Developing novel analgesics to treat chronic pain: inhibiting voltage gated sodium channel 1.7 with spider-venom peptides

Abstract

Chronic pain is responsible for great physical, mental and economic loss. While there are diverse methods available for managing pain, such as NSAID, opioids and local anesthetics, common issues associated such as addiction and tolerance highlights the unmet demand for a novel analgesic aimed at a novel target.Numerous reports on individuals suffering from loss of function mutations in SCN9A marker, which leads to complete inability to feel pain, yet otherwise normal physiology, has led to the recognition of voltage gated sodium channel 1.7 (Na V 1.7) as a prime pain target.Other reports of gain of function mutations that lead to conditions such as paroxysmal extreme pain disorder and primary erythromelalgia confirms the validity of this target.However the presence of nine closely related subtypes of voltage gated sodium channels, Na V 1.1-Na V 1.9 introduces complications as any cross inhibition may lead to detrimental results. For example inhibition of Na V 1.5 subtype could result in cardiac arrhythmia to complete cardiac arrest and death. Therefor it is of utmost importance that any pharmaceutical agent used to inhibit Na V 1.7 is highly specific for Na V 1.7 and display no cross reactivity on other voltage gated channels.Spiders are one of the most successful terrestrial predators; their venoms have evolved over many millions of years to selectively and potently inhibit nervous system targets in order to rapidly paralyze their prey. Spider-venom peptidomes have been identified as one of the largest known libraries of compounds with very high potency and specificity towards nervous system targets.Spider toxins listed in the Arachnoserver database have been classified into 12 families of sodium channel toxins (NaSpTx), denoted NaSpTx Families 1-12, based on the level of sequence conservation and intercystine spacing. NaSpTx family 2 is the largest toxin family; it comprises 34 peptides of theraphosid origin.This thesis primarily aims at discovering novel inhibitors of Na V 1.7. However the intriguing fact that people suffering form congenital indifference to pain also suffers form anosmia, has lead to efforts of understanding the role of Na V 1.7 in olfaction. This study has been iii completed with the finding that Na V 1.7 is located along axons of olfactory neurones. These findings have been published and research article is attached as chapter 4.During the initial discovery phase of this project, an assay guided fractionation method was used to identify Na V 1.7 inhibitors form six spider venoms. This resulted in nine fully sequenced peptides where four of these belonged to NaSpTx family 2. This included the well-known β/ω-TRTX-Tp1a (Protoxin1). Three novel peptides discovered were named β-TRTX-Pe1a, µ-TRTX-Pe1b, and U-TRTX-Pa1a.A complete alanine scan of β/ω-TRTX-Tp1a on Na V 1.7 was conducted using a Xenopus laevis oocyte based tethered toxin method. Residues that were important for Na V 1.7 binding were mapped onto the three-dimensional structure of β/ω-TRTX-Tp1a. Three novel toxin peptides β-TR...