Engineering Highly Potent and Selective Microproteins Against Nav1.7 Sodium Channel for Treatment of Pain

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.