateral aplasia of the vas deferens (CBAVD). The ΔF508CFTR mutation of the CFTR gene was stably transfected into the NIH/3T3 cell line and used for functional studies on QPatch.

Nicotinic α7 currents are challenging on automated platforms due to the fast desensitizing properties of the channel. This property calls for a cell line with exceedingly good expression of the channel. Sophion has previously successfully tested a rat cell line GH4-C1 expressing α7 however this cell line expresses the isoform of the rat and the cell line is not commercially available (Friis et al, 2009). Galantos has cloned a human isoform into a CHO background and this report describes the data obtained with this cell line. Nicotinic α7 is of interest since it has been found to be involved in many normal and pathological conditions. It is important in memory formation, arousal, and attention, nicotine addiction, and nicotine-induced reversal of age-related memory deficits. α7 activation leads to neuroprotection against amiloid-β-induced neurotoxicity, which suggests that the receptor may play an important role in Alzheimer’s disease. Also their role in hippocampal auditory gating has linked the receptor to schizophrenia.

Small-conductance (K_Ca2) and intermediate-conductance (K_Ca3.1) calcium-activated K⁺ channels are voltage-independent and share a common calcium/calmodulin-mediated gating mechanism. Existing positive gating modulators like EBIO, NS309, or SKA-31 activate both K_Ca2 and K_Ca3.1 channels with similar potency or, as in the case of CyPPA and NS13001, selectively activate K_Ca2.2 and K_Ca2.3 channels. We performed a structure-activity relationship (SAR) study with the aim of optimizing the benzothiazole pharmacophore of SKA-31 toward K_Ca3.1 selectivity. We identified SKA-111 (5-methylnaphtho[1,2-d]thiazol-2-amine), which displays 123-fold selectivity for K_Ca3.1 (EC₅₀ 111 ± 27 nM) over K_Ca2.3 (EC₅₀ 13.7 ± 6.9 μM), and SKA-121 (5-methylnaphtho[2,1-d]oxazol-2-amine), which displays 41-fold selectivity for K_Ca3.1 (EC₅₀ 109 nM ± 14 nM) over K_Ca2.3 (EC₅₀ 4.4 ± 1.6 μM). Both compounds are 200- to 400-fold selective over representative KV (K_V1.3, K_V2.1, K_V3.1, and K_V11.1), Na_V (Na_V1.2, Na_V1.4, Na_V1.5, and Na_V1.7), as well as Ca_V1.2 channels. SKA-121 is a typical positive-gating modulator and shifts the calcium-concentration response curve of KCa3.1 to the left. In blood pressure telemetry experiments, SKA-121 (100 mg/kg i.p.) significantly lowered mean arterial blood pressure in normotensive and hypertensive wild-type but not in K_Ca3.1−/− mice. SKA-111, which was found in pharmacokinetic experiments to have a much longer half-life and to be much more brain penetrant than SKA-121, not only lowered blood pressure but also drastically reduced heart rate, presumably through cardiac and neuronal KCa2 activation when dosed at 100 mg/kg. In conclusion, with SKA-121, we generated a K_Ca3.1-specific positive gating modulator suitable for further exploring the therapeutical potential of K_Ca3.1 activation.

New cardiac safety testing guidelines are being developed as part of the FDA’s Comprehensive in vitro Proarrhythmia Assay (CiPA) initiative, which aims to remove the reliance on screening against the hERG channel by expanding the panel to include other human ventricular ion channels such as Nav1.5, Ca_V1.2, K_V4.3/KChiP2.2, K_ir2.1 and K_V7.1/KCNE1. In addition, the CiPA working groups have recently identified two additional ion channel assay readouts required for in silico models to reliably predict proarrhythmia. The first is a ‘late’ Na_V1.5 assay, as inhibition of persistent inward current can affect repolarisation and mitigate proarrhythmia (e.g. Ranolazine). The second is a kinetic hERG assay that measures drug trapping using the Milnes voltage protocol(1) and improves the prediction of proarrhythmia risk(2). Here we describe validation of these additional CiPA assays on the gigaseal QPatch48 automated patch clamp platform.

As an alternative to standard pharmacological procedures for Na_V1.5(Late) assays, we present a more reliable and accurate Na_V1.5(Late) assay on QPatch that removes the requirement for activators like veratridine and ATX-II and delivers improved cardiac safety screening reliability and cost.

• High fidelity QPatch recordings of small amplitude Na_V1.5 (Late) currents from Long QT3 syndrome mutant channels.
• Stable recordings without pharmacological activators allow more reliable drug potency assessment.
• Pharmacologically validated with sodium channel blockers with a preference for the Na_V1.5(Late).

The voltage dependent sodium channel, Na_V1.5, was tested on QPatch 16X in single-hole and multi-hole mode. Single-hole mode is a classic patch clamp experiment where one cell is in whole-cell configuration, whereas multi-hole mode comprises up to 10 cells in whole-cell configuration. The multi-hole mode therefore tests the summed current of up to ten cells.
In this study, we wanted to determine the best test protocol to distinguish between two antiarrythmic drugs with different modes of action: flecainide, which blocks open Na_v channels, and lidocaine, which blocks inactivated Na_v channels. Therefore four different voltage protocols were set up to test for: 1) steady-state inactivation, 2) open-channel block, 3) recovery from inactivation, 4) state- versus use-dependence. Each protocol was tested in both single-hole and multi-hole mode to compare the capabilities of the QPatch 16X in the two modes.

The sodium channel Na_V1.1, highly expressed in fast spiking interneurons (FSIs), initiates the interneuron’s action potentials and promotes γ-oscillations, which are important for memory encoding and other cognitive functions. An impaired function of FSIs is associated with disorders like autism, schizophrenia, Alzheimer’s and others. Potentiators of Na_V1.1 can restore γ-oscillations by recovering reduced FSI function and they have been shown to minimize cognitive dysfunction in transgenic mice with decreased levels of Na_V1.1 in parvalbumin-positive neurons.

Human intoxication with the seafood poison ciguatoxin, a dinoflagellate polyether that activates voltage-gated sodium channels (Na_V), causes ciguatera, a disease characterised by gastrointestinal and neurological disturbances. We assessed the activity of the most potent congener, Pacific ciguatoxin-1 (P-CTX-1), on Na_V1.1–1.9 using imaging and electrophysiological approaches. Although P-CTX-1 is essentially a non-selective NaV toxin and shifted the voltage-dependence of activation to more hyperpolarising potentials at all NaV subtypes, an increase in the inactivation time constant was observed only at Na_V1.8, while the slope factor of the conductance-voltage curves was significantly increased for Na_V1.7 and peak current was significantly increased for Na_V1.6. Accordingly, P-CTX-1-induced visceral and cutaneous pain behaviours were significantly decreased after pharmacological inhibition of Na_V1.8 and the tetrodotoxin-sensitive isoforms Na_V1.7 and Na_V1.6, respectively. The contribution of these isoforms to excitability of peripheral C- and A-fibre sensory neurons, confirmed using murine skin and visceral single-fibre recordings, reflects the expression pattern of Na_V isoforms in peripheral sensory neurons and their contribution to membrane depolarisation, action potential initiation and propagation.

High-throughput automated patch clamp (APC) is quickly emerging as the new gold standard technology for screening compounds against ion channel targets. The latest APC platforms, such as the Qube, enable true giga-seal quality data generation, comparable to conventional patch clamp.

Traditional plate-based screening technologies rarely allowed for analysis of multiple parameters to detect potential differences in the mechanism of action of novel compounds. Using multiple data analysis cursors in the Sophion Analyzer software, it’s possible to gain valuable insight into compound activities on a particular ion channel target, which may translate into different results in further in vivo experiments. This also increases the value of APC data, as multiple parameters can be extracted from each well for both data quality control as well as hit detection.

Here we present Qube data for a 10,000 compound pilot screen for a sodium channel activator project, with analysis for multiple hit parameters, demonstrating the ability of the assay to detect novel compounds with different mechanisms of action.

Purinergic homomeric P2X3 and heteromeric P2X2/3 receptors are ligand-gated cation channels activated by ATP. Both receptors are predominantly expressed in nociceptive sensory neurons and an increase in extracellular ATP concentration under pathological conditions such as tissue damage or visceral distension induces channel opening, membrane depolarization and initiation of pain signaling. Hence these receptors are considered important therapeutic targets for pain management and development of selective antagonists is currently progressing. To advance the search for novel analgesics, we have generated a panel of monoclonal antibodies directed against hP2X3. We have found that these antibodies produce distinct functional effects depending on the homomeric or heteromeric composition of the target, its kinetic state and the duration of antibody exposure. The most potent antibody, 12D4, showed an estimated IC50 of 16 nM on hP2X3 after short-term exposure (up to 18 minutes), binding to the inactivated state of the channel to inhibit activity. By contrast, with the same short-term application, 12D4 potentiated the slow inactivating current mediated by the heteromeric hP2X2/3 channel. Extending the duration of exposure to approximately 20 hours resulted in a profound inhibition of both homomeric hP2X3 and heteromeric hP2X2/3 receptors, an effect mediated by efficient antibody-induced internalization of the channel from the plasma membrane. The therapeutic potential of mAb12D4 was assessed in the formalin, CFA and visceral pain models. The efficacy of 12D4 in the visceral hypersensitivity model indicates that antibodies against P2X3 may have therapeutic potential in visceral pain indications.

Drug-induced block of the cardiac hERG (human Ether-a`-go-go-Related Gene) potassium channel delays cardiac repolarization and increases the risk of Torsade de Pointes (TdP), a potentially lethal arrhythmia. A positive hERG assay has been embraced by regulators as a non-clinical predictor of TdP despite a discordance of about 30%. To test whether assaying concomitant block of multiple ion channels (Multiple Ion Channel Effects or MICE) improves predictivity we measured the concentration-responses of hERG, Na_v1.5 and Ca_v1.2 currents for 32 torsadogenic and 23 non-torsadogenic drugs from multiple classes. We used automated gigaseal patch clamp instruments to provide higher throughput along with accuracy and reproducibility. Logistic regression models using the MICE assay showed a significant reduction in false positives (Type 1 errors) and false negatives (Type 2 errors) when compared to the hERG assay. The best MICE model only required a comparison of the blocking potencies between hERG and Ca_v1.2.

Opioid receptor stimulants are analgesics used in patients with and without cancer; however, they often cause constipation, resulting in poor adherence and deterioration of the quality of life. Hence, suitable treatments for constipation are required. In this study, we investigated the pharmacological mechanisms of action of mashiningan (MNG), a Kampo medicine used to treat constipation, and evaluated the effect of MNG on opioid-induced constipation in rats. MNG (100 or 300 mg/kg) was orally administered to normal or codeine phosphate (CPH)-induced constipation in rats, and its effect was evaluated on the basis of fecal counts, characteristics, and weight. Small intestinal fluid secretion was measured after treatment with MNG alone or co-administration with a cystic fibrosis transmembrane conductance regulator (CFTR) -specific inhibitor (CFTRinh-172). The effects of MNG on the CFTR and type-2 chloride channel were determined using patch clamp or short-circuit current experiments, respectively. MNG increased the fecal weight and proportion of soft feces in normal rats. CPH-induced constipation in rats decreased fecal counts and weight, whereas MNG prevented these effects and increased the proportion of soft feces. MNG increased the electronic chloride current, and this effect was inhibited by the CFTRinh-172 in the CFTR assay. Furthermore, MNG increased small intestinal fluid secretion, and this effect was abolished by co-administration with the CFTRinh-172. MNG improved opioid-induced constipation in rats, and this improvement may have been mediated by increasing intestinal fluid secretion via CFTR chloride channel activation. Therefore, MNG is expected as a medicine of the treatment for constipation in patients taking opioids.

γ-Aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the central nervous system (CNS) and the binding of GABA to ionotropic GABA receptors (GABAAR) is a crucial process in the healthy brain. An imbalance of GABA secretion or the malfunction of the receptor is associated with multiple disease areas like anxiety disorders, seizures and schizophrenia. Pharmacological manipulation of the receptor has, therefore, a large therapeutic potential, which is underscored by the amount of available treatment possibilities and the ongoing search for alternatives thereof.
GABAA receptors are ligand-gated ion-channels that consist of 5 membrane-spanning subunits and are permeable to Cl- ions. So far, 16 different subunits have been identified in humans (ɑ1-6, ꞵ1-3, γ1-3, δ, ε, γ, θ, π) and their composition within the GABA receptor leads to different pharmacological responses. Here, we show pharmacological modulation of the GABAAR using our high-throughput automated patch clamp (APC) systems QPatch and Qube 384. Our study includes a characterization of the heterogeneous GABAAR population of cultured primary hippocampal astrocytes and an evaluation of the GABAAR clone ɑ5ꞵ3γ2. In addition, we utilize the well-characterized GABAAR response to establish a novel method for ligand release, namely the light-stimulated release of ruthenium-bipyridine-triphenylphosphine(RuBi)-caged GABA using light stimulation on the Qube 384 platform.

Glycyrrhiza extract has been used for the treatment of oral and gastric ulcers, but the analgesic mechanism remains unknown. In the present study, we investigated the effects of isoliquiritigenin, an active ingredient of Glycyrrhiza, on Na_v channels in vitro and nociceptive behaviors in vivo. In an autopatch-clamp study, isoliquiritigenin inhibited the currents of Na_v1.1, Na_v1.3, Na_v1.6, Na_v1.7, and Na_v1.8 in a channel expression system. In small- and medium-sized cultured trigeminal ganglion neurons, the compound suppressed Na_v currents in many neurons (78%) and K_v currents in all neurons, dose-dependently. In current-clamp mode, isoliquiritigenin blocked action potential generation in many neurons (64%), but it conversely accelerated action potential generation in the remaining neurons. The opposing effects on action potentials were reproduced in a computational simulation of a modified Hodgkin-Huxley-based model, based on the electrophysiological data. In behavioral experiments, local treatment with isoliquiritigenin suppressed nociceptive behaviors in response to oral ulcer development or nociceptive TRP channel agonists in the oral mucosa and hind paw. These results suggest that isoliquiritigenin exerts an analgesic effect predominantly via inhibitory action on Na_v channels on sensory nociceptive fibers. This pharmacological mechanism indicates that isoliquiritigenin is useful for pain relief and provides scientific evidence for Glycyrrhiza at the ingredient level.

A series of isoindolinone compounds have been developed showing good in vitro potency on the K_v1.5 ion channel. By modification of two side chains on the isoindolinone scaffold, metabolically stable compounds with good in vivo PK profile could be obtained leaving the core structure unsubstituted. In this way, low microsomal intrinsic clearance (CLint) could be achieved despite a relatively high logD. The compounds were synthesized using the Ugi reaction, in some cases followed by Suzuki and Diels–Alder reactions, giving a diverse set of compounds in a small number of reaction steps.

For every heartbeat, movement and thought, ion channels have to open and close, and thus, it is not surprising that malfunctioning of these membrane proteins leads to serious diseases. Today, only 7% of all marketed drugs act on ion channels but the systematic exploitation of this important target class has started mainly enabled by novel screening technologies. Thus, the discovery of selective and state-dependent drugs is on the horizon, hopefully leading to effective novel medicines.

Free intracellular calcium ([Ca²⁺]_i), in addition to being an important second messenger, is a key regulator of many cellular processes including cell membrane potential, proliferation, and apoptosis. In many cases, the mobilization of ([Ca²⁺]_i) is controlled by intracellular store activation and calcium influx. We have investigated the effect of several ion channel modulators, which have been used to treat a range of human diseases, on ([Ca²⁺]_i) release, by ratiometric calcium imaging. We show that six such modulators [amiodarone (Ami), dofetilide, furosemide (Fur), minoxidil (Min), loxapine (Lox), and Nicorandil] initiate release of ([Ca²⁺]_i) in prostate and breast cancer cell lines, PC3 and MCF7, respectively. Whole-cell currents in PC3 cells were inhibited by the compounds tested in patch-clamp experiments in a concentration-dependent manner. In all cases ([Ca²⁺]_i) was increased by modulator concentrations comparable to those used clinically. The increase in ([Ca²⁺]_i) in response to Ami, Fur, Lox, and Min was reduced significantly (P < 0.01) when the external calcium was reduced to nM concentration by chelation with EGTA. The data suggest that many ion channel regulators mobilize ([Ca²⁺]_i). We suggest a mechanism whereby calcium-induced calcium release is implicated; such a mechanism may be important for understanding the action of these compounds.

Internal addition of compounds targeting the chloride channel CIC-1. A robust assay with good pharmacology and high Z-score

• A robust assay of ClC-1 with Z’ constantly >0.5
• Biophysical and pharmacological characteristics as expected for ClC-1
• Qube offers the possibility to apply compounds both intra- and extracellularly
• Advanced analysis of compound effects on channel kinetics are available at a few click

In this report, we show a ClC-1 assay on Qube that shows biophysical characteristics as expected for ClC-1. We further introduce Sophion`s Analyzer software that allows rapid analysis of large data sets to answer advanced electrophysiological questions, in the present case: What is the mode of action of a novel, unknown compound?

Pain places a devastating burden on patients and society and current pain therapeutics exhibit limitations in efficacy, unwanted side effects and the potential for drug abuse and diversion. Although genetic evidence has clearly demonstrated that the voltage-gated sodium channel, Na_V1.7, is critical to pain sensation in mammals, pharmacological inhibitors of Na_V1.7 have not yet fully recapitulated the dramatic analgesia observed in Na_V1.7-null subjects. Using the tarantula venom-peptide ProTX-II as a scaffold, we engineered a library of over 1500 venom-derived peptides and identified JNJ63955918 as a potent, highly selective, closed-state Na_V1.7 blocking peptide. Here we show that JNJ63955918 induces a pharmacological insensitivity to pain that closely recapitulates key features of the Na_V1.7-null phenotype seen in mice and humans. Our findings demonstrate that a high degree of selectivity, coupled with a closed-state dependent mechanism of action is required for strong efficacy and indicate that peptides such as JNJ63955918 and other suitably optimized Na_V1.7 inhibitors may represent viable non-opioid alternatives for the pharmacological treatment of severe pain.

Cannabis sativa contains many related compounds known as phytocannabinoids. The main psychoactive and non-psychoactive compounds are Δ9-tetrahydrocannabidiol (THC) and cannabidiol (CBD), respectively. Much of the evidence for clinical efficacy of CBD-mediated anti-epileptic effects has been from case reports or smaller surveys. The mechanisms for CBD’s anticonvulsant effects are unclear and likely involve non-cannabinoid receptor pathways. CBD is reported to modulate several ion channels, including sodium channels (Nav). Evaluating therapeutic mechanisms and safety of CBD demands a richer understanding of its interactions with central nervous system targets. Here, we used voltage-clamp electrophysiology of HEK-293 cells and iPSC neurons to characterize the effects of CBD on Nav channels. Our results show that CBD inhibits hNav1.1-1.7 currents, with an IC50 of 1.9–3.8 μM, suggesting that this inhibition could occur at therapeutically relevant concentrations. A steep Hill slope of ~3 suggested multiple interactions of CBD with Nav channels. CBD exhibited resting-state blockade, became more potent at depolarized potentials, and also slowed recovery from inactivation, supporting the idea that CBD binding preferentially stabilizes inactivated Nav channel states. We also found that CBD inhibits other voltage-dependent currents from diverse channels, including bacterial homomeric Nav channel (NaChBac) and voltage-gated potassium channel subunit Kv2.1. Lastly, the CBD block of Nav was temperature-dependent, with potency increasing at lower temperatures. We conclude that CBD’s mode of action likely involves (1) compound partitioning in lipid membranes, which alters membrane fluidity affecting gating, and (2) undetermined direct interactions with sodium and potassium channels, whose combined effects are loss of channel excitability.

Neuropathic pain is a debilitating, chronic condition with a significant unmet need for effective treatment options. Recent studies have demonstrated that in addition to neurons, non-neuronal cells such as microglia contribute to the initiation and maintenance of allodynia in rodent models of neuropathic pain. The Ca²⁺– activated K⁺channel, K_Ca3.1 is critical for the activation of immune cells, including the CNS-resident microglia. In order to evaluate the role of K_Ca3.1 in the maintenance of mechanical allodynia following peripheral nerve injury, we used senicapoc, a stable and highly potent K_Ca3.1 inhibitor. In primary cultured microglia, senicapoc inhibited microglial nitric oxide and IL-1β release. In vivo, senicapoc showed high CNS penetrance and when administered to rats with peripheral nerve injury, it significantly reversed tactile allodynia similar to the standard of care, gabapentin. In contrast to gabapentin, senicapoc achieved efficacy without any overt impact on locomotor activity. Together, the data demonstrate that the K_Ca3.1 inhibitor senicapoc is effective at reducing mechanical hypersensitivity in a rodent model of peripheral nerve injury.

