High-throughput screening of animal venoms for identification of compounds active on the cardiac Nav1.5 channel

Abstract

Dysfunctions of the cardiac sodium channels (Nav1.5) have been associated with many cardiac diseases such as long QT syndrome or Brugada syndrome. In order to study the role of this channel in diseases or to restore function, molecules that specifically target this sodium channel are needed. Highly specific molecules for a given isoform of sodium channel are hard to discover with usual chemical libraries. Animal venoms, and especially spider venoms, contain each tens of peptides acting on ion channels and represent therefore interesting starting compound libraries for drug discovery. By screening spider venoms on Nav, we aimed to identify new toxins targeting specifically Nav1.5 channel which can be used as: – tools to study role of Nav1.5; – as pharmacological drug to prevent Nav1.5-related arrhythmias. For that purpose, 16 venoms isolated from spiders were first fractionated to generate libraries. Each fraction has been tested on several stable Nav cell lines using Syncropatch384PE system (Nanion) to ensure selectivity. False-positive fractions were excluded based on detection of material in HPLC and mass spectrometry analyses. We identified 10 native fractions active on Nav1.5. Among them, 5 inhibit sodium currents and 5 slow-down inactivation with increase in late sodium current. Theses fractions have been re-fractionated with a complementary purification approach until identification of isolated peptides. Validation of effects of these peptides on properties of Nav1.5 currents is ongoing. Sequences of positive hits are currently under study and chemical syntheses will be performed to get synthetic versions of these peptides. Complete pharmacological characterization of synthetic peptides is in progress. We developed a complete pipeline using automated patch-clamp that allows us to isolate and validate new toxins specific for human Nav1.5 sodium channel from large libraries of spider venoms.