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

Anatoly Shcherbatko,Andrea Rossi,Davide Foletti,Guoyun Zhu,Oren Bogin,Meritxell Galindo Casas,Mathias Rickert,Adela Hasa-Moreno,Victor Bartsevich,Andreas Crameri,Alexander R Steiner,Robert Henningsen,Avinash Gill,Jaume Pons,David L Shelton,Arvind Rajpal,Pavel Strop

doi:10.1074/jbc.m116.725978

Abstract

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 subtype-selective 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, because they naturally evolved to be potent ion channel blockers. Here, we report the engineering of highly potent and selective inhibitors of the 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 the Nav1.7 channel. The resulting microproteins are highly potent (IC50 to Nav1.7 of 2.5 nm) and selective. We achieved 80- 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 3000 nm The lead molecules have the potential for future clinical development as novel therapeutics in the treatment of pain.

Highlights

Ion channels are an important class of targets for drugs with critical clinical need
Overview of the Microprotein Engineering Process—To engineer microproteins with high potency and selectivity toward Nav1.7, we utilized a combination of directed evolution [25], saturation mutagenesis [34], and chemical modification
To explore the sequence space not sampled by the directed evolution, we performed an exhaustive saturation mutagenesis on the 2670 variant

Summary

Experimental Procedures

Design of Phylogenetic Libraries and Production of Microproteins by Recombinant Expression—A total of 13 original libraries were designed following phylogenetic approach, with low to high diversities (2– 4 mutations per clone) and analyzed before expression. The extracellular recording solution contained (in mM) 137 NaCl, 4 KCl, 10 HEPES, 1.8 CaCl2, 1 MgCl2, 10 glucose, 10 sucrose, adjusted to pH 7.4. Results are presented as means Ϯ S.E. Manual Patch Clamp Electrophysiology—Coverslips carrying cells were placed in a recording chamber and were constantly perfused at a rate of 0.5 ml/min with extracellular solution containing (in mM) 140 NaCl, 5 KCl, 2 CaCl2, 2 MgCl2, 10 HEPES, and 10 glucose (pH 7.4, 320 mosmol). For an automated patch clamp recording on the QPatch HT system, the cells were dissociated with enzymatic detachment solution Detachin (Genlantis, San Diego) and resuspended in serum-free medium supplemented with 25 mM HEPES and 1% penicillin/streptomycin (Mediatech, Manassas, VA). HEK293 cells stably expressing human Nav1.7 were grown in DMEM (catalog no. 10-01-CM, Mediatech, Manassas, VA), 10% FBS (JRScientific, Woodland, CA), 500 ␮g/ml G418 sulfate (Mediatech)

Results

20 Ϯ 2 nM

Discussion