The ongoing pain associated with arthritis reduces the quality of human life. Injection of complete Freund’s adjuvant (CFA) into the knee of mice induced inflammation and a concomitant decrease in their natural digging behaviour within 24-hours. After inflammation, retrogradely labelled knee-innervating dorsal root ganglion neurons showed a decreased action potential threshold and increased transient receptor potential vanilloid-1 (TRPV1) expression (validated with immunohistochemistry). Subsequently, systemic administration of a TRPV1 antagonist normalized mouse digging behaviour within 30-minutes, likely due to inhibition of pain signalling. To test the translational potential of these results, we recorded from mouse sensory neurons incubated overnight with synovial fluid from humans with osteoarthritis experiencing chronic pain (OA-SF). We find that OA-SF directly sensitizes mouse knee neurons by decreasing the action potential threshold and increasing the resting membrane potential implicating voltage-gated sodium (Na_v) and potassium (K_v) channels. Using Ca²⁺-imaging we also find that OA-SF increases TRPV1 and TRPM8 activity compared to synovial fluid from healthy individuals, highlighting the importance of TRP channels in OA pain. Taken together, our data suggest that inflammatory mediators in OA-SF excite sensory neurons and hence drive knee pain. This in vitro, translational model can be incorporated into clinical trials to unbiasedly assess the efficacy of drugs on sensory neurons and hence identify drivers of OA pain.

RIP2 kinase was recently identified as a therapeutic target for a variety of autoimmune diseases. We have reported previously a selective 4-aminoquinoline-based RIP2 inhibitor GSK583 and demonstrated its effectiveness in blocking downstream NOD2 signaling in cellular models, rodent in vivo models, and human ex vivo disease models. While this tool compound was valuable in validating the biological pathway, it suffered from activity at the hERG ion channel and a poor PK/PD profile thereby limiting progression of this analog. Herein, we detail our efforts to improve both this off-target liability as well as the PK/PD profile of this series of inhibitors through modulation of lipophilicity and strengthening hinge binding ability. These efforts have led to inhibitor 7, which possesses high binding affinity for the ATP pocket of RIP2 (IC₅₀ = 1 nM) and inhibition of downstream cytokine production in human whole blood (IC₅₀ = 10 nM) with reduced hERG activity (14 μM).

Growing functional and genetic evidence points to an emerging role for two-pore domain potassium channels (K2Ps) in numerous pathophysiologies. K2Ps carry background (or leak) potassium current and primarily function to maintain cellular resting membrane potential. However, K2Ps have proved a challenging superfamily to modulate with small molecules and there is currently a lack of pharmacological tools which target K2Ps. This has limited the investigation of K2Ps precise physiological function and efforts to generate therapeutics which act upon K2Ps. Our initial aim was to develop a screening system which would allow the identification of small molecule K2P activators. We initially showed that the identification of channel activators can be compromised in systems in which the target (K2P) is over-expressed at high levels. To avoid this, we used baculovirus (‘BacMam’) to transduce K2P expression in mammalian cells. ‘BacMam’ allows the precise titration of expression of the gene of interest. This enabled us to develop cell systems in which we were able to intricately and robustly select a level of K2P expression (via functional studies) optimized for the identification of channel activators. An example assay development using the K2P TRAAK is described. TRAAK is known to be expressed in nociceptors and has been shown to be a genetic predictor of post-surgical neuropathic pain. An appropriate level of TRAAK channel function was chosen after ‘BacMam’ optimization and channel function measured via thallium flux. The pharmacology of previously described TRAAK activators is also shown. A representative sub-group of K2Ps (TWIK-1, THIK-1, TASK-3, TASK-2, TREK-2) was screened using the BacMam/ Thallium flux assay against the LifeArc Index set (11k compounds). This allowed us to assess which K2Ps were ‘ligandable’ or ‘activatable’ by small molecules. Multiple novel activators were identified, and development is on-going. We also screened a library of drug-like molecules against the same group of K2Ps and identified Terbinafine as a novel activator of the TASK-3 channel. The selectivity of this molecule across the K2P superfamily was assessed and structure-activity relationship studies showed that relatively small changes to the molecule had significant effects on activity and selectivity.

Voltage-gated sodium channels (Na_v) are crucial to the initiation and propagation of action potentials (APs) in electrically excitable cells, and during the past decades they have received considerable attention due to their therapeutic potential. Here, we report for the first time the synthesis and the electrophysiological evaluation of 16 ligands based on a 2-methylbenzamide scaffold that have been identified as Na_v1.1 modulators. Among these compounds, N,N′-(1,3-phenylene)bis(2-methylbenzamide) (3a) has been selected and evaluated in ex-vivo experiments in order to estimate the activation impact of such a compound profile. It appears that 3a increases the Na_v1.1 channel activity although its overall impact remains moderate. Altogether, our preliminary results provide new insights into the development of small molecule activators targeting specifically Na_v1.1 channels to design potential drugs for treating CNS diseases.

Spider venoms are a rich source of ion channel modulators with therapeutic potential. Given the analgesic potential of subtypeselective inhibitors of voltage-gated sodium (Na_V) channels, we screened spider venoms for inhibitors of human Na_V1.7 (hNa_V1.7) using a high-throughput fluorescent assay. Here, we describe the discovery of a novel Na_V1.7 inhibitor, m-TRTX-Tp1a (Tp1a), isolated from the venom of the Peruvian green-velvet tarantula Thrixopelma pruriens. Recombinant and synthetic forms of this 33-residue peptide preferentially inhibited hNa_V1.7. hNa_V1.6. hNa_V1.2 . hNa_V1.1. hNa_V1.3 channels in fluorescent assays. N_aV1.7 inhibition was diminished (IC₅₀ 11.5 nM) and the association rate decreased for the C-terminal acid form of Tp1a compared with the native amidated form(IC₅₀ 2.1 nM), suggesting that the peptide C terminus contributes to its interaction with hNaV1.7. Tp1a had no effect on human voltage-gated calcium channels or nicotinic acetylcholine receptors at 5 mM. Unlike most spider toxins that modulate Na_V channels, Tp1a inhibited hNa_V1.7 without significantly altering the voltage dependence of activation or inactivation. Tp1a proved to be analgesic by reversing spontaneous pain induced in mice by intraplantar injection in OD1, a scorpion toxin that potentiates hNa_V1.7. The structure of Tp1a as determined using NMR spectroscopy revealed a classic inhibitor cystine knot (ICK) motif. The molecular surface of Tp1a presents a hydrophobic patch surrounded by positively charged residues, with subtle differences from other ICK spider toxins that might contribute to its different pharmacological profile. Tp1a may help guide the development of more selective and potent hNa_V1.7 inhibitors for treatment of chronic pain.

Rat basophilic leukaemia (RBL) cells endogenously express calcium-release-activated-calcium (CRAC) channels (Hoth and Penner, 1993). CRAC channels are activated by depletion of intracellular calcium stores, probably via the involvement of STIM-1 (stromal interaction molecule) sensing the depletion of the stores and travelling to the cell membrane activating the channel (Zhang et al, 2005; Vig et al, 2006). Several approaches can be used to deplete calcium stores ultimately leading to activation of Icrac and most of these have been tried on the QPatch. This document describes the results of these experiments and discusses the best conditions for studying Icrac with the QPatch.

• High fidelity voltage and current clamp recordings of human iPS cell-derived cardiomyocytes (Cor.4U®) on Qube
• Demonstration of the presence of INa, IK and ICa
• High throughput current clamp recordings
• Pharmacological characterization of action potentials and isolated currents
• Using Sophion Analyzer software it is possible to easily extract key parameters relevant for studying compound effects on action potentials

Voltage-gated sodium (Na_V) channels have an essential role in the initiation and propagation of action potentials in excitable cells, such as neurons. Of these channels, Na_V1.7 has been indicated as a key channel for pain sensation. While extensive efforts have gone into discovering novel Na_V1.7 modulating compounds for the treatment of pain, none has reached the market yet. In the last two years, new compound screening technologies have been introduced, which may speed up the discovery of such compounds. The Sophion Qube® is a next-generation 384-well giga-seal automated patch clamp (APC) screening instrument, capable of testing thousands of compounds per day. By combining high-throughput screening and follow-up compound testing on the same APC platform, it should be possible to accelerate the hit-to-lead stage of ion channel drug discovery and help identify the most interesting compounds faster. Following a period of instrument beta-testing, a Na_V1.7 high-throughput screen was run with two Pfizer plate-based compound subsets. In total, data were generated for 158,000 compounds at a median success rate of 83%, which can be considered high in APC screening. In parallel, IC₅₀ assay validation and protocol optimization was completed with a set of reference compounds to understand how the IC₅₀ potencies generated on the Qube correlate with data generated on the more established Sophion QPatch® APC platform. In summary, the results presented here demonstrate that the Qube provides a comparable but much faster approach to study Na_V1.7 in a robust and reliable APC assay for compound screening.

Human induced pluripotent stem cells (hiPSCs) can be differentiated into many cell types, including neurons and cardiomyocytes, and therefore constitute a novel way to model human diseases for drug testing in vitro1. Ion channels represent attractive therapeutic targets in the nervous and cardiovascular systems2 rendering electrophysiological studies of hiPSCs interesting for their usage in drug discovery. However, such studies have traditionally been limited by the labour-intensive and low throughput nature of patch clamp electrophysiology2.

Here we present the electrophysiological characterization of hiPSC-derived motor neurons using our automated patch clamp (APC) platforms, Qube 384 and QPatch. Our results include a measure of channel expression versus time in culture, the pharmacological dissection of endogenous ion channels (e.g. voltage-gated Na+ (NaV) and voltage-gated K+ (KV) channels), identification of ligand-gated receptors and recordings of action potentials using current clamp. The major challenge when investigating neurons using APC platforms is the requirement to dissociate the cells from their neuronal network while maintaining cell viability and membrane integrity3. By optimization of the harvest- and whole-cell protocols we have overcome this obstacle resulting in success rates of up to 60% using our 384-well APC system. Utilizing the high throughput nature of our system we tested in parallel two disease models, using hiPSC neurons derived from Spinal Muscular Atrophy (SMA) or Amyotrophic Lateral Sclerosis (ALS) patients, together with control cells from healthy subjects and isogenic control cells. The results show a general overactivity of Na+ channels in both cell lines, which could be rescued by a point mutation in the superoxide dismutase (SOD1) gene in the isogenic ALS cell line.

Our results demonstrate the feasibility of conducting electrophysiological characterization and screening of hiPSC-derived neurons on APC platforms like Qube 384 and QPatch, thus paving the way for high throughput ion channel-targeted screening of drugs for neurological disorders.

GluK1, a kainate subtype of ionotropic glutamate receptors, exhibits an expression pattern in the CNS consistent with involvement in pain processing and migraine. Antagonists of GluK1 have been shown to reduce pain signaling in the spinal cord and trigeminal nerve, and are predicted to provide pain and migraine relief. We developed an ultra-high-throughput small-molecule screen to identify antagonists of GluK1. Using the calcium indicator dye fluo-4, a multimillion-member small-molecule library was screened in 1536-well plate format on the FLIPR (Fluorescent Imaging Plate Reader) Tetra against cells expressing a calcium-permeable GluK1. Following confirmation in the primary assay and subsequent counter-screen against the endogenous Par-1 receptor, 6100 compounds were selected for dose titration to assess potency and selectivity. Final triage of 1000 compounds demonstrating dose-dependent inhibition with IC₅₀ values of less than 12 µM was performed in an automated whole-cell patch clamp electrophysiology assay. Although a weak correlation between electrophysiologically active and calcium-imaging active compounds was observed, the identification of electrophysiologically active compounds with a range of kinetic profiles revealed a broad spectrum of mechanisms of action.

Ion channels represent highly attractive targets for drug discovery and are implicated in a diverse range of disorders, in particular in the central nervous and cardiovascular systems. Moreover, assessment of cardiac ion-channel activity of new chemical entities is now an integral component of drug discovery programmes to assess potential for cardiovascular side effects. Despite their attractiveness as drug discovery targets ion channels remain an under-exploited target class, which is in large part due to the labour-intensive and low-throughput nature of patch-clamp electrophysiology. This Review provides an update on the current state-of-the-art for the various automated electrophysiology platforms that are now available and critically evaluates their impact in terms of ion-channel screening, lead optimization and the assessment of cardiac ion-channel safety liability.

• Constantly high success rates (99% in three experiments)
• Giga-ohm seal resistances in almost all wells
• Very low data spread
• All CV values of DMSO control below 10%
• No false positives with cut-off criteria 3 X SD
• Z’ constantly greater 0.75
• Discrimination between different modes of inhibition (state-dependency)
• Up to 384 individual IC50 values (3072 data points) determined in just one experiment
• Using Qube in combination with stacker it would be possible to determine up to 7680 IC₅₀ (60,000 data points) per 24 hours.

Cardiac drug discovery is hampered by the reliance on non-human animal and cellular models with inadequate throughput and physiological fidelity to accurately identify new targets and test novel therapeutic strategies. Similarly, adverse drug effects on the heart are challenging to model, contributing to costly failure of drugs during development and even after market launch. Human induced pluripotent stem cell derived cardiac tissue represents a potentially powerful means to model aspects of heart physiology relevant to disease and adverse drug effects, providing both the human context and throughput needed to improve the efficiency of drug development. Here we review emerging technologies for high throughput measurements of cardiomyocyte physiology, and comment on the promises and challenges of using iPSC-derived cardiomyocytes to model disease and introduce the human context into early stages of drug discovery. This article is part of a Special Issue entitled: Cardiomyocyte biology: Integration of Developmental and Environmental Cues in the Heart edited by Marcus Schaub and Hughes Abriel.

Proper function of ion channels is crucial for all living cells. Ion channel dysfunction may lead to a number of diseases, so-called channelopathies, and a number of common diseases, including epilepsy, arrhythmia, and type II diabetes, are primarily treated by drugs that modulate ion channels. A cornerstone in current drug discovery is high throughput screening assays which allow examination of the activity of specific ion channels though only to a limited extent. Conventional patch clamp remains the sole technique with sufficiently high time resolution and sensitivity required for precise and direct characterization of ion channel properties. However, patch clamp is a slow, labor-intensive, and thus expensive, technique. New techniques combining the reliability and high information content of patch clamping with the virtues of high throughput philosophy are emerging and predicted to make a number of ion channel targets accessible for drug screening. Specifically, genuine HTS parallel processing techniques based on arrays of planar silicon chips are being developed, but also lower throughput sequential techniques may be of value in compound screening, lead optimization, and safety screening. The introduction of new powerful HTS electrophysiological techniques is predicted to cause a revolution in ion channel drug discovery.

The citrus flavanone hesperetin (HSP) has been proposed for the treatment of several human pathologies, but its cardiovascular actions remain unexplored. Here we studied the effects of HSP on the human cardiac voltage-gated Na+ channel (hNaV1.5) and compared it to its effects on a recombinant hNaV1.5 channel baring a mutation (R1623Q) associated with lethal ventricular arrhythmias in the Long QT syndrome type 3 (LQT3).

METHODS: Whole-cell patch clamp experiments were used to record Na+-currents (INa) in HEK293T cells expressing hNaV1.5 wild type (WT) or mutant channels.

RESULTS: HSP blocked thehNaV1.5 channels in voltage-dependent manner with an IC50 ≈ 100 μM. Its inhibition was decreased by disruption of the F1760 residue. HSP preferentially accelerated the inactivation phase of INa and decreased the Na+ net influx into the cell. The effects of HSP on the inactivation phase and voltage-dependent inhibition of INa were more marked in the LQT3 mutant.

CONCLUSIONS: HSP could be used as a template to develop drugs against cardiac arrhythmias in LQT3.

The QPatch 16 automated patch clamp system was used to establish and record currents from HEK293 cells stably expressing CaV3.2 channels. Whole cell currents were measured upon application of a voltage-step protocol to obtain I-V curves.

Performing automated patch clamp experiments with different intracellular solutions is somehow difficult. Using QPatch with its multi well reservoir is it possible to test eight different solutions on the same cell line. In this study the multi well reservoir has been used for experiments on the voltage-dependent large conductance Ca2+-activated K+ channels, often referred to as BK channels. A human embryonic kidney cell line (HEK293) expressing the BK channel was used.
BK channels are large conductance calcium and voltage gated potassium channels, which allow potassium fluxwhen activated with intra cellular calcium and/or membrane potential (Strøbæk et al., 1996).

It is common that potential customers ask Sophion Bioscience to use the QPatch to test some of their compounds on specific ion channels before buying a system. In this application report, we present some of the results obtained from a validation study done for a big pharmaceutical company. Currents from the N-type voltage gated calcium channels (Ca_v2.2) were studied using the QPatch. A blocker of the Ca_v2.2 current, compound A, was applied in increasing concentrations to study the dose-response effects and estimate IC₅₀ values. Furthermore, flunarizine which is known to block T-type Ca²⁺/Na+ channels, and have low potency on N-type Ca channels, was tested.

hKir2.1 exhibit strong inward rectification with major current activity at very negative potentials. The channel has a fundamental role in controlling and maintaining the resting membrane potential. hKir2.1 is encoded by the KCNJ2 gene and mutation in this gene can cause cardiac arrhythmia.

The QPatch16 automated patch clamp system was used to establish and record currents from HEK293 cells stably expressing Ca_V3.2 channels. Whole cell currents were measured upon application of a voltage-step protocol to obtain I-V curves.

The QPatch16 automated patch clamp system was used to establish and record currents from HEK293 cells stably expressing Ca_V3.2 channels. Whole cell currents were measured upon application of a voltage-step protocol to obtain I-V curves.

The development of screening assays for the Ca_V1.2 channel has been challenging due to the tendency of Ca_V1.2 expressing cell lines to exhibit declining current levels (rundown) during the experiment^1,2. Here we report a robust Ca_V1.2 assay yielding high success rates, low rundown and reliable pharmacology.

• Pharmacology and current-voltage relationship in accordance with literature values
• Success rates up to 94%
• Stable currents with rundown as low as (1.2 ± 0.9)% per minute
• The CiPA protocol yields stable currents with rundown as low as (1 ± 1)% per minute

This study presents results produced on the QPatch with GH4-C1 cells expressing the homomeric nicotinic ligand gated α7 ion channel (nAChR α7). nAChR α7 is linked to many normal and pathological conditions. It is important in memory formation, arousal, and attention, nicotine addiction, and nicotine-induced reversal of age-related memory deficits. nAChR α7 activation leads to neuroprotection against amiloid-β-induced neurotoxicity, which suggests that the receptor may play an important role in Alzheimer’s disease. Also their role in hippocampal auditory gating has linked the receptor to schizophrenia. These findings makes this particular channel an interesting target for pharmaceutical
companies. The fast kinetics of the α7 channel makes this a difficult ion channel to work on even in conventional patch clamp rigs. At Sophion we have developed assays that captures this current in a reproducible way enabling automated technology to be used in the search for blockers and positive allosteric modulators of this channel.

Pathogenic T cell effector memory (TEM) cells drive many autoimmune disorders and are uniquely dependent on the Kv1.3 channel. A number of venom derived knottin (cysteine-rich mini-protein) inhibitors of Kv1.3 are being developed as potential drug candidates but can suffer from manufacturing difficulties, short half-lives and a lack of specificity. We have developed a novel molecular format wherein a peripheral CDR loop of an antibody has been replaced by a knottin. In this novel KnotBody^TM format, the knottin benefits from the improved therapeutic functionality of an antibody and the antibody gains additional diversity by the addition of a scaffold which is pre-disposed to blockade of ion channels.

A proof-of-concept fusion protein of one structural domain within another was initially achieved by inserting a trypsin inhibiting knottin (EETI-II) flanked by diverse repertoire of short linker sequences into the CDR2 position of naïve antibody light chain sequences. Functional KnotBody^TM molecules were selected from this library using phage display technology on the basis of retained trypsin binding, with the correct folding of both domains confirmed using X-ray crystallography.

To further demonstrate the benefits of this novel format, the modular nature of the KnotBody^TM binding surface was exploited to: (i) improve existing knottin binding by introducing additional V_H contacts; (ii) create a bispecific molecule by introducing a V_H chain that binds to a different target; (iii) substitute the proof-of-concept knottin (EETI-II, a trypsin inhibitor) with ShK, a Kv1.3 ion channel blocking toxin; (iv) develop a panel of low-nM Kv1.3 inhibitors with selectivity exceeding 3000-fold over the Kv1.1 channel, a closely related Kv family member.

Pathogenic T cell effector memory (TEM) cells drive many autoimmune disorders and are uniquely dependent on the Kv1.3 channel. A number of venom derived knottin (cysteine-rich mini-protein) inhibitors of Kv1.3 are being developed as potential drug candidates, but can suffer from manufacturing difficulties, short half-lives and a lack of specificity. We have developed a novel molecular format wherein a peripheral CDR loop of an antibody has been replaced by a knottin. In this novel KnotBody^TM format, the knottin benefits from the improved therapeutic functionality of an antibody and the antibody gains additional diversity by the addition of a scaffold which is pre-disposed to blockade of ion channels.

A proof-of-concept fusion protein of one structural domain within another was initially achieved by inserting a trypsin inhibiting knottin (EETI-II) flanked by diverse repertoire of short linker sequences into the CDR2 position of naïve antibody light chain sequences. Functional KnotBody^TM molecules were selected from this library using phage display technology on the basis of retained trypsin binding, with the correct folding of both domains confirmed using X-ray crystallography.

