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

Author(s): Anatoly Shcherbatko1, Andrea Rossi1, Davide Foletti2, Guoyun Zhu1, Oren Bogin3, Meritxell Galindo-Casas1, Mathias Rickert4, Adela Hasa-Moreno1, Victor Bartevitch5, Andreas Crameri6, Alexander R. Steiner7, Robert Henningsen7, Avinash Gill8, Jaume Pons1, David L. Shelton1, Arvind Rajpal9 and Pavel Strop1*

The prominent role of voltage-gated sodium channel 1.7 (Nav1.7) 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 Nav1.7 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 Nav1.7 channel. The resulting microproteins are highly potent (IC50 to Nav1.7 of 2.5 nM) and selective. We achieved 80-fold and 20-fold selectivity over the closely related Nav1.2 and Nav1.6 channels, respectively, and the IC50 on skeletal (Nav1.4) and cardiac (Nav1.5) sodium channels is above 3000nM. The lead molecules have the potential for future clinical development as novel therapeutics in the treatment of pain.