Engineering of a Spider Peptide via Conserved Structure-Function Traits Optimizes Sodium Channel Inhibition In Vitro and Anti-Nociception In Vivo

Abstract

Venom peptides are potent and selective modulators of voltage-gated ion channels that regulate neuronal function both in health and in disease. We previously identified the spider venom peptide Tap1a from the Venezuelan tarantula <i>Theraphosa apophysis</i> that targeted multiple voltage-gated sodium and calcium channels in visceral pain pathways and inhibited visceral mechano-sensing neurons contributing to irritable bowel syndrome. In this work, alanine scanning and domain activity analysis revealed Tap1a inhibited sodium channels by binding with nanomolar affinity to the voltage-sensor domain II utilising conserved structure-function features characteristic of spider peptides belonging to family NaSpTx1. In order to speed up the development of optimized Na_V-targeting peptides with greater inhibitory potency and enhanced <i>in vivo</i> activity, we tested the hypothesis that incorporating residues identified from other optimized NaSpTx1 peptides into Tap1a could also optimize its potency for Na_Vs. Applying this approach, we designed the peptides Tap1a-OPT1 and Tap1a-OPT2 exhibiting significant increased potency for Na_V1.1, Na_V1.2, Na_V1.3, Na_V1.6 and Na_V1.7 involved in several neurological disorders including acute and chronic pain, motor neuron disease and epilepsy. Tap1a-OPT1 showed increased potency for the off-target Na_V1.4, while this off-target activity was absent in Tap1a-OPT2. This enhanced potency arose through a slowed off-rate mechanism. Optimized inhibition of Na_V channels observed <i>in vitro</i> translated <i>in vivo</i>, with reversal of nocifensive behaviours in a murine model of Na_V-mediated pain also enhanced by Tap1a-OPT. Molecular docking studies suggested that improved interactions within loops 3 and 4, and C-terminal of Tap1a-OPT and the Na_V channel voltage-sensor domain II were the main drivers of potency optimization. Overall, the rationally designed peptide Tap1a-OPT displayed new and refined structure-function features which are likely the major contributors to its enhanced bioactive properties observed <i>in vivo</i>. This work contributes to the rapid engineering and optimization of potent spider peptides multi-targeting Na_V channels, and the research into novel drugs to treat neurological diseases.