To further demonstrate the benefits of this novel format, the modular nature of the KnotBody^TM binding surface was exploited to: (i) improve existing knottin binding by introducing additional V_H contacts; (ii) create a bispecific molecule by introducing a V_H chain that binds to a different target; (iii) substitute the proof-of-concept knottin (EETI-II, a trypsin inhibitor) with ShK, a Kv1.3 ion channel blocking toxin; (iv) develop a panel of low-nM Kv1.3 inhibitors with selectivity exceeding 3000-fold over the Kv1.1 channel, a closely related Kv family member.

This report presents QPatch studies based on whole-cell current recordings from the ligand-gated ion channel hGABAA receptors (α1β3γ2), expressed in HEK-293 cells from Millipore. GABAA receptors are anionic channels and upon activation under physiological conditions, an inward chloride driven current is recorded. The channels were targeted with an agonist; GABA (γ-amino-butyric acid) and bicuculline (an antagonist).

Over the last six decades, voltage-gated sodium (Nav) channels have attracted a great deal of scientific and pharmaceutical interest, driving fundamental advances in both biology and technology. The structure and physiological function of these channels have been extensively studied; clinical and genetic data have uncovered their implication in diseases such as epilepsy, arrhythmias, and pain, bringing them into focus as current and future drug targets. While different techniques have been established to record the activity of Nav channels, proper determination of their properties still presents serious challenges, depending upon the experimental conditions and the desired subtype of channel to be characterized. The aim of this unit is to review the characteristics of Nav channels, their properties, the cells in which they can be studied, and the currently available techniques. Topics covered include the determination of Nav-channel biophysical properties as well as the use of toxins to discriminate between subtypes using electrophysiological or optical methods. Perspectives on the development of high-throughput screening assays with their advantages and limitations are also discussed to allow a better understanding of the challenges encountered in voltage-gated sodium channel preclinical drug discovery.

Voltage-gated sodium channels are transmembrane proteins that are responsible for the rapid depolarization that elicits the upstroke of action potentials in neurons and cardiac cells. Molecular cloning techniques have identified nine related voltage-gated sodium channels subtypes with distinct biophysical properties, interacting proteins, and cellular patterns of expression that are involved in electrical signaling. The goal of many research groups is to target specific sodium channels subtypes to develop selective inhibitors that could, in theory, produce safer and more efficacious drugs. To address compound sodium subtype profiling, we used a two-pulse
state-dependent protocol to develop a sub-type specific sodium channel (hNav1.1 to hNav1.8) panel on the QPatch HT patch clamp system. Additionally, we tested three stimulus protocols on hNav1.5 with known sodium inhibitors: 1) CiPA protocol for cardiac safety testing (tonic), 2) two-pulse state-dependent protocol (inactivation-dependent), 3) three-pulse protocol (tonic, inactivation-dependent, and V1/2 dependent inactivation). The CiPA protocol identified state-independent compounds (e.g. Flecainide) within three-fold of published
literature values but didn’t identify state-dependent compounds such as Lidocaine. The two-pulse protocol identified both state-dependent and independent compounds at pulse 2 with IC50 values within 4-fold published literature values. Data from the first two pulses of the three-pulse protocol had comparable pharmacology to the two-pulse protocol. The functional activity from V_1/2 inactivation (a delayed 3rd pulse) was less predictive of state-dependent inhibition than the two-pulse protocol but may reflect the in vivo inhibition. The hNav1.x panel using the two-pulse protocol can be useful to profile target identification, selectivity and state-dependence of novel compounds. (QPatch Automated Patch Clamp System is manufactured by Sophion Bioscience).

The Na_V 1.7 channel is highly expressed in dorsal root ganglia of the sensory nervous system and plays a central role in the pain signalling process. We investigated a library prepared from original venoms of 117 different animals to identify new selective inhibitors of this target.

Series resistance compensation for fast activating ion channels can help remove voltage clamp errors and increase clamp accuracy. All Sophions patch clamp amplifiers utilize a patented technique enabling up to 100% RS compensation. In addition, Sophion amplifiers are equipped with automatic clip detection to avoid loss of cells due to fatal oscillations, thereby increasing data throughput while maintaining high quality recordings. Here we show that voltage clamp errors can be up to 20-30 mV when uncompensated and in general that errors occur if not using RS compensation. However, we also show that the increase of accuracy due to use of RS compensation comes with a price of a lower success rate, and that the settings are critical to maximize success rates while avoiding or decreasing errors. Last we demonstrate the importance of automated clip detection.

• The nicotinic acetylcholine receptor alpha1 (nAChR α1) is a fast desensitizing channel that makes the development of a stable high throughput assay on automated patch clamp systems challenging.
• The solution stacking feature on Qube reduces ligand exposure times below 1 s. Using this feature, it is now possible to evoke stable acetylcholine signals for agonist concentrations at least up to EC₅₀.
• An assay for nAChR α1 endogenously expressed in TE671 cells with 5 µM acetylcholine showed the following characteristics:
  • Overall success rate of 89%
  • Overall whole-cell seal resistance = 1.2 ± 0.4 GΩ (SD)
  • Coefficient of variance for rows and columns in DMSO control ≤12%
  • IC₅₀(max/min)(tetracaine) of a non-cumulative concentration response experiment = 1.5
  • Z’ greater than 0.70
• The assay stability is also owed to unique architecture of the QChip where microfluidic channels ensure rapid and complete solution exchange of compound and agonist.

There is increasing interest for cardiomyocytes as models for studying cardiac cellular physiology and preclinical drug safety testing. Stem cell-derived cardiomyocytes have the potential for such a model and have the possibility for modeling human diseases. The present investigation is the first to describe current properties from stem cell-derived cardiomyocytes using multi-hole recordings with planar automated patch clamp technology. In our study pluripotent stem cell-derived cardiomyocytes were biophysically and pharmacologically characterized. The cells are differentiated in large numbers and cryo-preserved, which make them suitable for automated patch clamping and facilitate their use in drug screening. We tested the cells in two different recording modes; single-hole and multi-hole, respectively. For multi-hole recordings up to ten cells are patched at the same time and the total current is measured per site. This recording mode can be useful for small currents (e.g. endogenous) and typically increases the success rate for useful data. For all experiments the whole-cell configuration was used and three different types of currents were studied; Na⁺, Ca2⁺ and K⁺. Using specific voltage protocols biophysical characteristics of each current was described and compared from single-hole and multi-hole experiments. We showed that currents recorded from these pluripotent stem cell-derived cardiomyocytes are similar to human cardiomyocytes and the response to known pharmacology is as expected. The V0.5 values, I-V relationships, current kinetics and IC₅₀ values determined for known blockers (TTX, nifedipine and cisapride) were comparable for the two recording modes. Clearly the success rate for usable data per measurement plate was significantly increased with the multi-hole technology. This is the first time current properties of stem cell-derived cardiomyocytes have been described from multi-hole recordings with planar automated patch clamp. Our study has shown that automated patch clamp is ready for stem cell-derived exploration.

The QPatch HTX is the newest member of the of the QPatch family of high-end fully automated patch clamp systems from Sophion Biosciences. The QPatch HTX builds on the proven technology from the QPatch 16 and QPatch HT, where cells are patched on a silicon chip with integrated microfluidic flow channels made in glass. The chip, called a QPlate, allows true gigaseals to be formed and high quality data to be recorded. For QPatch HTX modifications has been made to the QPlate and to the amplifier. To diminish problems with low-expressing cell lines, the QPlate HTX contains multiple holes per recording site and to accommodate the increased current amplitudes, the amplifier has been built with a broader current range.
In this study we present data obtained during the assay validation and optimization of a high throughput screening assay using a HEK293 cell line expressing the voltage gated sodium channel Na_v1.2a. This stable cell line is characterized by a very low functional expression of the Na_v1.2a channel with approximately 30% expressing cells in the population resulting in a relatively low success rate in data recording on QPatch HT. We found that with the QPlate HTX the success rate could be dramatically improved and upon assay optimization on QPatch HTX the effective success rate approaches 100%. We present data to compare throughput and assay quality between the QPatch HTX in multi-hole mode and traditional single cell patch clamp on QPatch HT.
Furthermore we have evaluated the compound profiling properties of the QPatch HTX by characterizing known blockers against the Na_v1.2a channel – a challenging channel for electrophysiological recording due the extremely low time constants of the response elicited upon voltage stimulation of the channel. Surprisingly, our biophysical and pharmacological data suggest that the data quality obtained on the QPatch HTX system is overall comparable to equivalent data obtained on the QPatch HT system.

Small-conductance Ca²⁺-activated K⁺ (SK) channels mediate afterhyperpolarization in neurons and dampen the firing frequency of action potentials. Calmodulin is constitutively associated with the SK2 channels and serves as the Ca²⁺ sensor for the SK-calmodulin complex. The SK2 channel subtype plays a key role in the regulation of excitability of Purkinje cells in the cerebellum. Given their importance in Purkinje cells, SK2 channels are a promising drug target for ataxia, a movement disorder.

Automated patch clamp (APC) machines, such as QPatch, have been used in the pharmaceutical industry to study drug interaction with varieties of ion channels. Over the past decade, technology has contributed significantly to pharmaceutical research and drug discovery. Here, we report to use QPatch as a tool for testing and evaluating the positive allosteric modulators of SK2 channels. A stable cell line of the rat SK2 channel tagged with GFP was established through transfection of HEK293 cells followed by puromycin selection and enrichment using repeated GFP fluorescence-activated cell sorting.

Positive allosteric modulators were tested under whole-cell voltage-clamp configuration with automated QPatch. The compounds that positively modulate the SK2 channels in the automated QPatch was further tested with the inside-out patch manual recordings. The results from automated whole-cell recordings and inside-out patch manual recordings were consistent.

The human ether-à-go-go related gene (hERG) carries one of the major repolarizing currents in the heart myocytes and block of this channel by certain drugs may lead to prolongation of the cardiac action potential and the potentially lethal arrhythmia torsade de pointes (TdP). Therefore current guidelines are that potency against hERG should be assessed in preclinical drug development. However, not all compounds inhibiting hERG are proarrhythmogenic and more integrated effects of the rug must be taken into consideration.

The comprehensive in vitro proarrhythmic assay (CiPA) initiative have introduced a new paradigm for assessing
proarrhythmic risk based on a thorough understanding of the underlying cellular mechanisms leading to a more predictive estimation of the risk carried by the specific compound.

One pillar of the CiPA paradigm is to measure the major
ionic currents involved in cardiac depolarization and repolarization using the high throughput automated patch clamp (APC) systems. Until recently this has meant that the measurements were carried out at ambient temperature a but recently automated patch clamp instruments with temperature control have become available making it possible to perform up to 384 parallel recordings at controlled temperatures ranging from 10 to 40°C.

Here we estimate potencies and binding kinetics to the hERG channel which are important parameters for the outcome of the risk assessment for a panel of clinical drugs using a Qube APC system equipped with temperature control.

The QPatch HTX automated patch clamp technology was developed to 1) increase throughput in ion channel drug screening by parallel operation of 48 multi-hole patch clamp sites, each comprising 10 individual patch clamp holes, in a single measurements site on a QPlate X, and 2) diminish problems with low-expressing cell lines. Thus, parallel recording from 10 cells represents a 10-fold signal amplification, and it increases the success rate at each site substantially.
To further increase throughput we explored the possibility of simultaneous recording of a number of ion channel currents. Two or three cell lines, each expressing a specific ion channel, were applied at each site simultaneously. The ion channel currents were separated temporally or pharmacologically by proper choices of voltage protocols or ion channel inhibitors. Using this strategy we were successful in recording currents from specific ion channel populations. This strategy, which ensured the exact same conditions for the cell lines with respect to Ringer solutions, temperature, pH and osmolarity, allowed a doubling or even tripling of the throughput. We conducted a series of QPatch HTX experiments with a combination of ion channels involved in cardiac risk assessment: hERG, K_v1.5, KvLQT1/minK and Na_v1.5. Specifically, tests were set up for recordings of 1) IV-relationships and concentration-responses for K_v1.5 and Na_v1.5 in parallel by using multiple voltage protocols, and 2) pharmacological properties of specific blockers of hERG, KvLQT1/mink and Nav1.5 in parallel. We present biophysical and pharmacological data obtained with QPatch HTX using multiple voltage protocols and cell lines in combination, and compare them to traditional single-hole data obtained with QPatch HT.

The QPatch HTX automated patch clamp technology was developed to 1) increase throughput in ion channel drug screening by parallel operation of 48 multi-hole patch clamp sites, each comprising 10 individual patch clamp holes, in a single measurements site on a QPlate X, and 2) diminish problems with low-expressing cell lines. Thus, parallel recording from 10 cells represents a 10-fold signal amplification, and it increases the success rate at each site substantially. To further increase throughput we explored the possibility of simultaneous recording of a number of ion channel currents. Two or three cell lines, each expressing a specific ion channel, were applied at each site simultaneously. The ion channel currents were separated temporally or pharmacologically by proper choices of voltage protocols or ion channel inhibitors. Using this strategy we were successful in recording currents from specific ion channel populations. This strategy, which ensured the exact same conditions for the cell lines with respect to Ringer solutions, temperature, pH and osmolarity, allowed a doubling or even tripling of the throughput. We conducted a series of QPatch HTX experiments with a combination of ion channels involved in cardiac risk assessment: hERG, Kv1.5, KvLQT1/minK and Nav1.5. Specifically, tests were set up for recordings of 1) IV-relationships and concentration-responses for Kv1.5 and Nav1.5 in parallel by using multiple voltage protocols, and 2) pharmacological properties of specific blockers of hERG, KvLQT1/mink and Nav1.5 in parallel. We present biophysical and pharmacological data obtained with QPatch HTX using multiple voltage protocols and cell lines in combination, and compare them to traditional single-hole data obtained with QPatch HT.

The prominent role of voltage-gated sodium channel 1.7 (Na_v1.7) in nociception was revealed by remarkable human clinical and genetic evidence. Development of potent and subtypeselective inhibitors of this ion channel is crucial for obtaining therapeutically useful analgesic compounds. Microproteins isolated from animal venoms have been identified as promising therapeutic leads for ion channels, since they naturally evolved to be potent ion channel blockers. Here we report the engineering of highly potent and selective inhibitors of Na_v1.7 channel based on tarantula Ceratotoxin-1 (CcoTx1). We utilized a combination of directed evolution, saturation mutagenesis, chemical modification, and rational drug design to obtain higher potency and selectivity to Na_v1.7 channel. The resulting microproteins are highly potent (IC₅₀ to Na_v1.7 of 2.5 nM) and selective. We achieved 80-fold and 20-fold selectivity over the closely related Na_v1.2 and Na_v1.6 channels, respectively, and the IC₅₀ on skeletal (Na_v1.4) and cardiac (Na_v1.5) sodium channels is above 3000nM. The lead molecules have the potential for future clinical development as novel therapeutics in the treatment of pain.

A large number of mammalian cell lines are commercially available to be used as expression systems for membrane or cytoplasmal proteins. A number of voltage and ligand gated ion channels of potential interest for the pharmaceutical industry are endogenously expressed in several CNS and non-CNS cell lines including TTX-sensitive Na+ channels, Ca2+-release activated Ca2+ (CRAC) channels, inward rectifier K+ channels, acid-sensing ion channels (ASIC) and muscarinic alpha-adrenergic receptors mAChR). We have explored the applicability of five commonly employed cell lines from American Type Culture Collection (ATTC) for use with Sophions QPatchTM automated patch clamp systems (QPatch 16 and QPatch HT) and characterized the ion channel types that they endogenously express. Specificly we have explored:

• Suitability for automated patch clamp studies
  (‘patchability’)
• Background ionic currents that may interfere
  with currents of experimentally expressed ion
  channels
• Possible use for characterizing ion channels of
  interest without the need to experimentally
  introduce their genes (expression)

The tests have led to the development of a number of simple standard operation procedures (SOPs) for employment of the cell lines in QPatch characterizations of ion channels.

Pluripotent stem cell-derived cardiomyocytes, iCells® Cardiomyocytes, were biophysically and pharmacologically characterized with planer automated patch clamp using the QPatch. iCells Cardiomyocytes are differentiated from human induced pluripotent stem cells. They are differentiated in large numbers and cryo-preserved, which make them highly suitable for automated patch clamping and facilitates their use in drug screening. Here we present the results obtained during assay optimization of the cell culture, assay set-up on the QPatch as well as biophysical and pharmacological validation of the cardiac currents. Implementing optimal cell culture routines, the iCells Cardiomyocytes demonstrated spontaneous rhythmical contractions indicating functional properties of adult cardiomyocytes. We tested the cells in two different QPatch recording modes; single-hole recordings and multi-hole recordings where up to ten cells are patched at the same time and the total current is measured per site. Three different types of currents were studied including sodium, calcium and potassium. By using specific buffer solutions and changing the voltage protocols it was possible to characterize calcium, and sodium currents from the same cell. Our study showed that iCells Cardiomyocytes can successfully be applied to the QPatch. The recorded currents are similar to human cardiomyocytes and the response to known pharmacology is as expected. Using the QPatch we believe that iCells Cardiomyocytes have great potential for safety screening and other cardiovascular investigations early in the drug discovery process.

Human-induced pluripotent stem cells (hiPSCs) can be differentiated into many cell types, including neurons and cardiomyocytes, and therefore constitute a novel way to model human diseases¹. However, hiPSC-based applications are often challenged by the variability among differentiation batches, even when using iPSC lines originating from a single donor, and by heterogeneity among specific cell types derived from iPSCs². Here we demonstrate the application of high throughput electrophysiological measurements using our automated patch clamp (APC) platform, Qube 384, for characterization of iPSC-derived cortical neurons.

Generic cortical neurons were differentiated from iPSCs using small molecules under dual-SMAD inhibition conditions³. The cells were handled and differentiated in parallel, and a total of three independent differentiation batches were carried out. We conducted an electrophysiological analysis of the endogenous ion channel expression in the three different batches of iPSC neurons. In agreement with sequencing data, the measurements revealed a good reproducibility between the three iPSC batches with 60 % – 70 % of the investigated cells expressing voltage-gated Na⁺ (Na_v) channels and 60 % – 80 % expressing voltage-gated K⁺ (Kv) channels. Furthermore, filtering the measurements for the ion channel of interest, allowed a thorough biophysical and pharmacological investigation of the individual ion channels.

Finally, we compared iPSC-derived neurons from a patient (proband) carrying a missense mutation in cyclin-dependent kinase-like 5 (CDKL5) (c.871T>C, p.Cys291Arg), who was clinically diagnosed to present with classic CDKL5 Deficiency Disorder (CDD), with cells derived from an isogenic control. Using immunostaining, whole transcriptome analysis, and electrophysiological measurements we found that proband neurons were larger and had a more immature morphology, suggesting that they do not differentiate as efficiently as the control neurons.

Rat basophilic leukaemia (RBL) cells endogenously express calcium-release-activated-calcium (CRAC) channels (1). CRAC channels are activated by depletion of intracellular calcium
stores via the involvement of STIM-1 (stromal interaction molecule) sensing the depletion of the stores and travelling to the cell membrane activating the channel (2, 3). Several approaches can be used to deplete calcium stores, ultimately leading to activation of ICRAC. The present work was to develop assays using different depletion strategies on the
QPatch automated patch clamp platform.

Human-induced pluripotent stem cells (hiPSCs) were developed a decade ago and hold great promise for disease modelling, drug discovery and personalized medicine, especially in cardiac and neurological diseases1. Dysfunctional ion channels are one of the major targets in these diseases and thus, as the iPSC technology improves, a requirement arises for instrumentation that can characterize electrophysiological properties of iPSCs in a high-throughput fashion. Here, we demonstrate the use of the automated patch clamp (APC) platforms Qube 384 and QPatch in three characterization studies of hiPSC motor neurons derived from patients and healthy individuals. The results include:

• APC recording of hiPSC-derived motor neurons with high success rates on Qube (60%) and QPatch (30%)
• Biophysical characterization of voltage-gated channels, Na⁺ (Na_V) and K⁺ (K_V), in healthy hiPSCs
• Measurements of a ligand-gated channel (GABAA receptor) in healthy control cells
• Screening of hiPSC motor neurons derived from Spinal Muscular Atrophy (SMA) and Amyotrophic Lateral Sclerosis (ALS) patients
• Comparison of hiPSC motor neurons derived from two different healthy individuals

The loss of dopaminergic neurons in the substantia nigra plays an important role in the development of the Parkinson’s disease The symptoms of this disease typically occur after around 80 of these neurons degenerated This cell decay can be caused or promoted by genetic defects or environmental factors
including chemical compounds like pesticides For proper testing of neurotoxic effects on these neurons as well as for the development of neuroprotective drugs, assays based on animal primary cells lack predictivity due to mostly weak correlation between animal and human data. Therefore, models based on
human neuronal cells have a high potential to overcome the limitations of animal models One interesting neuronal cell line is the LUHMES (Lund human mesencephalic) line, which consists of immortalized fetal human mesencephalic cells that can be differentiated into fully postmitotic dopaminergic neurons in 6 days Scholz et al 2011 We here describe functional properties of these cells as a fundament for the
development of LUHMES based pharmacological assays.

