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 Theraphosa apophysis 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 NaV-targeting peptides with greater inhibitory potency and enhanced in vivo activity, we tested the hypothesis that incorporating residues identified from other optimized NaSpTx1 peptides into Tap1a could also optimize its potency for NaVs. Applying this approach, we designed the peptides Tap1a-OPT1 and Tap1a-OPT2 exhibiting significant increased potency for NaV1.1, NaV1.2, NaV1.3, NaV1.6 and NaV1.7 involved in several neurological disorders including acute and chronic pain, motor neuron disease and epilepsy. Tap1a-OPT1 showed increased potency for the off-target NaV1.4, while this off-target activity was absent in Tap1a-OPT2. This enhanced potency arose through a slowed off-rate mechanism. Optimized inhibition of NaV channels observed in vitro translated in vivo, with reversal of nocifensive behaviours in a murine model of NaV-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 NaV 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 in vivo. This work contributes to the rapid engineering and optimization of potent spider peptides multi-targeting NaV channels, and the research into novel drugs to treat neurological diseases.
Highlights
Animal venoms are an exquisite source of bio-active peptides that modulate human neurophysiology
Chronic visceral pain is a highly prevalent problem associated to irritable bowel syndrome (IBS) that affects 11% of the global population (Chey et al, 2015; Enck et al, 2016), and in which NaV channel subtypes were shown to participate via signalling visceral pain in IBS (Feng et al, 2015; Osteen et al, 2016; Erickson et al, 2018; Grundy et al, 2018; Salvatierra et al, 2018)
Spider peptides belonging to NaSpTx1 are known to bind to voltage-sensor domain II (VSDII) of NaV channels and induce gating-modifying effects that inhibit channel activation (Xiao et al, 2011; Cai et al, 2015)
Summary
Animal venoms are an exquisite source of bio-active peptides that modulate human neurophysiology. Alterations in NaV channel function contribute to a range of neurological disorders including chronic pain, epilepsy, and motor neuron disease (Kubat Oktem et al, 2016; Catterall, 2017; Cardoso and Lewis, 2018; Saba et al, 2019; Cardoso, 2020). These alterations are presented as remodelling of the expression and excitability of the subtypes NaV1.1, NaV1.3, NaV1.6, NaV1.7, NaV1.8 and NaV1.9 (Cardoso and Lewis, 2018; Cardoso, 2020). The peptides ProTx-II, HwTx-IV and Tap1a showed analgesic effects in painful diabetic neuropathy, spared nerve injury-induced neuropathy and chronic visceral pain (Liu et al, 2014; Tanaka et al, 2015; Flinspach et al, 2017; Cardoso et al, 2021), respectively, and the peptide Hm1a reduced seizures in Dravet syndrome (Richards et al, 2018)
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