Inwardly rectifying IK1 potassium currents of the heart control the resting membrane potential of ventricular cardiomyocytes during diastole and contribute to their repolarization after each action potential. Mutations in the gene encoding Kir2.1 channels, which primarily conduct ventricular IK1, are associated with inheritable forms of arrhythmias and sudden cardiac death. Therefore, potential iatrogenic inhibition of Kir2.1-mediated IK1 currents is a cardiosafety concern during new drug discovery and development. Kir2.1 channels are part of the panel of cardiac ion channels currently considered for refined early compound risk assessment within the Comprehensive in vitro Proarrhythmia Assay initiative. In this study, we have validated a cell-based assay allowing functional quantification of Kir2.1 inhibitors using whole-cell recordings of Chinese hamster ovary cells stably expressing human Kir2.1 channels. We reproduced key electrophysiological and pharmacological features known for native IK1, including current enhancement by external potassium and voltage- and concentration-dependent blockade by external barium. Furthermore, the Kir inhibitors ML133, PA-6, and chloroquine, as well as the multichannel inhibitors chloroethylclonidine, chlorpromazine, SKF-96365, and the class III antiarrhythmic agent terikalant demonstrated slowly developing inhibitory activity in the low micromolar range. The robustness of
this assay authorizes medium throughput screening for cardio safety purposes and could help to enrich the currently limited Kir2.1 pharmacology.

Background

Eleclazine (GS-6615) is a sodium channel blocker designed to improve the selectivity for cardiac late Na⁺ current (I_Na) over peak I_Na.

Objectives

The goals of this study were to investigate the inhibition of late INa by eleclazine using a sample of long QT syndrome type 3 (LQT3) and overlap LQT3/Brugada syndrome mutant channels; to compare the apparent binding rates for eleclazine with those for other class 1 antiarrhythmic agents; and to investigate the binding site.

Methods

Wild-type human cardiac voltage-gated sodium channel (hNa_V1.5) and 21 previously reported variants were studied using patch clamp recordings from a heterologous expression system.

Results

Eleclazine inhibited anemone toxin II–enhanced late INa from wild-type hNa_V1.5 with a drug concentration that causes 50% block of 0.62 ± 0.12 μM (84-fold selectivity over peak INa). The drug concentration that causes 50% block of eleclazine to inhibit the enhanced late INa from LQT3 mutant channels ranged from 0.33 to 1.7 μM. At predicted therapeutic concentrations, eleclazine and ranolazine inhibited peak INa to a similar degree as assessed with 4 overlap LQT3/Brugada syndrome mutations. Eleclazine was found to interact with hNa_V1.5 significantly faster than ranolazine and 6 other class 1 antiarrhythmic agents. Engineered mutations (F1760A/Y1767A) located within the local anesthetic binding site decreased the inhibition of late I_Na and peak I_Na by eleclazine.

Conclusion

At predicted therapeutic concentrations, eleclazine elicits potent inhibition of late INa across a cohort of Na_V1.5 mutant channels. These properties are consistent with a class 1b antiarrhythmic agent that associates with unusually rapid binding/unbinding rates.

Atrial fibrillation (AF) is the most common type of arrhythmia. Current pharmacological treatment for AF is moderately effective and/or increases the risk of serious ventricular adverse effects. To avoid ventricular adverse effects, a new target has been considered, the small conductance calcium-activated K⁺ channels (K_Ca2.X, SK channels). In the heart, K_Ca2.X channels are functionally more important in atria compared to ventricles, and pharmacological inhibition of the channel confers atrial selective prolongation of the cardiac action potential and converts AF to sinus rhythm in animal models of AF. Whether antiarrhythmic drugs (AADs) recommended for treating AF target K_Ca2.X channels is unknown. To this end, we tested a large number of AADs on the human K_Ca2.2 and K_Ca2.3 channels to assess their effect on this new target using automated whole-cell patch clamp. Of the AADs recommended for treatment of AF only dofetilide and propafenone inhibited hK_Ca2.X channels, with no subtype selectivity. The calculated IC₅₀ were 90±10 µmol/l vs 60±10 µmol/l for dofetilide and 42±4 µmol/l vs 80±20 µmol/l for propafenone (hK_Ca2.3 vs hK_Ca2.2). Whether this inhibition has clinical importance for their antiarrhythmic effect is unlikely, as the calculated IC₅₀ values are very high compared to the effective free therapeutic plasma concentration of the drugs when used for AF treatment, 40,000-fold for dofetilide and 140-fold higher for propafenone.

Society urgently needs new, effective medicines for the treatment of tuberculosis. To kick-start the required hit-to-lead campaigns, the libraries of pharmaceutical companies have recently been evaluated for starting points. The GlaxoSmithKline (GSK) library yielded many high-quality hits, and the associated data were placed in the public domain to stimulate engagement by the wider community. One such series, the spiro compounds, are described here. The compounds were explored by a combination of traditional in-house research and open source methods. The series benefits from a particularly simple structure and a short associated synthetic chemistry route. Many members of the series displayed striking potency and low toxicity, and highly promising in vivo activity in a mouse model was confirmed with one of the analogues. Ultimately the series was discontinued due to concerns over safety, but the associated data remain public domain, empowering others to resume the series if the perceived deficiencies can be overcome.

There is an unmet need in severe asthma where approximately 40% of patients exhibit poor β-agonist responsiveness, suffer daily symptoms and show frequent exacerbations. Antagonists of the Ca²⁺-activated-Cl⁻ channel, TMEM16A, offers a new mechanism to bronchodilate airways and block the multiple contractiles operating in severe disease. To identify TMEM16A antagonists we screened a library of ~580,000 compounds. The anthelmintics niclosamide, nitazoxanide and related compounds were identified as potent TMEM16A antagonists that blocked airway smooth muscle depolarization and contraction. To evaluate whether TMEM16A antagonists resist use- and inflammatory-desensitization pathways limiting β-agonist action, we tested their efficacy under harsh conditions using maximally contracted airways or airways pretreated with a cytokine cocktail. Stunningly, TMEM16A antagonists fully bronchodilated airways, while the β-agonist isoproterenol showed only partial effects. Thus, antagonists of TMEM16A and repositioning of niclosamide and nitazoxanide represent an important additional treatment for patients with severe asthma and COPD that is poorly controlled with existing therapies. It is of note that drug repurposing has also attracted wide interest in niclosamide and nitazoxanide as a new treatment for cancer and infectious disease. For the first time, we identify TMEM16A as a molecular target for these drugs and thus provide fresh insights into their mechanism for the treatment of these disorders in addition to respiratory disease.

The molecular determinants of abnormal propagation of action potentials along axons and ectopic conductance in demyelinating diseases of the central nervous system, like multiple sclerosis (MS), are poorly defined. Widespread interruption of myelin occurs in several mouse models of demyelination, rendering them useful for research. Herein, considerable myelin loss is shown in the optic nerves of cuprizone-treated demyelinating mice. Immuno-fluorescence confocal analysis of the expression and distribution of voltage-activated K⁺ channels (K_V1.1 and 1.2 a subunits) revealed their spread from typical juxta-paranodal (JXP) sites to nodes in demyelinated axons, albeit with a disproportionate increase in the level of K_V1.1 subunit. Functionally, in contrast to monophasic compound action potentials (CAPs) recorded in controls, responses derived from optic nerves of cuprizone-treated mice displayed initial synchronous waveform followed by a dispersed component. Partial restoration of CAPs by broad spectrum (4-aminopyridine) or K_V1.1-subunit selective (dendrotoxin K) blockers of K⁺ currents suggest enhanced K_V1.1-mediated conductance in the demyelinated optic nerve. Biophysical profiling of K⁺ currents mediated by recombinant channels comprised of different K_V1.1 and 1.2 stoichiometries revealed that the enrichment of KV1 channels K_V1.1 subunit endows a decrease in the voltage threshold and accelerates the activation kinetics. Together with the morphometric data, these findings provide important clues to a molecular basis for temporal dispersion of CAPs and reduced excitability of demyelinated optic nerves, which could be of potential relevance to the patho-physiology of MS and related disorders.

The molecular determinants of abnormal propagation of action potentials along axons and ectopic conductance in demyelinating diseases of the central nervous system, like multiple sclerosis (MS), are poorly defined. Widespread interruption of myelin occurs in several mouse models of demyelination, rendering them useful for research. Herein, considerable myelin loss is shown in the optic nerves of cuprizone-treated demyelinating mice. Immuno-fluorescence confocal analysis of the expression and distribution of voltage-activated K⁺ channels (K_V1.1 and 1.2 α subunits) revealed their spread from typical juxta-paranodal (JXP) sites to nodes in demyelinated axons, albeit with a disproportionate increase in the level of K_V1.1 subunit. Functionally, in contrast to monophasic compound action potentials (CAPs) recorded in controls, responses derived from optic nerves of cuprizone-treated mice displayed initial synchronous waveform followed by a dispersed component. Partial restoration of CAPs by broad spectrum (4-aminopyridine) or K_V1.1-subunit selective (dendrotoxin K) blockers of K⁺currents suggest enhanced K_V1.1-mediated conductance in the demyelinated optic nerve. Biophysical profiling of K⁺ currents mediated by recombinant channels comprised of different K_V1.1 and 1.2 stoichiometries revealed that the enrichment of K_V1 channels K_V1.1 subunit endows a decrease in the voltage threshold and accelerates the activation kinetics. Together with the morphometric data, these findings provide important clues to a molecular basis for temporal dispersion of CAPs and reduced excitability of demyelinated optic nerves, which could be of potential relevance to the pathophysiology of MS and related disorders.

The paper focuses on the scaffold hopping-based discovery and characterization of novel nicotinic alpha 7 receptor positive modulator (α7 nAChR PAM) ligands around the reference molecule (A-867744). First, substantial efforts were carried out to assess the importance of the various pharmacophoric elements on the in vitro potency (SAR evaluation) by chemical modifications. Subsequently, several new derivatives with versatile, heteroaromatic central cores were synthesized and characterized. A promising, pyrazole-containing new chemotype with good physicochemical and in vitro parameters was identified. Retrospective analysis based on homology modeling was also carried out. Besides its favorable in vitro characteristics, the most advanced derivative 69 also showed in vivo efficacy in a rodent model of cognition (scopolamine-induced amnesia in the mouse place recognition test) and acceptable pharmacokinetic properties. Based on the in vivo data, the resulting molecule with advanced drug-like characteristics has the possibility to improve cognitive performance in a biologically relevant dose range, further strengthening the view of the supportive role of α7 nACh receptors in the cognitive processes.

The cytosolic metalloenzyme leukotriene A₄ hydrolase (LTA4H) is the final and rate-limiting enzyme in the biosynthesis of pro-inflammatory leukotriene B₄ (LTB₄). Preclinical studies have validated this enzyme as an attractive drug target in chronic inflammatory diseases. Despite several attempts, no LTA4H inhibitor has reached the market, yet. Herein, we disclose the discovery and preclinical profile of LYS006, a highly potent and selective LTA4H inhibitor. A focused fragment screen identified hits that could be cocrystallized with LTA4H and inspired a fragment merging. Further optimization led to chiral amino acids and ultimately to LYS006, a picomolar LTA4H inhibitor with exquisite whole blood potency and long-lasting pharmacodynamic effects. Due to its high selectivity and its ability to fully suppress LTB₄ generation at low exposures in vivo, LYS006 has the potential for a best-in-class LTA4H inhibitor and is currently investigated in phase II clinical trials in inflammatory acne, hidradenitis suppurativa, ulcerative colitis, and NASH.

Late sodium current (late I_Na) is enhanced during ischemia by reactive oxygen species (ROS) modifying the Na_v 1.5 channel, resulting in incomplete inactivation. Compound 4 (GS-6615, eleclazine) a novel, potent, and selective inhibitor of late I_Na, is currently in clinical development for treatment of long QT-3 syndrome (LQT-3), hypertrophic cardiomyopathy (HCM), and ventricular tachycardia–ventricular fibrillation (VT–VF). We will describe structure–activity relationship (SAR) leading to the discovery of 4 that is vastly improved from the first generation late I_Na inhibitor 1 (ranolazine). Compound 4 was 42 times more potent than 1 in reducing ischemic burden in vivo (S–T segment elevation, 15 min left anteriorior descending, LAD, occlusion in rabbits) with EC₅₀ values of 190 and 8000 nM, respectively. Compound 4represents a new class of potent late I_Na inhibitors that will be useful in delineating the role of inhibitors of this current in the treatment of patients.

A novel series of pyrazolyltetrahydropyran N-type calcium channel blockers are described. Structural modifications of the series led to potent compounds in both a cell-based fluorescent calcium influx assay and a patch clamp electrophysiology assay. Representative compounds from the series were bioavailable and showed efficacy in the rat CFA and CCI models of inflammatory and neuropathic pain.

Spider venoms are rich sources of peptidic ion channel modulators with important therapeutical potential. We screened a panel of 60 spider venoms to find modulators of ion channels involved in pain transmission. We isolated, synthesized and pharmacologically characterized Cd1a, a novel peptide from the venom of the spider Ceratogyrus darlingi. Cd1a reversibly paralysed sheep blowflies (PD50 of 1318 pmol/g) and inhibited human Ca_v2.2 (IC₅₀ 2.6 μM) but not Ca_v1.3 or Ca_v3.1 (IC₅₀ > 30 μM) in fluorimetric assays. In patch clamp electrophysiological assays Cd1a inhibited rat Ca_v2.2 with similar potency (IC₅₀ 3 μM) without influencing the voltage dependence of Cav2.2 activation gating, suggesting that Cd1a doesn’t act on Ca_v2.2 as a classical gating modifier toxin. The Cd1a binding site on Ca_v2.2 did not overlap with that of the pore blocker ω-conotoxin GVIA, but its activity at Ca_v2.2-mutant indicated that Cd1a shares some molecular determinants with GVIA and MVIIA, localized near the pore region. Cd1a also inhibited human Na_v1.1–1.2 and Na_v1.7–1.8 (IC₅₀ 0.1–6.9 μM) but not Na_v1.3–1.6 (IC₅₀ > 30 μM) in fluorimetric assays. In patch-clamp assays, Cd1a strongly inhibited human Na_v1.7 (IC₅₀ 16 nM) and produced a 29 mV depolarising shift in Na_v1.7 voltage dependence of activation. Cd1a (400 pmol) fully reversed Na_v1.7-evoked pain behaviours in mice without producing side effects. In conclusion, Cd1a inhibited two anti-nociceptive targets, appearing to interfere with Ca_v2.2 inactivation gating, associated with the Ca_v2.2 α-subunit pore, while altering the activation gating of Na_v1.7. Cd1a was inactive at some of the Na_v and Ca_v channels expressed in skeletal and cardiac muscles and nodes of Ranvier, apparently contributing to the lack of side effects at efficacious doses, and suggesting potential as a lead for development of peripheral pain treatments.

Despite the importance of the hERG channel in drug discovery and the sizable number of antagonist molecules discovered, only a few hERG agonists have been discovered. Here we report a novel hERG agonist; SKF-32802 and a structural analog of the agonist NS3623, SB-335573. These were discovered through a similarity search of published hERG agonists. SKF-32802 incorporates an amide linker rather than NS3623’s urea, resulting in a compound with a different mechanism of action. We find that both compounds decrease the time constant of open channel kinetics, increase the amplitude of the envelope of tails assay, mildly increased the amplitude of the IV curve, bind the hERG channel in either open or closed states, increase the plateau of the voltage dependence of activation and modulate the effects of the hERG antagonist, quinidine. Neither compound affects inactivation nor deactivation kinetics, a property unique among hERG agonists. Additionally, SKF-32802 induces a leftward shift in the voltage dependence of activation. Our structural models show that both compounds make strong bridging interactions with multiple channel subunits and are stabilized by internal hydrogen bonding similar to NS3623, PD-307243 and RPR26024. While SB-335573 binds in a nearly identical fashion as NS3623, SKF-32802 makes an additional hydrogen bond with neighboring threonine 623. In summary, SB-335573 is a type 4 agonist which increases open channel probability while SKF-32802 is a type 3 agonist which induces a leftward shift in the voltage dependence of activation.

Class II antiarrhythmics or β-blockers are antisympathetic nervous system agents that act by blocking β-adrenoceptors. Despite their common clinical use, little is known about the effects of β-blockers on free intracellular calcium (Ca2+ i), an important cytosolic second messenger and a key regulator of cell function. We investigated the role of four chemical analogs, commonly prescribed β-blockers (atenolol, metoprolol, propranolol, and sotalol), on Ca2+ i release and whole-cell currents in mammalian cancer cells (PC3 prostate cancer and MCF7 breast cancer cell lines). We discovered that only propranolol activated free Ca2+ i release with distinct kinetics, whereas atenolol, metoprolol, and sotalol did not. The propranolol-induced Ca2+ i release was significantly inhibited by the chelation of extracellular calcium with ethylene glycol tetraacetic acid (EGTA) and by dantrolene, an inhibitor of the endoplasmic reticulum (ER) ryanodine receptor channels, and it was completely abolished by 2-aminoethoxydiphenyl borate, an inhibitor of the ER inositol-1,4,5-trisphosphate (IP3) receptor channels. Exhaustion of ER stores with 4-chloro-m-cresol, a ryanodine receptor activator, or thapsigargin, a sarco/ER Ca2+ ATPase inhibitor, precluded the propranolol-induced Ca2+ i release. Finally, preincubation of cells with sotalol or timolol, nonselective blockers of β-adrenoceptors, also reduced the Ca2+ i release activated by propranolol. Our results show that different β-blockers have differential effects on whole-cell currents and free Ca2+ i release and that propranolol activates store-operated Ca2+ i release via a mechanism that involves calcium-induced calcium release and putative downstream transducers such as IP3 The differential action of class II antiarrhythmics on Ca2+ i release may have implications on the pharmacology of these drugs.

The treatment of epilepsy remains difficult mostly since almost 30% of patients suffer from pharmacoresistant forms of the disease. Therefore, there is an urgent need to search for new antiepileptic drug candidates. Previously, it has been shown that 4-alkyl-5-substituted-1,2,4-triazole-3-thione derivativatives possessed strong anticonvulsant activity in a maximal electroshock-induced seizure model of epilepsy. In this work, we examined the effect of the chemical structure of the 1,2,4-triazole-3-thione-based molecules on the anticonvulsant activity and the binding to voltage-gated sodium channels (VGSCs) and GABA_A receptors. Docking simulations allowed us to determine the mode of interactions between the investigated compounds and binding cavity of the human VGSC. Selected compounds were also investigated in a panel of ADME-Tox assays, including parallel artificial membrane permeability assay (PAMPA), single cell gel electrophoresis (SCGE) and cytotoxicity evaluation in HepG2 cells. The obtained results indicated that unbranched alkyl chains, from butyl to hexyl, attached to 1,2,4-triazole core are essential both for good anticonvulsant activity and strong interactions with VGSCs. The combined in-vivo, in-vitro and in-silico studies emphasize 4-alkyl-5-substituted-1,2,4-triazole-3-thiones as promising agents in the development of new anticonvulsants.

The chloride (Cl^–) channel cystic fibrosis transmembrane conductance regulator (CFTR) is defective in cystic fibrosis (CF), and mutation of its encoding gene leads to various defects such as retention of the misfolded protein in the endoplasmic reticulum, reduced stability at the plasma membrane, abnormal channel gating with low open probability, and thermal instability, which leads to inactivation of the channel at physiological temperature. Pharmacotherapy is one major therapeutic approach in the CF field and needs sensible and fast tools to identify promising compounds. The high throughput screening assays available are often fast and sensible techniques but with lack of specificity. Few works used automated patch clamp (APC) for CFTR recording, and none have compared conventional and planar techniques and demonstrated their capabilities for different types of experiments. In this study, we evaluated the use of planar parallel APC technique for pharmacological search of CFTR-trafficking correctors and CFTR function modulators. Using optimized conditions, we recorded both wt- and corrected F508del-CFTR Cl^– currents with automated whole-cell patch clamp and compared the data to results obtained with conventional manual whole-cell patch clamp. We found no significant difference in patch clamp parameters such as cell capacitance and series resistance between automated and manual patch clamp. Also, the results showed good similarities of CFTR currents recording between the two methods. We showed that similar stimulation protocols could be used in both manual and automatic techniques allowing precise control of temperature, classic I/V relationship, and monitoring of current stability in time. In conclusion, parallel patch-clamp recording allows rapid and efficient investigation of CFTR currents with a variety of tests available and could be considered as new tool for medium throughput screening in CF pharmacotherapy.

The α7 nicotinic acetylcholine receptor (a7 nAChR) is an important and challenging target for drug discovery in the area of neuropsychiatric disorders. The current screening for chemicals targeting α7 nAChRs is primarily achieved by the use of low throughput assay two-electrode voltage clamp (TEVC) in non-mammalian Xenopus oocytes. Automated patch clamp system has emerged as an attractive approach compared to conventional electrophysiology. To develop a mammalian cell-based functional assay in an automated electrophysiology system, we in this study generated a stable expression of α7 nAChRs in GH3 cells that originated from a rat pituitary tumor cell line and utilized automated QPatch 16 to test a set of tool compounds and chemicals identified as α7 agonists by TEVC. For the improvement of evaluating weak or partial α7 nAChRs agonists, we achieved enhancement of the signal-to-noise ratio by the addition of a positive allosteric modulator PNU-120596, which only activates α7 current in the presence of agonist. This improved assay was further validated by using known α7 partial agonists, such as RG3487, EVP-6124, and A-P90. Using this validated assay, we were able to identify a novel agonist 140507C that partially activates α7 nAChRs. Taken together, our results validate the use of QPatch 16 for evaluation α7 partial agonists, demonstrating its utility as an effective tool for α7 ion channel drug discovery.

The ionotropic glutamate receptor GluK1 (previously known as GluR5) is a kainate subtype of the glutamate receptor family and is comprised of a ligand binding domain and an associated ion channel. The receptor is expressed throughout the central nervous system with particularly strong expression in the dorsal root ganglion and trigeminal ganglion; antagonists for the receptor could be treatments for pain disorders, as well as acute migraine.

Calcium (Ca²⁺) is an important second messenger that is involved in critical processes in cell homeostasis and signal transduction. The ratio between extra- and intracellular Ca²⁺ contributes to setting the plasma membrane potential. Intracellularly, Ca²⁺ is stored in organelles such as Mitochondria and the Endoplasmic reticulum (ER). Depletion of these stores triggers the opening of Ca²⁺ – permeable channels that allow entry of Ca²⁺ into the cell along the electrochemical gradient. This process is called store-operated calcium entry (SOCE). Activation of SOCE happens as a secondary result of ER depletion (ref). ER is being depleted by an inositol-triphosphate (IP3) pathway and phospholipase C cascade, via the IP3 receptor. SOCE can experimentally be activated by elevating intracellular levels of IP3 in the presence of a Ca²⁺ chelator.

The activation of SOCE can directly be measured as a change in clamp current when performing patch-clamp experiments. In this study the automated patch clamp system, Qube 384 was used to record Icrac. Qube 384 is a second-generation automated patch clamp device capable of testing thousands of compounds per day whilst providing true giga-ohm seal quality data. Using the Qube 384 in a drug discovery cascade enables acquisition of mode of action data simultaneous with hit detection during the primary screen, thereby minimizing the need for extended follow-up validations studies.

In this study, we present an assay for STIM1/Orai1. Firstly, we present that Orai1 current can be activated either by exchange of the intracellular solution or alternative by de facto caging an agonist in the IC in the on-cell configuration. Secondly, we pharmacologically validate the assay using the trivalent lanthanide cations blocker lanthanum (La). With this, we demonstrate that Qube 384 also is a versatile patch clamp system, even though it is fully automated and can be running unattended.

The intermediate-conductance Ca²⁺-activated K+ channel KCa3.1 (also known as KCNN4, IK1, or the Ga´rdos channel) plays an important role in the activation of T and B cells, mast cells, macrophages, and microglia by regulating membrane potential, cellular volume, and calcium signaling. KCa3.1 is further involved in the proliferation of dedifferentiated vascular smooth muscle cells and fibroblast and endothelium-derived hyperpolarization responses in the vascular endothelium. Accordingly, KCa3.1 inhibitors are therapeutically interesting as immunosuppressants and for the treatment of a wide range of fibroproliferative disorders, whereas KCa3.1 activators constitute a potential new class of endothelial function preserving antihypertensives. Here, we report the development of QPatch assays for both KCa3.1 inhibitors and activators. During assay optimization, the Ca²⁺ sensitivity of KCa3.1 was studied using varying intracellular Ca²⁺ concentrations. A free Ca²⁺ concentration of 1 lM was chosen to optimally test inhibitors. To identify activators, which generally act as positive gating modulators, a lower Ca²⁺ concentration (*200 nM) was used. The QPatch results were benchmarked against manual patch-clamp electrophysiology by determining the potency of several commonly used KCa3.1 inhibitors (TRAM-34, NS6180, ChTX) and activators (EBIO, riluzole, SKA-31). Collectively, our results demonstrate that the QPatch provides a comparable but much faster approach to study compound interactions with KCa3.1 channels in a robust and reliable assay.

Voltage-gated sodium channels (Na_Vs) are key therapeutic targets for pain, epilepsy and cardiac arrhythmias. Here we describe the development of a no-wash fluorescent sodium
influx assay suitable for high-throughput screening and characterization of novel drug leads. Addition of red-violet food dyes (peak absorbance range 495–575 nm) to assays in HEK293 cells heterologously expressing hNa_V1.1–1.8 effectively quenched background fluorescence of the sodium indicator dye Asante NaTRIUM Green-2 (ANG-2; peak emission 540 nm), negating the need for a wash step. Ponceau 4R (1 mM) was identified as a suitable quencher, which had no direct effect on NaV channels as assessed by patch-clamp experiments, and did not alter the pharmacology of the Na_V1.1–1.7 activator veratridine (EC50 10–29 μM) or the Na_V1.1–1.8 inhibitor tetracaine (IC50’s 6–66 μM). In addition, we also identified that the food dyes Ponceau 4R, Brilliant Black BN, Allura Red and Amaranth are effective at quenching the background fluorescence of the calcium indicator dyes fluo-4, fura-2 and fura-5F, identifying them as potential inexpensive alternatives to no-wash calcium ion indicator kits. In summary, we have developed a no-wash fluorescent sodium influx assay suitable for high-throughput screening based on the sodium indicator dye ANG-2 and the quencher Ponceau 4R.

Ligand-gated ion channels are of particular interest to the pharmaceutical industry for the treatment of diseases from a variety of therapeutic areas including CNS disorders, respiratory disease and chronic pain. Ligand-gated ion channels have historically been investigated using fluorescence-based and low throughput patch-clamp techniques. However, with the development of the Qube 384 automated patch-clamp system, the rapid exchange of liquid and direct measurement of ion channel currents on a millisecond timescale is now possible at a greater throughput than previously possible. Here, we have used the Qube platform to develop assays against two ligand-gated families: 1) the P2X receptor and 2) the GABAA receptor families. The P2X family is comprised of 7 family members, which are cation permeable and gated by the binding of extracellular ATP. We have assessed both agonist and antagonist pharmacology of 4 members of the P2X family, P2X1, P2X2, P2X3 and P2X4, as well as two species homologs, rP2X3 and gpP2X3. The GABAA α1β3γ2 receptor is a chloride permeable ion channel gated by the binding of GABA. We utilized stacked liquid addition to assess the open state kinetics of the channel and to investigate the effects of a positive allosteric modulator on channel function. As such, we have successfully characterized and developed assays for both the P2X receptor and GABAA receptor families and present EC/IC50 data for antagonists and positive allosteric modulators.

High conductance, calcium-activated potassium (BK) channels (K_Ca1.1) are important in regulating physiologic responses in many types of tissues and, as such, present opportunities for development of new therapeutic agents. Both channel agonists and inhibitors could have therapeutic utility, depending on medical application under consideration. However, characterization of molecular pharmacology of BK channels is incomplete and has been difficult to accomplish because of paucity of chemical leads that are acceptable templates for Medicinal Chemistry investigation. Only through continued prosecution of new high-throughput screening campaigns can this situation be rectified. Examples are presented of BK channel agonist and inhibitor discovery paradigms which will be useful for progressing BK channel future drug discovery strategies.

Electroporation is a technique allowing chemicals, drugs, or DNA to be introduced into the cell by applying an electrical field to the cells, thereby increasing the permeability of the cell membrane. The Neon^® Transfection System is a novel benchtop electroporation device that employs the pipette tip as an electroporation chamber to easily and efficiently transfect mammalian cells.

In this study, native HEK 293 cells were transfected with a plasmid encoding the human glycine receptor α1 (hGlyRα1), to demonstrate how a Qube can be used for:

1. optimization of electroporation transfection

2. pharmacological evaluation of transfected cells with varying transfection levels

Based on our recent finding that α1 selective GABA-A receptor potentiator—zolpidem—(a hypnotic drug) exerts antipsychotic-like effects in rats, we developed a series of fluorinated imidazo[1,2-a]pyridine derivatives as potential novel antipsychotic agents. The selected compounds displayed high affinity and positive allosteric modulator properties at the GABA-A receptor, enhanced metabolic stability and lack of hepatotoxicity. The most promising compound 2-(2-(4-fluorophenyl)-6-methylimidazo[1,2-a]pyridin-3-yl)-N,N-dimethylethanamide (26) showed antipsychotic-like activity in amphetamine-induced hyperlocomotion test in rats (MED = 1 mg/kg) and was characterized by a longer duration of antipsychotic-like activity as compared to zolpidem. These results are an encouraging example of a compound with non-dopaminergic mechanism of action displaying antipsychotic activity and are a point of entry for the future studies in this field.

Bruton’s tyrosine kinase (Btk) is a Tec family kinase with a well-defined role in the B cell receptor (BCR) and Fcγ receptor (FcR) signaling pathways, which makes it a uniquely attractive target for the treatment of autoimmune diseases, such as rheumatoid arthritis (RA). We reported a series of compounds bearing 7H-pyrrolo [2,3-d]pyrimidin-4-amine scaffold that potently inhibited Btk in vitro. Analysis of the structure-activity relationships (SAR) and drug-like profiles led to the discovery of the optimal compound B16. B16 preferentially inhibited Btk (IC50 = 21.70 ± 0.82 nM) over closely related kinases with moderate selectivity. Cell-based tests also confirmed that B16 significantly inhibited Btk Y223 auto-phosphorylation and PLCγ2 Y1217 phosphorylation. MTT revealed that B16 displayed weak suppression against normal LO2, HEK293 and THP-1 cell lines with IC50 values over 30 μM. Moreover, B16 showed very weak potential to block the hERG channel (IC50 = 11.10 μM) in comparison to ibrutinib (IC50 = 0.97 μM). Owing to its favorable physicochemical properties (ClogP = 2.53, aqueous solubility ≈ 0.1 mg/mL), pharmacokinetic profiles (F = 49.15%, t1/2 = 7.02 h) and reasonable CYP450 profile, B16 exhibited potent anti-arthritis activity and similar efficacy to ibrutinib in reducing paw thickness in CIA mice. In conclusion, B16 is a potent, selective and durable inhibitor of Btk and has the potential to a safe and efficacious treatment for arthritis.

A series of novel berberine derivatives, 4,7,12,12a-tetrahydro-5H-thieno[3′,2’:3,4]pyrido[1,2-b]isoquinolines was designed, synthesized, and biologically evaluated for their anti-diabetic activity. Following the evaluation in two types of cells, compounds 4aa, 4bq, and 4bv stimulated glucose consumption (1.8- to 2.3-fold), reduced gluconeogenesis (60–85%), inhibited mitochondria respiratory chain complex I and activated AMPK indirectly. In a db/db mice model, compounds 4bq and 4bv lowered fasting blood glucose at a dose of 120 mg/kg/day. In addition, compounds 4bq and 4bv were found to possess improved pharmacokinetic profiles (bioavailability 45 and 106%, respectively) compared to berberine. Compounds 4bq and 4bv exhibited no obvious hERG inhibition (IC₅₀ > 10 μM).

When determining the potencies of drugs in single-cell assays it is a major concern that the employed assay is affected by factors such as the density of the applied cell suspension. It is envisaged that a high density of cells may lead to titration of test compound molecules (by receptor-binding and adsorption) by cells in the vicinity of the targeted cell. This would cause a rightward shift in the concentration-response relationship, and consequently, incorrectly increased IC₅₀ values. We have tested whether IC₅₀ values determined with the QPatch 16 automated patch-clamp system are dependent on the density of the applied cell suspension. We assume that cell density determines the number of cells present in the extracellular flow channel.

It is predicted that increasing the cell suspension density may reduce the time it takes to position a cell on the patch-clamp site. The cell positioning step is accomplished by suction. We tested the effect of the cell suspension density using a wide range of densities (from 0.5 to 16 million cells per ml) on the cell positioning time. For the tests we used two expression systems: (1) CHO cells expressing hERG potassium channels, and (2) HEK cells expressing Na_v1.2a sodium channels. We used test compounds with very different oilwater partition coefficient (log(P)): astemizole and verapamil with log(P) values of 6.43 and 3.45, respectively, (source: www.syrres.com/esc/physdemo) for hERG assays, and tetrodotoxin (TTX) with log(P) of -6.21 for the Na_v1.2a assay.

Embryonic stem cell-derived cardiomyocytes have many of the phenotypic properties of authentic cardiomyocytes, and great interest has been shown in their possibilities for modeling human diseases. For drug discovery stem cell derived cardiomyocytes provide an exciting new tool for e.g. cardiac target discovery and cardiac safety studies. Axiogenesis has developed the Cor.At® cells. These cells are cardiomyocytes derived from transgenic mouse embryonic stem cells and are 99.9% pure. This report presents recordings of three essential currents; I_K, I_Ca, and I_Na from the Cor.At® cells obtained with the QPatch system.

Fully differentiated, cell lineages formed from human induced pluripotent stem cells (iPSC) are powerful cellular models for action potential based screening of compounds. Parallel recordings from up to 48 iPS derived cardiomyocytes are made on the QPatch HT with full analysis of action potential features including action potential duration, upstroke velocity and membrane firing potential. In this report we describe the validation of pharmacology and biophysics of iPSC from Axiogenesis on the QPatch in current clamp mode.

Recent increasing evidence suggests that the currently-used platforms in vitro I_Kr and APD, and/or in vivo QT assays are not fully predictive for TdP, and do not address potential arrhythmia (VT and/or VF) induced by diverse mechanisms of action. In addition, other cardiac safety liabilities such as functional dysfunction of excitation-contraction coupling (contractility) and structural damage (morphological damage to cardiomyocytes) are also major causes of drug attrition, but current in vitro assays do not cover all these liabilities. We organized the Consortium for Safety Assessment using Human iPS cells (CSAHi; http://csahi.org/en/), based on the Japan Pharmaceutical Manufacturers Association (JPMA), to verify the application of human iPS/ES cell-derived cardiomyocytes in drug safety evaluation. The main goal of the CSAHi HEART team has been to propose comprehensive screening strategies to predict a diverse range of cardiotoxicities by using recently introduced platforms (multi-electrode array (MEA), patch clamp, cellular impedance, motion field imaging [MFI], and Ca transient systems) while identifying the strengths and weaknesses of each. Our study shows that hiPS-CMs used in these platforms have pharmacological responses more relevant to humans in comparison with existent hERG, APD or Langendorff (MAPD/contraction) assays, and not only MEA but also other methods such as impedance, MFI, and Ca transient systems would offer paradigm changes of platforms for predicting drug-induced QT risk and/or arrhythmia or contractile dysfunctions. Furthermore, we propose a potential multi-parametric platform in which field potential (MEA)-Ca transient-contraction (MFI) could be evaluated simultaneously as an ideal novel platform for predicting a diversity of cardiac toxicities, namely whole effects on the excitation-contraction cascade.

We have prepared three alkaloids from the Agelas sponges, clathrodin, hymenidin and oroidin, and a series of their synthetic analogues, and evaluated their inhibitory effect against six isoforms of the K_v1 subfamily of voltage-gated potassium channels, K_v1.1-K_v1.6, expressed in Chinese Hamster ovary (CHO) cells using automated patch clamp electrophysiology assay. The most potent inhibitor was the (E)-N-(3-(2-amino-1H-imidazol-4-yl)allyl)-4,5-dichloro-1H-pyrrole-2-carboxamide (6g) with IC₅₀ values between 1.4 and 6.1 μM against K_v1.3, K_v1.4, K_v1.5 and K_v1.6 channels. All compounds tested displayed selectivity against K_v1.1 and K_v1.2 channels. For confirmation of their activity and selectivity, compounds were additionally evaluated in the second independent system against K_v1.1-K_v1.6 and K_v10.1 channels expressed in Xenopus laevis oocytes under voltage clamp conditions where IC₅₀ values against K_v1.3-K_v1.6 channels for the most active analogues (e.g. 6g) were lower than 1 μM. Because of the observed low sub-micromolar IC₅₀ values and fairly low molecular weights, the prepared compounds represent good starting points for further optimisation towards more potent and selective voltage-gated potassium channel inhibitors.

The sodium ion channel Na_v1.5 is expressed as in integral membrane protein and contains a tetrodotoxin-resistant voltage-gated sodium channel subunit.The encoded protein is found primarily in cardiac muscle and is responsible for the initial upstroke of the action potential in an electrocardiogram. Mutations in the gene are associated with long QT syndrome type 3, Brugada syndrome, primary cardiac conduction disease and idiopathic ventricular fibrillation.

The slowly activating, delayed rectifier K+ channel is among other activities important for regulating the repolarization phase of cardiac action potentials. The KvLQT1/minK channel consists of two transmembrane proteins. Mutations in the genes KCNQ1/KCNE1 coding for KvLQT1/minK are associated with predisposition to deafness, cardiac arrhythmia syndromes including long QT syndrome, atrial fibrillation and sudden infant death syndrome.

The voltage gated potassium channel K_V1.5 is a homotetrameric protein present in the heart. It is a delayed rectifier, participating in the early phase of the heart action potential. This report shows data from CHO cells stably expressing K_V1.5 tested on the QPatch platform.

Na_V1.7 has moved in the focus of the drug discovery industry in the last years as numerous studies provided strong proof for the efficacy to alleviate both neuropathic and inflammatory pain (Sun, Cohen, & Dehnhardt, 2014). We demonstrate high reproducibility and precision of Na_V1.7 data recorded on Qube

95% of all tested cells had a GΩ seal in the beginning and
61% at the end of a long, 70 min experiment

V_1/2 is stable over time with only a slight shift

Experiments can be performed in physiological ringers,
however fluoride in the internal solution significantly
increases success rate without any altering biophysical
parameters.

Functional validation and performance data for the CHO hERG DUO cell line available as a QCell from Sophion. This cell line is made in collaboration with B’SYS GmbH (Switzerland).

CHO-hERG DUO is a QCell, a cell line that can be provided by Sophion. The performance of this particular cell line was evaluated on multi-hole QPlates on QPatch HTX. We have evaluated several features – success rates, stability, pharmacology and biophysical properties of the assay. The report also contains introduction to some of the methodology that is used when running multi-hole experiments on the QPatch.

CHO-hERG DUO is a QCell, a cell line that can be provided by Sophion. The performance of this particular cell line was evaluated on multi-hole QPlates on QPatch HTX. We have evaluated several features – success rates, stability, pharmacology and biophysical properties of the assay. The report also contains introduction to some of the methodology that is used when running multi-hole experiments on the QPatch.

In this report the applications of the CHO TREx-P2X₃ cell line (hereafter called CHO P2X3) are described. The purpose was to determine the EC₅₀ value for αβ-methyl ATP, and the IC₅₀ value of PPADS on the P2X₃ receptors. All data presented was obtained with QPatch, and analysed with the QPatch assay software.

L-type voltage gated calcium channels are involved in cardiac arrhythmia and hypertension and are therefore possible therapeutic targets. Current from Cav1.2 expressed in CHO-K1 was tested on QPatch in both single-hole and multi-hole mode. Success rates, IV characteristics, steady state inactivation and three different known compounds were tested to see if the biophysical and pharmaceutical properties were as expected. Furthermore it was tested how well the cells could be frozen down for “direct use” on the QPatch.

The risk of fatal arrhythmias is the major concern for using chloroquine (CQ) or hydroxychloroquine (HCQ) to treat coronavirus disease 2019 (COVID‐19), but the reported number of life‐threatening arrhythmic events or deaths is relatively small. The objective of this study was to assess the arrhythmogenic risk of these two drugs using a multiscale heart simulation, which allows testing even at high concentrations, including those that cause fatal arrhythmias. We measured the inhibitory action of CQ, HCQ, and HCQ with 30 μM azithromycin (AZ) on six ion currents (fast [INa] and late [INa,L] components of the sodium current, L‐type calcium current [ICa,L], rapid [IKr/hERG], and slow [IKs] components of delayed rectifier potassium, and inward rectifier potassium [IK1]) over a wide range of concentrations using the automated patch‐clamp system. Using the concentration–inhibition relationship that was thus obtained, we simulated the drug effects while increasing the concentration until the life‐threatening arrhythmia, Torsade de Pointes (TdP), was observed. The obtained threshold concentrations for TdP were 12.5, 35, and 22.5 μM for CQ, HCQ, and HCQ with AZ, respectively. Adding therapeutic concentrations of mexiletine or verapamil successfully prevented the occurrence of TdP, and verapamil was more effective. CQ, HCQ, and HCQ with AZ thresholds for TdP were larger than both antiviral concentrations that were reported by in vitro experiments and free plasma concentrations that were attained by the clinically used dosage. The current simulation data provided a safety margin to the currently used clinical dose for CQ and HCQ/AZ.

Electrically excitable cells, such as neurons, exhibit tremendous diversity in their firing patterns, a consequence of the complex collection of ion channels present in any specific cell. Although numerous methods are capable of measuring cellular electrical signals, understanding which types of ion channels give rise to these signals remains a significant challenge. Here, we describe exogenous probes which use a novel mechanism to report activity of voltage-gated channels. We have synthesized chemoselective derivatives of the tarantula toxin guangxitoxin-1E (GxTX), an inhibitory cystine knot peptide that binds selectively to Kv2-type voltage gated potassium channels. We find that voltage activation of K_v2.1 channels triggers GxTX dissociation, and thus GxTX binding dynamically marks K_v2 activation. We identify GxTX residues that can be replaced by thiol- or alkyne-bearing amino acids, without disrupting toxin folding or activity, and chemoselectively ligate fluorophores or affinity probes to these sites. We find that GxTX–fluorophore conjugates colocalize with K_v2.1 clusters in live cells and are released from channels activated by voltage stimuli. K_v2.1 activation can be detected with concentrations of probe that have a trivial impact on cellular currents. Chemoselective GxTX mutants conjugated to dendrimeric beads likewise bind live cells expressing K_v2.1, and the beads are released by channel activation. These optical sensors of conformational change are prototype probes that can indicate when ion channels contribute to electrical signaling.

The rising interest in Kv7 modulators originates from their ability to evoke fundamental electrophysiological perturbations in a tissue-specific manner. A large number of therapeutic applications are, in part, based on the clinical experience with two broad-spectrum Kv7 agonists, flupirtine and retigabine. Since precise molecular structures of human Kv7 channel subtypes in closed and open states have only very recently started to emerge, computational studies have traditionally been used to analyze binding modes and direct the development of more potent and selective Kv7 modulators with improved safety profiles. Herein, the synthetic and medicinal chemistry of small molecule modulators and the representative biological properties are summarized. Furthermore, new therapeutic applications supported by in vitro and in vivo assay data are suggested.

Planar silicon chips with 1-2-μm etched holes (average resistance: 2.04 ± 0.02 MΩ in physiological buffer, n = 274) have been developed for patch-clamp recordings of whole-cell currents from cells in suspension. An automated 16-channel parallel screening system, QPatch 16, has been developed using this technology. A single-channel prototype of the QPatch system was used for validation of the patch-clamp chip technology. We present here data on the quality of patch-clamp recordings and from actual drug screening studies of human potassium channels expressed in cultured cell lines. Using Chinese hamster ovary (CHO) and human embryonic kidney cells (HEK), gigaseals of 4.1 ± 0.4 GΩ (n = 146) and high-quality whole-cell current recordings were obtained from hERG and KCNQ4 potassium channels. Success rates for gigaseal recordings varied from 40 to 95%, and 67% of the whole-cell configurations lasted for >20 min. Cells were maintained in suspension up to 4 h in a cell storage facility that is integrated in the QPatch 16. No decline in patchability was observed during this time course. A series of screens was conducted with known inhibitors of the hERG and KCNQ4 potassium channels. Dose-response relationship characterizations of verapamil and rBeKm-1 blockage of hERG currents provided IC50 values similar to values reported in the literature.

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC–CM) are providing new, highly predictive strategies to assess cardiotoxicity in vitro and can thus reduce costs for cardiac safety assessment in drug development. Different technologies are available to assess compound effects on cardiomyocytes, out of all, the patch clamp technique remains the gold standard as it allows to study compound effects both on individual currents but also on the entire ion channel ensemble in form of an action potential (AP). However, such studies have traditionally been limited by the labour-intensive and low-throughput nature of patch clamp electrophysiology.

Here we present the electrophysiological characterization of hiPSC–CM (Cor.4U) cells using our automated patch clamp (APC) platform Qube 384. Electrophysiological investigation of hiPSC-CM requires a high quality set up that offers the possibility to both record APs in current clamp mode and to isolate individual ion channel currents using voltage clamp. Qube is a 384-channel automated patch clamp system that fulfils all these requirements.

Our data illustrate that stem cell technology in combination with Qube’s high throughput capability holds great potential to accelerate cardiac safety studies.

Human induced pluripotent stem cell-derived cardiomyocytes
(hiPSC – CM) are providing new, highly predictive strategies to assess cardiotoxicity in vitro and can thus reduce costs for cardiac safety assessment in drug development (Ma et al., 2011).
In the present study, we characterized Cor.4U® cells using Sophion’s 48-channel QPatch both in voltage and in current clamp mode.

Electrophysiological investigation of hiPSC-CM requires a high quality set up that offers the possibility to both record action potentials in current clamp mode and to isolate individual ion channel currents using voltage clamp.

The QPatch fulfills these requirements and does not rely on the use of a seal enhancer. Many automated patch clamp systems rely on the use of fluoride in the internal solution to gain sufficiently high seal resistances. However, fluoride is also known to chelate Ca²⁺, inhibit proteases and hyperpolarize the gating of sodium channels and may thus affect the recordings (Cummins et al., 2009).

As the number of cardiomyocytes are usually limited, it is critical to minimize the cell consumption per experiment. QPatch is endowed with a feature that helps to reduce the number of cells needed per experiment. Using this feature, 200 μl of cell suspension is sufficient to run a 48-well QPlate.

Human induced pluripotent stem cells (hiPSCs) can be differentiated into multiple cell types, including neurons and cardiomyocytes, which can be used as human disease models for in vitro drug development. Ion channels represent highly attractive therapeutic targets in the nervous and the cardiovascular system2, rendering electrophysiological studies of hiPSCs important for their usage in drug discovery. However, such studies have traditionally been limited by the labour-intensive and low-throughput nature of patch clamp electrophysiology.

Here we present the electrophysiological characterization of hiPSC-derived motor neurons using our automated patch clamp (APC) platforms, Qube 384 and QPatch. Our observations include channel expression versus time in culture, the pharmacological dissection of endogenous ion channels (e.g. NaV and KV), identification of ligand-gated receptors and recordings of action potentials.

Utilizing the high-throughput nature of our systems we tested in parallel two disease models, using hiPSC neurons derived from Spinal Muscular Atrophy (SMA) or Amyotrophic Lateral Sclerosis (ALS) patients, together with control cells from healthy subjects and isogenic control cells. The results indicate an over-expression of Na+ channels in both cell lines, which could be rescued by a point mutation in the superoxide dismutase (SOD1) gene in the isogenic ALS cell line.

The QPatch™ technology has been developed to significantly increase throughput in ion channel drug screening. It is based on planar glass-coated silicon chips with micro-etched patch-clamp holes. Extra andintracellular Ringer solutions are applied by miniature flow channels, which ensures laminar flow and short fluid exchange times (<100 ms). The walls of the flow channels are covered with non-polymer materials (glass and silicon) to minimize problems with non-specific binding of ‘sticky’ compounds. Below is shown the QPatch-16, the first instrument based on the QPatch technology. It runs 16 parallel whole-cell patch-clamp experiments simultaneously with success rates of 50-80 %. Cells are maintained in growth medium in an onboard cell storage facility for up to 4 hours until shortly before an experiment. At that time they are automatically spun down in a miniature centrifuge, washed, and resuspended in Ringer’s solution before being applied to the patch-clamp site. We have determined the IC50 values for 32 hERG channel blockers in a blind test and made a comparison with IC50 values obtained in conventional
manual patch-clamp.

The rodent neuroblastoma cell line, ND7-23, is used to express voltage-dependent sodium (Na_v) and other neuronal ion channels resistant to heterologous expression in Chinese hamster ovary (CHO) or human embryonic kidney (HEK) cells. Their advantage is that they provide endogenous factors and signaling pathways to promote ion channel peptide folding, expression, and function at the cell surface and are also amenable to automated patch clamping. However, ND7-23 cells exhibit endogenous tetrodotoxin (TTX)-sensitive Na_v currents, and molecular profiling has revealed the presence of Na_v1.2, Na_v1.3, Na_v1.6, and Na_v1.7 transcripts, but no study has determined which subtypes contribute to functional channels at the cell surface. We profiled the repertoire of functional Nav channels endogenously expressed in ND7-23 cells using the QPatch automated patch clamp platform and selective toxins and small molecules. The potency and subtype selectivity of the ligands (Icagen compound 68 from patent US-20060025415-A1-20060202, 4,9 anhydro TTX, and Protoxin-II) were established in human Na_v1.3, Na_v1.6, and Na_v1.7 channel cell lines before application of selective concentrations to ND7-23 cells. Our data confirm previous studies that >97% of macroscopic Na_v current in ND7-23 cells is carried by TTX-sensitive channels (300 nM TTX) and that Na_v1.7 is the predominant channel contributing to this response (65% of peak inward current), followed by Nav1.6 (∼20%) and negligible Na_v1.3 currents (∼2%). In addition, our data are the first to assess the Na_v1.6 potency (50% inhibitory concentration [IC₅₀] of 33 nM) and selectivity (50-fold over Na_v1.7) of 4,9 anhydro TTX in human Na_v channels expressed in mammalian cells, confirming previous studies of rodent Na_v channels expressed in oocytes and HEK cells.

Alpha7 nicotinic acetylcholine receptor channels are important ligand-gated ion channels that are fast desensitizing, cation selective and have been implicated in the pathophysiology of schizophrenia and Alzheimer’s disease. We report here high quality 7 parallel patch clamp recordings using the QPatch automated patch clamp system. The QPatch patch clamps up to 48 cells in parallel with the same high fidelity as conventional patch clamp. EC₅₀ and IC₅₀ values were comparable to values obtained with conventional patch clamp. The EC₅₀ value for acetylcholine (ACh) on the QPatch with area under the curve (AUC) analysis was 26M compared to a value of 29M determined from conventional patch clamp experiments. Sequential additions of ACh can be made with minimal decay of the peak amplitude. The competitive7 antagonistmethyllycaconitine (MLA) blocked currents with an IC₅₀ value of 0.25nM which is similar to published IC₅₀ values for MLA. Finally, two different classes of positive allosteric modulators represented by PNU-120596 and NS-1738 elicited characteristic responses, thus allowing accurate characterization of modulation and measurements of potency. These results demonstrate that 7 nicotinic acetylcholine receptor channels can be studied reliably in a higher throughput, parallel manner with the QPatch automated patch clamp system.

Cystic fibrosis transmembrane conductance regulator (CFTR) functions as a chloride channel and controls the regulation of other transport pathways. Mutations in the CFTR gene have been found to cause cystic fibrosis (CF) and congenital bilateral aplasia of the vas deferens (CBAVD).
Here we demonstrate that reliable recordings with the chloride conducting ion channel CFTR can be performed on QPatch using fluoride as activator of adenylate cyclase. We have shown the ability to determine the IC50 for CFTRinh-172 in close correspondence to the literature values.
We can conclude that the overall success in obtaining stable seals and completed experiments are at a level that clearly identifies the CHO–hCFTR assay as feasible and with high quality results on QPatch.

A number of potassium channels including members of the KCNQ family and the Ca²⁺ activated IK and SK, but not BK, are strongly and reversibly regulated by small changes in cell volume. It has been argued that this general regulation is mediated through sensitivity to changes in membrane stretch. To test this hypothesis we have studied the regulation of KCNQ1 and BK channels after expression in Xenopus oocytes. Results from cell-attached patch clamp studies (∼50 μm2 macropatches) in oocytes expressing BK channels demonstrate that the macroscopic volume-insensitive BK current increases with increasing negative hydrostatic pressure (suction) applied to the pipette. Thus, at a pipette pressure of−5.0±0.1 mmHgthe increase amounted to 381±146% (mean±s.e.m., n =6, P <0.025). In contrast, in oocytes expressing the strongly volume-sensitive KCNQ1 channel, the current was not affected by membrane stretch. The results indicate that (1) activation of BK channels by local membrane stretch is not mimicked by membrane stress induced by cell swelling, and (2) activation of KCNQ1 channels by cell volume increase is not mediated by local tension in the cell membrane. We conclude that stretch and volume sensitivity can be considered two independent regulatory mechanisms.

Correct cell handling is important for optimal experimental results. This report gives advice on cell handling in order that you can obtain the best cells possible for use on the QPatch or Qube instruments. Here we describe how to culture your cells and how to reach optimal cell confluence for both cell propagation (“mother flasks”) and for experimental use. Detachment enzymes recommended by Sophion are also described together with methods for harvesting and storing cells.

The voltage-sensitive L-type Ca²⁺-channel (LTCC) Ca_V1.2 is widely expressed in vascular smooth muscle tissue and the heart muscle^1-3. The opening of the channels leads to an increase of intracellular calcium, which acts as second messenger and thereby affects a variety of cellular processes⁴ including heart muscle contraction and neurotransmitter release. Ca_V1.2 is therefore an important target in e.g. safety pharmacology screening. The channels are known to require a large depolarization for their activation and once activated they display a long-lasting current flow, which typically can be blocked by low micromolar concentrations of e.g. dihydropyridines, phenylalkylamines and benzothiazepines^5,6.
In this study, currents from CHO-hCa_V1.2 were recorded on the high-throughput platform Qube 384 in multi-hole mode (10 patch holes per well) at both a customer’s site and at Sophion. Success rates, rundown, sealing properties and the pharmacological effects of two compounds were determined.

The voltage-sensitive L-type Ca²⁺-channel (LTCC) Ca_V1.2 is a crucial component for controlling intracellular activity and thereby essential in the cardiovascular and neuronal system. It is widely expressed in vascular smooth muscle tissue and the heart muscle. The opening of the channels leads to an increase of intracellular calcium, which act as second messenger, and thereby affects a variety of cellular processes including heart muscle contraction and Ca_V1.2 is therefore an important target in e.g. safety pharmacology screening. Ca_V1.2 channels are known to require a large depolarization for their activation and once activated they display a long-lasting current flow, which typically can be blocked by low micromolar concentrations of e.g. dihydropyridines, phenylalkylamines and benzothiazepines. In these studies, currents from HEK-hCa_V1.2 were recorded on the high-throughput platform Qube 384 in both single-hole and multi-hole mode. Success rates, IV characteristics and the pharmacological effects of three different compounds were determined.

Substantial efforts have been recently committed to develop coronavirus disease-2019 (COVID-19) medications, and Hydroxychloroquine alone or in combination with Azithromycin has been promoted as a repurposed treatment. Although these drugs may increase cardiac toxicity risk, cardiomyocyte mechanisms underlying this risk remain poorly understood in humans. Therefore, we evaluated the proarrhythmia risk and inotropic effects of these drugs in the cardiomyocyte contractility-based model of the human heart. We found Hydroxychloroquine to have a low proarrhythmia risk, whereas Chloroquine and Azithromycin were associated with high risk. Hydroxychloroquine proarrhythmia risk changed to high with low level of K⁺, whereas high level of Mg²⁺ protected against proarrhythmic effect of high Hydroxychloroquine concentrations. Moreover, therapeutic concentration of Hydroxychloroquine caused no enhancement of elevated temperature-induced proarrhythmia. Polytherapy of Hydroxychloroquine plus Azithromycin and sequential application of these drugs were also found to influence proarrhythmia risk categorization. Hydroxychloroquine proarrhythmia risk changed to high when combined with Azithromycin at therapeutic concentration. However, Hydroxychloroquine at therapeutic concentration impacted the cardiac safety profile of Azithromycin and its proarrhythmia risk only at concentrations above therapeutic level. We also report that Hydroxychloroquine and Chloroquine, but not Azithromycin, decreased contractility while exhibiting multi-ion channel block features, and Hydroxychloroquine’s contractility effect was abolished by Azithromycin. Thus, this study has the potential to inform clinical studies evaluating repurposed therapies, including those in the COVID-19 context. Additionally, it demonstrates the translational value of the human cardiomyocyte contractility-based model as a key early discovery path to inform decisions on novel therapies for COVID-19, malaria, and inflammatory diseases.

Cardiomyocytes derived from human induced pluripotent stem cells (hiPSC-CM) form spontaneously beating syncytia in-vitro. We evaluated whether hiPSC-CM are a compelling model of human cardiac pharmacology useful for early drug development. Methods: We measured hiPSC-CM beating frequency using Ca-sensitive dyes and a high-throughput screening system. We quantified the effects of 640 drugs with various structures and pharmacologies. Results: When tested at 1 µM, most drugs without direct effects on heart rhythm or with effects at high concentrations do not change frequency, indicating specificity. In contrast, the preparation detects compounds with direct activity on heart rhythm, demonstrating sensitivity. In particular, β-adrenergic agonists increase frequency and the model differentiates β2 from β1 agonists, as well as partial from full agonists. Phosphodiesterase inhibitors have subtype-specific actions and PDE4 is particularly important in controlling frequency. The preparation is sensitive to cardiac ion channel blockers: L-type calcium channel blockers, Class-I and Class-III antiarrhythmics change frequency but drugs acting on KATP channels do not. The assay detects compounds blocking the cardiac rapid delayed-rectifier K channel and is an alternative to the classic “hERG test”. Conclusion: hiPSC-CM are a useful in-vitro cardiac model in drug development since they respond appropriately to drugs that modify heart rate in humans.

Introduction

Cardiac sodium channel antagonists have historically been used to treat cardiac arrhythmias by preventing the re-entry of the electrical impulse that could occur following myocardial damage. However, clinical studies have highlighted a significant increase in mortality associated with such treatment. Cardiac sodium channel antagonist activity is now seen as an off-target pharmacology that should be mitigated during the drug development process. The aim of this study was to examine the correlation between in vitro/ex vivo assays that are routinely used to measure Na_V1.5 activity and determine the translatability of the individual assays to QRS prolongation in the clinic.

Methods

A set of clinical compounds with known Na_V1.5 activity was profiled in several in vitro/ex vivo assays (binding, membrane potential, patch clamp and the Langendorff isolated heart). Clinical data comprising compound exposure levels and changes in QRS interval were obtained from the literature. Sensitivity/specificity analysis was performed with respect to the clinical outcome.

Results

The in vitro assays showed utility in predicting QRS prolongation in the clinic. Optimal thresholds were defined for each assay (binding: IC₂₀; membrane potential: IC10; patch clamp: IC₂₀) and sensitivity (69–88%) and specificity (53–84%) values were shown to be similar between assay formats.
Discussion

The data provide clear statistical insight into the translatability of Na_V1.5 antagonism data generated in vitro to potential clinical outcomes. These results improve our ability to understand the liability posed by such activity in novel development compounds at an early stage.

Increasing evidence suggests that ion channels and pumps not only regulate membrane potential, ion homeostasis, and electric signaling in excitable cells but also play important roles in cell proliferation, migration, apoptosis and differentiation. Consistent with a role in cell signaling, channel proteins and ion pumps can form macromolecular complexes with growth factors, and cell adhesion and other signaling molecules. And while cancer is still not being cataloged as a channelopathy, as the non-traditional roles of ion pumps and channels are being recognized, it is increasingly being suggested that ion channels and ion pumps contribute to cancer progression. Cancer cell migration requires the regulation of adhesion complexes between migrating cells and surrounding extracellular matrix (ECM) proteins. Cell movement along solid surfaces requires a sequence of cell protrusions and retractions that mainly depend on regulation of the actin cytoskeleton along with contribution of microtubules and molecular motor proteins such as mysoin.

This process is triggered and modulated by a combination of environmental signals, which are sensed and integrated by membrane receptors, including integrins and cadherins. Membrane receptors transduce these signals into downstream signaling pathways, often involving the Rho GTPase protein family. These pathways regulate the cytoskeletal rearrangements necessary for proper timing of adhesion, contraction and detachment of cells in order to find their way through extracellular spaces. Migration and adhesion involve continuous modulation of cell motility, shape and volume, in which ion channels and pumps play major roles. Research on cancer cells suggests that certain ion channels may be involved in aberrant tumor growth and channel inhibitors often lead to growth arrest. This review will describe recent research into the role of ion pumps and ion channels in cell migration and adhesion, and how they may contribute to tumor development.

The calcium-activated chloride channel anoctamin 1 (ANO1) is located within the 11q13 amplicon, one of the most frequently amplified chromosomal regions in human cancer, but its functional role in tumorigenesis has remained unclear. The 11q13 region is amplified in ∼15% of breast cancers. Whether ANO1 is amplified in breast tumors, the extent to which gene amplification contributes to ANO1 overexpression, and whether overexpression of ANO1 is important for tumor maintenance have remained unknown. We have found that ANO1 is amplified and highly expressed in breast cancer cell lines and primary tumors. Amplification of ANO1 correlated with disease grade and poor prognosis. Knockdown of ANO1 in ANO1-amplified breast cancer cell lines and other cancers bearing 11q13 amplification inhibited proliferation, induced apoptosis, and reduced tumor growth in established cancer xenografts. Moreover, ANO1 chloride channel activity was important for cell viability. Mechanistically, ANO1 knockdown or pharmacological inhibition of its chloride-channel activity reduced EGF receptor (EGFR) and calmodulin-dependent protein kinase II (CAMKII) signaling, which subsequently attenuated AKT, v-src sarcoma viral oncogene homolog (SRC), and extracellular signal-regulated kinase (ERK) activation in vitro and in vivo. Our results highlight the involvement of the ANO1 chloride channel in tumor progression and provide insights into oncogenic signaling in human cancers with 11q13 amplification, thereby establishing ANO1 as a promising target for therapy in these highly prevalent tumor types.

Novel biphenyloxy-alkyl derivatives of piperidine and azepane were synthesized and evaluated for their binding properties at the human histamine H3 receptor. Two series of compounds were obtained with a meta- and a para-biphenyl moiety. The alkyl chain spacer contained five and six carbon atoms. The highest affinity among all compounds was shown by 1-(6-(3-phenylphenoxy)hexyl)azepane (13) with a Ki value of 18 nM. Two para-biphenyl derivatives, 1-(5-(4-phenylphenoxy)pentyl)piperidine (14; Ki = 25 nM) and 1-(5-(4-phenylphenoxy)pentyl)azepane (16; Ki = 34 nM), classified as antagonists in a cAMP accumulation assay (IC₅₀ = 4 and 9 nM, respectively), were studied in detail. Compounds 14 and 16 blocked RAMH-induced dipsogenia in rats (ED₅₀ of 2.72 mg/kg and 1.75 mg/kg respectively), and showed high selectivity (hH4R vs hH3R > 600-fold) and low toxicity (hERG inhibition: IC₅₀ > 1.70 µM; hepatotoxicity IC₅₀ > 12.5 µM; non-mutagenic up to 10 µM). Furthermore, the metabolic stability was evaluated in vitro on human liver microsomes (HLMs) and/or rat liver microsomes (RLMs). Metabolites produced were analyzed and tentatively identified by UPLC-MS techniques. The results demonstrated easy hydroxylation of the biphenyl ring.

Voltage-gated sodium channels (VGSC) are in the spotlight of drug development as strong evidence is available linking different subtypes to various disease states. VGSC is responsible for the initiation and propagation of action potentials in excitable cells. During this process, the VGSC transitions from a closed to an open into an inactivated state. Interestingly, inhibitory compounds often exhibit different pharmacological profiles dependent upon the conformational state of the ion channel.

In the present work, the second generation QPatch (QPatch II; Sophion Bioscience) was used in combination with adaptive voltage protocols to investigate state-dependent inhibition of tetrodotoxin (TTX), amitriptyline and tetracaine on 8 different VGSC subtypes (Nav1.1-8). A first step was to determine the half-inactivation potential V½(inactivation) for each individual cell. This value was then used during the next steps as a preconditioning pulse. Such an adaptive protocol allowed to determine IC50 values for both the closed and the inactivated state and reduce heterogeneity of the cells. Both IC50 values and biophysical parameters of the different subtypes align well with literature values.

The new clone screening feature developed for QPatch HT and QPatch 16 allows running up to eight different cell lines (clones or subtypes) at the same time, thus ensuring that the exact same conditions – such as temperature, Ringer’s, pH etc. – are applied for each of the cell lines tested. On QPatch HT each of the eight cell lines are applied to six separate sites on the measurement plate, QPlate 48. After the experiment is finished the data for each cell type can be tracked and compared easily using the advanced analysis functionalities in the QPatch Assay Software. In our study, seven subtypes of the voltage gated sodium channel, Na_v1.1 αβ1, 1.2 α, 1.3 αβ1, 1.4 αβ1, 1.6 αβ1, 1.7 αβ1 and Na_v1.8 α, were tested in parallel on QPatch HT, using the cell cone screening feature. Experiments were designed to explore 1) TTX sensitivity, 2) IV-relationship for activation and inactivation, for the entire panel of Na_v channel subtypes in a single experiment. Thus several different voltage protocols were used in the same experiment.

The voltage dependent sodium channel is responsible for the upstroke and directed propagation of action potentials in nerve and muscle cells and is therefore a central ion channel in excitable tissues. The implication of voltage gated sodium channels in pain mediation and diseases such as epilepsy and cardiac arrhythmia has made them very important targets for drug discovery. Here we show eight subtypes of the voltage gated sodium channel tested in parallel on Qube, the Sophion 384-format automated patch clamp system designed for high throughput and high fidelity electrophysiological recordings. On Qube it is possible to run up to 16 different clones or cell lines simultaneously which ensures identical conditions across the experiments. Three types of experiments were designed to explore 1) TTX sensitivity, 2) IV-relationship for activation and inactivation and 3) pulse train suitable for screening for use dependent sodium channels blockers. For the screening example pharmacology was represented by tetracaine. For every run Nav channel subtypes Nav1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7 and 1.8 were tested side by side.

The multi-hole patch clamp technology for the QPatch enables gigaseal recordings of up to ten cells patch-clamped on a single measurement site. In this set of experiments, we have convincingly demonstrated that multi-hole QPatch experiments of fast and slow desensitizing ligand-gated ion channels perform as well as single-hole QPatch experiments with respect to both biophysical and pharmacological characteristics. In the QPlate X, the ten patch holes have a relatively wide spatial distribution to avoid intercellular contact and downstream space clamp issues. The wide spatial distribution could, on the other hand, potentially slow down the liquid exchange times. We examined the glutamate receptor GluR5, the nicotinic acetylcholine receptor nAChR α1, the acid-sensing ion channel ASIC1a, and the anionic γ-aminobutyric acid receptor A GABA-A α1β2γ2 with regards to agonist rise-time, reversal potential and pharmacological properties on the QPatch HTX in multi-hole mode and compared to results obtained in the classic single-hole mode. All data clearly demonstrate that while the amplitude of the elicited ion channel current is multiplied by a factor of 7-10, and the successrate in terms of usable current amplitude is increased, other significant biophysical properties of these ion channels remain unaltered.

The spread of antibiotic-resistant human pathogens and the declining number of novel antibiotics in the development pipeline is a global challenge that has fueled the demand for alternative options. The search for novel drug candidates has expanded to include not only antibiotics but also adjuvants capable of restoring antibiotic susceptibility in multidrug-resistant (MDR) pathogens. Insect-derived antimicrobial peptides (AMPs) can potentially fulfil both of these functions. We tested two coleoptericins and one coleoptericin-like peptides from the invasive harlequin ladybird Harmonia axyridis against a panel of human pathogens. The AMPs displayed little or no activity when tested alone but were active even against clinical MDR isolates of the Gram-negative ESKAPE strains when tested in combination with polymyxin derivatives, such as the reserve antibiotic colistin, at levels below the minimal inhibitory concentration. Assuming intracellular targets of the AMPs, our data indicate that colistin potentiates the activity of the AMPs. All three AMPs achieved good in vitro therapeutic indices and high intrahepatic stability but low plasma stability, suggesting they could be developed as adjuvants for topical delivery or administration by inhalation for anti-infective therapy to reduce the necessary dose of colistin (and thus its side effects) or to prevent development of colistin resistance in MDR pathogens.

Neuropathic pain arises because of neuronal injury. Unlike inflammatory pain which can be managed by classical nonsteroid anti-inflammatory drugs (NSAIDs), neuropathic pain is difficult to treat. The classical NSAIDs work through inhibition of cyclooxygenase 2 (COX2) enzyme. However, COX2 inhibitors are insufficient to treat neuropathic pain. Hence, it becomes important to explore for novel molecules acting through cell surface molecules like ion channels, for the treatment of neuropathic pain. We investigated multiple bromobenzothiophene carboxamides for their efficacy against neuropathic pain. Interestingly, AS6 was found to be very effective in treating neuropathic pain through inhibition of Kv4.3 ion channel. AS6 also reduced the COX2 overexpression associated with neuropathic pain. These results, as well as results from our previous study, indicate that AS6 can be a potent antinociceptive agent against both inflammatory and neuropathic pain.

The QPatch™ is a powerful ion channel screening system, which was developed through extensive automation of the conventional patch clamp technique. Whole-cell current measurements take place on a disposable 16-channel QPlate™ in MTP format. Later, also a 48-channel QPlate will be available. Automation has been achieved by (i) employing planar silicon chips rather than traditional glass micropipettes, by (ii) elimination and/or simplification of a number of time-consuming tasks, e.g.visual cell selection and pipette tip localisation, manual microscope-aided pipette positioning and cell contact establishment, and subsequent gigaseal and whole-cell formation, and by (iii) cell preparation directly on theQPatch platform. We here report whole-cell currentdata from cultured cell lines expressing a number of voltage- or ligand-gated ion channels. The data were obtained on either complete 16-channel QPlates oron single chip assemblies (1-channel prototype).

The human sodium channel hNaV1.5, encoded by the SCN5A gene, is crucial for the cardiac action potential upstroke and subsequent signal propagation in the heart. Inhibition of the sodium current by drugs decreases the rate of cardiomyocyte depolarization and the conduction velocity which may lead to serious implications. For these reasons, off-target effects on hNaV1.5 are considered a risk marker for drug candidates and the hNaV1.5 is one of the most used in vitro assays in cardiac safety testing and is also one of the most important currents in the CiPA paradigm.

Several drugs, known to inhibit the hNaV1.5 channel, are either use- or state-dependent and preferentially bind to the open and inactivated state, respectively. When experimenting on voltage-gated ion channels, it is imperative that the voltage applied to the cells is accurate. This is especially important when testing state-dependent compounds. In order to determine compound activity in the most accurate way, all tested cells should have an identical degree of inactivation, a state that will not be achieved by using the same voltage to all experiment sites but rather individual voltages adjusted to the cell’s individual biophysical properties need to be applied.
The Qube, as well as the new QPatch II, are high-throughput automated patch clamp platforms, suitable for studying a wide range of ion channels. Both systems are equipped with the option to run online adaptive protocols, which makes it possible to set each individual cell to a user-defined level of inactivation, e.g. the half-inactivation potential (V½) and use that value subsequently in e.g. a preconditioning pulse. Applying this new adaptive protocol feature, we determined NaV1.5 IC50 values for both the open and the inactivated state for a set of known sodium channel inhibitors. We could show that the use of individual V½ reduces data variability compared to standard methods and thereby improves the accuracy of drug evaluation.

Planar chip technology has strongly facilitated the progress towards fully automated electrophysiological systems that, in contrast to the traditional patch clamp technology, have the capability of parallel compound testing. The throughput has been increased from testing below 10 compounds per day to a realized capacity approaching high throughput levels. Many pharmaceutical companies have implemented automated planar chip electrophysiology in their drug discovery process, particularly at the levels of lead optimization, secondary screening and safety testing, whereas primary screening is generally not performed. In this review, we briefly discuss the technology and give examples from selected NeuroSearch ion channel programs, where one of the systems, the QPatch, has been evaluated for use in lead optimization and primary screening campaigns, where high information content was a requirement.

The suitability of an automated patch clamp for the characterization and pharmacological screening of calcium release–activated calcium (CRAC) channels endogenously expressed in RBL-2H3 cells was explored with the QPatch system. CRAC currents (ICRAC) are small, and thus precise recordings require high signal-to-noise ratios obtained by high seal resistances. Automated whole-cell establishment resulted in membrane resistances of 1728 ± 226 MΩ (n = 44). CRAC channels were activated by a number of methods that raise intracellular calcium concentration, including EGTA, ionomycin, Ins(1,4,5)P3, and thapsigargin. ICRAC whole-cell currents ranged from 30 to 120 pA with rise times of 40 to 150 s. An initial delay in current activation was observed in particular when ICRAC was activated by passive store depletion using EGTA. Apparent rundown of ICRAC was commonly observed, and the current could be reactivated by subsequent addition of thapsigargin. ICRAC was blocked by SKF-96365 and 2-APB with IC₅₀ values of 4.7 ± 1.1 μM (n = 9) and 7.5 ± 0.7 (n = 9) μM, respectively. The potencies of these blockers were similar to values reported for ICRAC in similar conventional patch-clamp experiments. The study demonstrates that CRAC channels can be rapidly and efficiently targeted with automated patch-clamp techniques for characterization of physiological and pharmacological properties.

Whole-cell voltage clamp electrophysiology using glass patch pipettes (1) is regarded as the gold standard for measurement of compound activity on ion channels. Despite the high quality of the data generated by this method, in its traditional format, patch clamping has limited use in drug screening due to very low throughput. Over the years, developments in microfabrication have driven the development of planar, multi-aperture technologies that are suitable for parallel, automated patch recording techniques. Here we present detailed methods for two common applications of the planar patch technology using one of the commercially available instruments. The results demonstrate (a) the high quality of whole-cell recordings obtainable from cell lines expressing human Na_v1.2 or hERG ion channels, (b) the advantages of the methodology for increasing throughput, and (c) examples of how these assays support ion channel drug discovery.

The CFTR gene encodes a cyclic AMP-regulated chloride channel, a pivotal protein responsible for epithelial chloride transport. In the lungs, chloride transport is vital in maintaining the hydration status of the airways such that mucus is runny and fluid, which fulfils a key role in protecting the airways from dangerous insults such as bacteria and other noxious particles, so it can be moved up and out of the lungs.

In cardiac muscle, sustained inward Na+ currents, also known as late Na⁺ currents (I_Na-L), occur under physiological conditions during the cardiac Action Potential AP (AP). In addition to extending the plateau duration before AP recovery, an increased INa-L can lead to the development of various triggers and substrates (early after depolarization, EAD) for arrhythmogenesis. Inhibition of I_Na-L current can prevent proarrythmia by reducing or reversing EAD even in the presence of a prolonged QTc interval (e.g. Ranolazine).

Therefore, I_Na-L is one of the seven ion channels selected for
evaluation in the comprehensive in vitro proarrythmia assay (CiPA).

Eurofins is a contributor to the Ion-Channel Work-Group of the
FDA/HESI/CIPA initiative, supporting validation of automated high-throughput methods on the QPatch HT to support the program consortium. These methods measure 1) enhancement or 2) inhibition of I_Na-L current, using either step pulse or ramp pulse protocol and reference pharmacology, ATXII. Three key parameters, I_Na-L current, leak current, time-matched vehicle control were measured. The data generated in this study demonstrate that measuring I_Na-L current charge measurement is superior to measuring I_Na-L current amplitude for both test and ramp pulse. We investigated the effects of CiPA-28 compounds on I_Na-L at the EC₅₀ of ATXII (30nM). With the un-blinding of CiPA Phase I (12) compounds, Chlorpromazine, Diltiazem, and Mexiletine were identified as hits. Our results indicate that none of the CiPA-28 compounds potentiated the I_Na-L current.

Electrophysiological measurements, in particular, patch clamping, have long been regarded as the “gold standard” for assaying ion channels. Despite its high information content, the technique suffers from laborious, manual processing by highly skilled workers and extremely low throughput. Recently, a number of researchers have started to automate patch clamping by either automating conventional micropipette-based patch clamping or developing planar microelectrode arrays. This article reviews the brief history of these emerging technologies and discusses the technical details, advantages, and disadvantages of each approach and technique. As will be evident from the discussion, two types of automated patch-clamping technologies are emerging. The first places emphasis on data quality, comparable to conventional patch clamping, and the second has much higher throughput. Future developments will include sophisticated patch-clamping devices with both high-quality data and high throughput capabilities and further integration of patch clamping with other cell-based assays.

Human induced pluripotent stem cells can be differentiated into dopaminergic neurons (Dopa.4U). Dopa.4U neurons expressed voltage-gated Na_V and K_V channels and showed neuron-like spontaneous electrical activity. In automated patch clamp measurements with suspended Dopa.4U neurons, delayed rectifier K⁺ current (delayed K_V) and rapidly inactivating A-type K⁺ current (fast K_V) were identified. Examination of the fast K_V current with inhibitors yielded IC₅₀ values of 0.4 mM (4-aminopyridine) and 0.1 mM (tetraethylammonium). In manual patch clamp measurements with adherent Dopa.4U neurons, fast K_Vcurrent could not be detected, while the delayed K_V current showed an IC₅₀ of 2 mM for 4-aminopyridine. The Na_V channels in adherent and suspended Dopa.4U neurons showed IC₅₀ values for tetrodotoxin of 27 and 2.9 nM, respectively. GABA-induced currents that could be observed in adherent Dopa.4U neurons could not be detected in suspended cells. Application of current pulses induced action potentials in approx. 70 % of the cells. Our results proved the feasibility of automated electrophysiological characterization of neuronal cells.

Most ligand-gated ion channels (LGICs) are characterized by fast transient currents in response to application of agonists. Typically the time constants for activation and subsequent desensitization amount to1-100 and 10-1000 ms, respectively. Consequently, recording of proper whole-cell LGIC currents with the patch-clamp technique requires a fast solution exchange system. Furthermore, characterization of LGIC blockers and modulators generally requires complex compound application protocols, because the effect of a test compound needs to be evaluated simultaneously with a tran- sient application of the agonist. These require- ments challenge a realistic electrophysiological characterization of LGICs. We employed the auto- mated QPatch 16 patch-clamp system, to charac- terize the effects of agonists, antagonists and acti- vators on two types of fast LGICs: (1) GABAA (γ- amino-butyric acid A) receptors and (2) ASIC (acid sensing ion channels) types 1a and 3.

Shaker-related Kv1 channels contain four channel-forming α subunits. Subfamily member Kv1.1 often occurs oligomerized with Kv1.2 α subunits in synaptic membranes, and so information was sought on the influence of their positions within tetramers on the channels’ properties. Kv1.1 and 1.2 α genes were tandem linked in various arrangements, followed by expression as single-chain proteins in mammalian cells. As some concatenations reported previously seemed not to reliably position Kv1 subunits in their assemblies, the identity of expressed channels was methodically evaluated. Surface protein, isolated by biotinylation of intact transiently transfected HEK-293 cells, gave Kv1.1/1.2 reactivity on immunoblots with electrophoretic mobilities corresponding to full-length concatenated tetramers. There was no evidence of protein degradation, indicating that concatemers were delivered intact to the plasmalemma. Constructs with like genes adjacent (Kv1.1-1.1-1.2-1.2 or Kv1.2-1.2-1.1-1.1) yielded delayed-rectifying, voltage-dependent K+ currents with activation parameters and inactivation kinetics slightly different from the diagonally positioned genes (Kv1.1-1.2-1.1-1.2 or 1.2–1.1-1.2-1.1). Pore-blocking petidergic toxins, α dendrotoxin, agitoxin-1, tityustoxin-Kα, and kaliotoxin, were unable to distinguish between the adjacent and diagonal concatamers. Unprecedentedly, external application of the pore-blocker tetraethylammonium (TEA) differentially inhibited the adjacent versus diagonal subunit arrangements, with diagonal constructs having enhanced susceptibility. Concatenation did not directly alter the sensitivities of homomeric Kv1.1 or 1.2 channels to TEA or the toxins. TEA inhibition of currents generated by channels made up from dimers (Kv1.1-1.2 and/or Kv1.2-1.1) was similar to the adjacently arranged constructs. These collective findings indicate that assembly of α subunits can be directed by this optimized concatenation, and that subunit arrangement in heteromeric Kv channels affects TEA affinity.

ICH guidelines state that compounds in drug discovery must be tested for inhibition of hERG cardiac
ion channel. It is often prudent to test compounds against a wider array of cardiac ion channels, e.g.
hNa_V1.5 and hCa_V1.2 (Kramer et al., 2013). The CiPA initiative will demand testing and additional ion
channel targets as well; namely hNa_V1.5 late current, hKir2.1, hKvLQT1 and Kv4.3 (Gintant et al., 2016).
These ion channel assays are all amenable to automated patch-clamp and have typically been run
using recombinant cell lines over-expressing an individual ion channel. The aim of this research was to
investigate whether human induced pluripotent stem cell-derived cardiomyocytes are a useful,
affordable and predictive cellular reagent for use on the QPatch automated patch-clamp system.

We applied a set of in silico and in vitro assays, compliant with the Comprehensive In Vitro Proarrhythmia Assay (CiPA) paradigm, to assess the risk of chloroquine (CLQ) or hydroxychloroquine (OH‐CLQ)‐mediated QT prolongation and Torsades de Pointes (TdP), alone and combined with erythromycin (ERT) and azithromycin (AZI), drugs repurposed during the first wave of coronavirus disease 2019 (COVID‐19). Each drug or drug combination was tested in patch clamp assays on seven cardiac ion channels, in in silico models of human ventricular electrophysiology (Virtual Assay) using control (healthy) or high‐risk cell populations, and in human‐induced pluripotent stem cell (hiPSC)‐derived cardiomyocytes. In each assay, concentration‐response curves encompassing and exceeding therapeutic free plasma levels were generated. Both CLQ and OH‐CLQ showed blocking activity against some potassium, sodium, and calcium currents. CLQ and OH‐CLQ inhibited I_Kr (half‐maximal inhibitory concentration [IC₅₀]: 1 µM and 3–7 µM, respectively) and I_K1 currents (IC₅₀: 5 and 44 µM, respectively). When combining OH‐CLQ with AZI, no synergistic effects were observed. The two macrolides had no or very weak effects on the ion currents (IC₅₀ > 300–1000 µM). Using Virtual Assay, both antimalarials affected several TdP indicators, CLQ being more potent than OH‐CLQ. Effects were more pronounced in the high‐risk cell population. In hiPSC‐derived cardiomyocytes, all drugs showed early after‐depolarizations, except AZI. Combining CLQ or OH‐CLQ with a macrolide did not aggravate their effects. In conclusion, our integrated nonclinical CiPA dataset confirmed that, at therapeutic plasma concentrations relevant for malaria or off‐label use in COVID‐19, CLQ and OH‐CLQ use is associated with a proarrhythmia risk, which is higher in populations carrying predisposing factors but not worsened with macrolide combination.

The QPatch 16 screening station is a second generation automated patch-clamp system based on planar silicon chip technology. QPatch 16 has previously been employed for a series of screening studies on a number of voltage-gated ion channels (e.g. hERG, KCNQ4, Na_v1.2, Na_v1.4 and Na_v1.5). Recently, ligand-gated ion channels (LGIC) including GABAA, nAChR and ASIC have been targeted with QPatch 16. We here report a study in which the GABAA receptor (α1 β2 γ2) was targeted with 4-5 concentrations of an agonist (GABA), an antagonist (bicuculline) and a modulator (chlordizepoxide). Subsequently we report a study on acid sensitive ion channels (ASIC1a) in which the effect of pH, i.e. protons which serve as the ligand, was examined.

Loss-of-function mutations of Na_v1.7 lead to congenital insensitivity to pain, a rare condition resulting in individuals who are otherwise normal except for the inability to sense pain, making pharmacological inhibition of NaV1.7 a promising therapeutic strategy for the treatment of pain. We characterized a novel mouse model of Na_v1.7-mediated pain based on intraplantar injection of the scorpion toxin OD1, which is suitable for rapid in vivo profiling of Na_v1.7 inhibitors. Intraplantar injection of OD1 caused spontaneous pain behaviors, which were reversed by co-injection with Na_vV1.7 inhibitors and significantly reduced in Na_v1.7−/− mice. To validate the use of the model for profiling Na_v1.7 inhibitors, we determined the Na_v selectivity and tested the efficacy of the reported NaV1.7 inhibitors GpTx-1, PF-04856264 and CNV1014802 (raxatrigine). GpTx-1 selectively inhibited NaV1.7 and was effective when co-administered with OD1, but lacked efficacy when delivered systemically. PF-04856264 state-dependently and selectively inhibited Na_v1.7 and significantly reduced OD1-induced spontaneous pain when delivered locally and systemically. CNV1014802 state-dependently, but non-selectively, inhibited NaV channels and was only effective in the OD1 model when delivered systemically. Our novel model of Na_v1.7-mediated pain based on intraplantar injection of OD1 is thus suitable for the rapid in vivo characterization of the analgesic efficacy of Na_v1.7 inhibitors.

Rare respiratory diseases (RRDs) are a heterogeneous group of disorders that collectively represent a significant health care burden. In recent years, strong advocacy and policy initiatives have led to advances in the implementation of research and clinical care for rare diseases. The development of specialized centers and research networks has facilitated support for affected individuals as well as emerging programs in basic, translational, and clinical research. In selected RRDs, subsequent gains in knowledge have informed the development of targeted therapies and effective diagnostic tests, but many gaps persist. There was therefore a desire to identify the elements contributing to an effective translational research program in RRDs. To this end, a workshop was convened in October 2015 with a focus on the implementation of effective transnational research networks and collaborations aimed at developing novel diagnostic and therapeutic tools. Key elements included an emphasis on molecular pathogenesis, the continuing engagement of patient advocacy groups and policy makers, the effective use of preclinical models in the translational research pipeline, and the detailed phenotyping of patient cohorts. During the course of the workshop, current logistical and knowledge gaps were identified, and new solutions or opportunities were highlighted.

A growing subset of β-secretase (BACE1) inhibitors for the treatment of Alzheimer’s disease (AD) utilizes an anilide chemotype that engages a key residue (Gly230) in the BACE1 binding site. Although the anilide moiety affords excellent potency, it simultaneously introduces a third hydrogen bond donor that limits brain availability and provides a potential metabolic site leading to the formation of an aniline, a structural motif of prospective safety concern. We report herein an alternative aminomethyl linker that delivers similar potency and improved brain penetration relative to the amide moiety. Optimization of this series identified analogues with an excellent balance of ADME properties and potency; however, potential drug–drug interactions (DDI) were predicted based on CYP 2D6 affinities. Generation and analysis of key BACE1 and CYP 2D6 crystal structures identified strategies to obviate the DDI liability, leading to compound 16, which exhibits robust in vivo efficacy as a BACE1 inhibitor.

Voltage-gated sodium (Na_V) channels are studied extensively due to their potential as targets for several indications, such as pain, epilepsy, cardiac and muscle paralysis. Some Na_V channel modulators show state-dependence and bind preferentially to the inactivated state of the channel. The potency of state-dependent compounds is known to vary depending on the percentage inactivation of the channels.

To calculate accurate compound activity the precise value for the VHalf of inactivation should be used for each cell. The adaptive protocol block for the Sophion Qube 384-well automated patch clamp platform has made it possible to separately define the voltage applied to individual wells for both the activation and inactivation of the channels. This enables the generation of more precise data for voltage-gated ion channels.

Revising the ICH regulatory guideline, S7B, for the Non-clinical Evaluation of the Potential for Delayed Ventricular repolarization (QT Interval Prolongation by Human Pharmaceuticals) has been suggested and Comprehensive In Vitro Proarrhythmia Assay (CiPA) activities are being implemented. We previously studied the applicability of the CiPA-recommended voltage protocols for hERG, Cav1.2, and late Nav1.5 current patch clamping which was announced last year (this research was presented at the 2019 Japanese Safety Pharmacology Society annual meeting). As a result, inter-facility differences were found for some compounds in the hERG assay on an automated patch-clamp system, QPatch. Such differences were also noted in enhancement ability of ATX-II for late Nav1.5 current. This time, we investigated the factors for the interfacility differences and studied more appropriate protocols. In addition, we present the results of the assays by the CiPA-recommended protocols for each ion channel. Manual patch clamp and QPatch system were selected as the platforms. For the voltage protocols, the CiPA-recommended ones were followed whereas the composition of intra- and extracellular buffers were modified. Preparation procedures and application methods of test solutions (such as application duration or number of replicates) for the QPatch assay were examined. Test compounds were selected from each of the low-, intermediate-, and high-risk categories of the “training drugs” in the CiPA in silico model. We present the comparison results of our data by the CiPA-recommended protocols and the data given by the CiPA. We also discuss buffer compositions, preparation procedures or application methods for test solutions to reduce the inter-facility differences. We would like to show and discuss the more appropriate protocols including the detailed experimental methods, which are not given in the CiPA-recommended protocols, and points to be modified.

Chronic pain is a serious debilitating condition that affects ∼20% of the world’s population. Currently available drugs fail to produce effective pain relief in many patients and have dose-limiting side effects. Several voltage-gated sodium (Na_V) and calcium (Ca_V) channels are implicated in the etiology of chronic pain, particularly Na_V1.1, Na_V1.3, Na_V1.7–Na_V1.9, Ca_V2.2, and Ca_V3.2. Numerous Na_V and Ca_V modulators have been described, but with few exceptions, they display poor potency and/or selectivity for pain-related channel subtypes. Here, we report the discovery and characterization of 2 novel tarantula-venom peptides (Tap1a and Tap2a) isolated from Theraphosa apophysis venom that modulate the activity of both Na_V and Ca_V3 channels. Tap1a and Tap2a inhibited on-target Na_V and Ca_V3 channels at nanomolar to micromolar concentrations and displayed moderate off-target selectivity for Na_V1.6 and weak affinity for Na_V1.4 and Na_V1.5. The most potent inhibitor, Tap1a, nearly ablated neuronal mechanosensitivity in afferent fibers innervating the colon and the bladder, with in vivo intracolonic administration reversing colonic mechanical hypersensitivity in a mouse model of irritable bowel syndrome. These findings suggest that targeting a specific combination of Na_V and Ca_V3 subtypes provides a novel route for treatment of chronic visceral pain.

Na_V1.1 (SCN1A) channels primarily located in gamma-aminobutyric acid (GABA)ergic fast-spiking interneurons are pivotal for action potential generation and propagation in these neurons. Inappropriate function of fast-spiking interneurons, leading to disinhibition of pyramidal cells and network desynchronization, correlates with decreased cognitive capability. Further, reduced functionality of Na_V1.1 channels is linked to various diseases in the central nervous system. There is, at present, however no subtype selective pharmacological activators of Na_V1.1 channels available for studying pharmacological modulation of interneuron function. In the current study we identified a small molecule Na_V1.1 activator, 3-Amino-5-(4-methoxyphenyl)thiophene-2-carboxamide, named AA43279, and provided an in vitro to in vivo characterization of the compound. In HEK-293 cells expressing human Na_V1.1 channels, AA43279 increased the Na_V1.1-mediated current in a concentration-dependent manner mainly by impairing the fast inactivation kinetics of the channels. In rat hippocampal brain slices, AA43279 increased the firing activity of parvalbumin-expressing, fast-spiking GABAergic interneurons and increased the spontaneous inhibitory post-synaptic currents (sIPSCs) recorded from pyramidal neurons. When tested in vivo, AA43279 had anti-convulsive properties in the maximal electroshock seizure threshold test. AA43279 was tested for off-target effects on 72 different proteins, including Na_V1.2, Na_V1.4, Na_V1.5, Na_V1.6 and Na_V1.7, and exhibited reasonable selectivity. Taken together, AA43279 might constitute a valuable tool compound for revealing biological functions of Na_V1.1 channels.

K⁺ channels containing Kv1.1 α subunits, which become prevalent at internodes in demyelinated axons, may underlie their dysfunctional conduction akin to muscle weakness in multiple sclerosis. Small inhibitors were sought with selectivity for the culpable hyper-polarising K⁺currents. Modelling of interactions with the extracellular pore in a Kv1.1 deduced-structure identified a diaryldi-(2-pyrrolyl)methane (DPM) as a suitable scaffold, with optimised alkyl ammonium side chains. The resultant synthesised candidate [2,2’-((5,5’(di-p-topyldiaryldi-(2-pyrrolyl)methane)bis(2,2’carbonyl)bis (azanediyl)) diethaneamine.2HCl] (8) selectively blocked Kv1.1 channels (IC₅₀ ~15 µM) recombinantly expressed in mammalian cells, induced a positive shift in the voltage-dependency of K⁺ current activation and slowed its kinetics. It inhibited preferentially channels containing 2 or more Kv1.1 subunits, regardless of their positioning in concatenated tetramers. In slices of corpus callosum from mice subjected to a demyelination protocol, this novel inhibitor improved neuronal conduction, highlighting its potential for alleviating symptoms in multiple sclerosis.

Kv4.3 α-subunits associate with ancillary β-subunits of the KChIP2 family in heart muscular tissue to channel transient outward (Ito) currents. Decreased expression or dysfunction of Kv4.3 channels following myocardial infarction or during heart failure can contribute to abnormal repolarization, which may result in ventricular arrhythmias. Therefore, drug-induced inhibition of Kv4.3/KChIP2-mediated Ito exposes to potential cardio safety liabilities which are important to document early during the drug discovery process. In this study, we have characterized basic electrophysiological properties pertaining to Ito currents in CHO cells expressing recombinant Kv4.3 and KChIP2.2 subunits. The currents obtained were validated pharmacologically by assessing the efficacy of small molecule and peptide toxin inhibitors known from manual patch-clamp studies to block Ito. We then examined the inhibitory activities of 28 drugs with clinically documented high, medium or low pro-arrhythmic risk encompassing the test- and validation-sets of the Comprehensive in vitro Pro-Arrhythmia (CiPA) panel. While none of the CiPA drugs inhibited Kv4.3 at submicromolar concentrations (IC50 < 1 µM), two drugs (loratadine and nitrendipine) displayed intermediate potency (1 µM < IC50 < 10 µM), and four drugs (bepridil, quinidine, chlorpromazine and astemizole) revealed weak, but significant (10 µM < IC50 < 30 µM) inhibitors.

Recent understanding of the systems that mediate complex disease states, has generated a search for molecules that simultaneously modulate more than one component of a pathologic pathway. Chronic pain syndromes are etiologically connected to functional changes (sensitization) in both peripheral sensory neurons and in the central nervous system (CNS). These functional changes involve modifications of a significant number of components of signal generating, signal transducing and signal propagating pathways. Our analysis of disease-related changes which take place in sensory neurons during sensitization led to the design of a molecule that would simultaneously inhibit peripheral NMDA receptors and voltage sensitive sodium channels. In the current report, we detail the selectivity of N,N-(diphenyl)-4-ureido-5,7-dichloro-2-carboxy-quinoline (DCUKA) for action at NMDA receptors composed of different subunit combinations and voltage sensitive sodium channels having different α subunits. We show that DCUKA is restricted to the periphery after oral administration, and that circulating blood levels are compatible with its necessary concentrations for effects at the peripheral cognate receptors/channels that were assayed in vitro. Our results demonstrate that DCUKA, at concentrations circulating in the blood after oral administration, can modulate systems which are upregulated during peripheral sensitization, and are important for generating and conducting pain information to the CNS. Furthermore, we demonstrate that DCUKA ameliorates the hyperalgesia of chronic pain without affecting normal pain responses in neuropathic and inflammation-induced chronic pain models.

Kv3.1 and Kv3.2 high voltage-activated potassium channels, which display fast activation and deactivation kinetics, are known to make a crucial contribution to the fast-spiking phenotype of certain neurons. Pharmacological experiments
show that the blockade of native Kv3 currents with low concentrations of etraethylammonium or 4-aminopyridine impairs
the expression of this firing phenotype. In particular, Kv3 channels are highly expressed by fast-spiking, arvalbuminpositive interneurons in corticolimbic brain circuits, which modulate the synchronization of cortical circuits and the generation of brain rhythms. Here, we describe a novel small molecule, (5R)-5-ethyl-3-(6-{[4-methyl-3-(methyloxy)phenyl]oxy}-3-pyridinyl)- 2,4-imidazolidinedione (AUT1), which modulates Kv3.1 and Kv3.2 channels in human recombinant and rodent native neurons. AUT1 increased whole currents mediated by human Kv3.1b and Kv3.2a channels, with a concomitant leftward shift in the voltage dependence of activation. A less potent effect was observed on hKv3.3 currents. In mouse somatosensory cortex slices in vitro, AUT1 rescued the fast-spiking phenotype of parvalbumin-positive–fast-spiking interneurons following an impairment of their firing capacity by blocking a proportion of Kv3 channels with a low concentration of tetraethylammonium. Notably, AUT1 had no effect on interneuron firing when applied alone. Together, these data confirm the role played by Kv3 channels in the regulation of the firing phenotype of somatosensory
interneurons and suggest that AUT1 and other Kv3 modulators could represent a new and promising therapeutic
approach to the treatment of disorders associated with dysfunction of inhibitory feedback in corticolimbic circuits, such as
schizophrenia.

Abstract

Ligand gated ion channels are involved in many pathophysiological processes and represent a relevant, although challenging, target for drug discovery. We propose an innovative electro-optical approach to their analysis able to derive membrane conductance values from the local membrane potential changes imposed by test current pulses and measured by fast voltage-sensitive fluorescent dyes. We exploited the potential of this proprietary method by developing a drug testing system called “ionChannel Optical High-content Microscope” (ionChannelΩ). This automated platform was validated by testing the responses of reference drugs on cells expressing different ligand-gated ion channels. Furthermore, a double-blind comparison with FLIPR and automated patch-clamp was performed on molecules designed to act as antagonists of the P2RX7 receptor. ionChannelΩ proved highly reliable in all tests, resulting faster and more cost-effective than electrophysiological techniques. Overall, ionChannelΩ is amenable to the study of ligand gated ion channels that are receiving less attention due to limitations in current assays.

There is currently a strong interest in using high-throughput in-vitro ion-channel screening data to make predictions regarding the cardiac toxicity potential of a new compound in both animal and human studies. A recent FDA think tank encourages the use of biophysical mathematical models of cardiac myocytes for this prediction task. However, it remains unclear whether this approach is the most appropriate. Here we examine five literature data-sets that have been used to support the use of four different biophysical models and one statistical model for predicting cardiac toxicity in numerous species using various endpoints. We propose a simple model that represents the balance between repolarisation and depolarisation forces and compare the predictive power of the model against the original results (leave-one-out cross-validation). Our model showed equivalent performance when compared to the four biophysical models and one statistical model. We therefore conclude that this approach should be further investigated in the context of early cardiac safety screening when in-vitro potency data is generated.

A single pot dipolar cycloaddition reaction / Cope elimination sequence was developed to access novel 1,4,6,7-tetrahydro-5H-[1,2,3]triazolo[4,5-c]pyridine P2X7 antagonists that contain a synthetically challenging chiral center. The structure-activity relationships of the new compounds are described. Two of these compounds, (S)-(2-fluoro-3-(trifluoromethyl)phenyl)(1-(5-fluoropyrimidin-2-yl)-6-methyl-1,4,6,7-tetrahydro-5H-[1,2,3]triazolo[4,5-c]pyridin-5-yl)methanone (compound 14) and (S)-(3-fluoro-2-(trifluoromethyl)pyridin-4-yl)(1-(5-fluoropyrimidin-2-yl)-6-methyl-1,4,6,7-tetrahydro-5H-[1,2,3]triazolo[4,5-c]pyridin-5-yl)methanone (compound 20), were found to have robust P2X7 receptor occupancy at low doses in rat with ED50 values of 0.06 and 0.07 mg/kg, respectively. Compound 20 had notable solubility compared to 14 and showed good tolerability in preclinical species. Compound 20 was chosen as a clinical candidate for advancement into Phase I clinical trials to assess safety and tolerability in healthy human subjects prior to the initiation of proof of concept studies for the treatment of mood disorders.

Ants (Hymenoptera: Formicidae) are diverse and ubiquitous, and their ability to sting is familiar to many of us. However, their venoms remain largely unstudied. We provide the first comprehensive characterization of a polypeptidic ant venom, that of the giant red bull ant, Myrmecia gulosa. We reveal a suite of novel peptides with a range of posttranslational modifications, including disulfide bond formation, dimerization, and glycosylation. One venom peptide has sequence features consistent with an epidermal growth factor fold, while the remaining peptides have features suggestive of a capacity to form amphipathic helices. We show that these peptides are derived from what appears to be a single, pharmacologically diverse, gene superfamily (aculeatoxins) that includes most venom peptides previously reported from the aculeate Hymenoptera. Two aculeatoxins purified from the venom were found to be capable of activating mammalian sensory neurons, consistent with the capacity to produce pain but via distinct mechanisms of action. Further investigation of the major venom peptide MIITX1-Mg1a revealed that it can also incapacitate arthropods, indicative of dual utility in both defense and predation. MIITX1-Mg1a accomplishes these functions by generating a leak in membrane ion conductance, which alters membrane potential and triggers neuronal depolarization. Our results provide the first insights into the evolution of the major toxin gene superfamily of the aculeate Hymenoptera and provide a new paradigm in the functional evolution of toxins from animal venoms.

The cardiac ion channel encoded by KCNH2, also known as the human ether-à-go-go-related gene (hERG) or KV11.1 can lead to cardiac arrhythmia when blocked and is thus a major off-target in drug discovery. Mutations in this channel can lead to congenital long QT syndrome (LQTS), associated with an irregular heartbeat, fainting and sudden cardiac arrest.
Automated patch clamp (APC) allows assessing the effects of compounds on the hERG channel in the preclinical development process, enabling an early evaluation in drug development. Unattended runs – as we show here on the Qube – are a crucial advantage by saving time and resources. We here report the results of an overnight run with CHO-hERG-Duo (B’SYS) cells at 25˚C, made possible by the Qube supplemented with stacker function and temperature control.

A novel series of 1,2,4-triazolyl 5-azaspiro[2.4]heptanes with high affinity and selectivity at the dopamine (DA) D3 receptor (D3R) is described. Some of these compounds also have high selectivity over the hERG channel and were characterized with respect to their pharmacokinetic properties both in vitro and in vivo during lead identification and early lead optimization phases. A few derivatives with overall favorable developability characteristics were selected for further late lead optimization studies.

Sodium ion channels are some of the fastest ion channels. With a high current and fast activation, it is very difficult for conventional amplifiers to correctly clamp these signals. The main challenge in providing a perfect clamp is the error introduced by the series resistance (Rs), which will introduce a voltage drop as the current increases rapidly (Sigworth 1983). This leads to delays in the signal and incorrect estimation of the current magnitude. In order to correctly compensate Rs fast enough, one will have to compromise on another cell parameter; cell capacitance (Cs) (Sherman 1999). In the QPatch system, it is possible to conduct experiments without direct Cs compensation, but still measure Cs for later use.

When compensating completely for Rs, oscillations may occur and this is mainly due to incorrect cell parameter estimations or that the cell simply changes slightly. Oscillations can often lead to reduction in the seal resistance and terminate the whole-cell configuration. In the QPatch system, critical current oscillations are detected and the Rs feedback loop is disconnected for the remaining of the sweep until new parameter estimations can be made.