Disulfide-rich animal venom peptides targeting either the voltage-sensing domain or the pore domain of voltage-gated sodium channel 1.7 (Na V 1.7) have been widely studied as drug leads and pharmacological probes for the treatment of chronic pain. However, despite intensive research efforts, the full potential of Na V 1.7 as a therapeutic target is yet to be realized. In this study, using evolved sortase A, we enzymatically ligated two known Na V 1.7 inhibitors−PaurTx3, a spider-derived peptide toxin that modifies the gating mechanism of the channel through interaction with the voltage-sensing domain, and KIIIA, a small cone snail-derived peptide inhibitor of the pore domain−with the aim of creating a bivalent inhibitor which could interact simultaneously with two noncompeting binding sites. Using electrophysiology, we determined the activity at Na V 1.7, and to maximize potency, we systematically evaluated the optimal linker length, which was nine amino acids. Our optimized synthetic bivalent peptide showed improved channel affinity and potency at Na V 1.7 compared to either PaurTx3 or KIIIA individually. This work shows that novel and improved Na V 1.7 inhibitors can be designed by combining a pore blocker toxin and a gating modifier toxin to confer desired pharmacological properties from both the voltage sensing domain and the pore domain.

Voltage-gated ion channels (VGICs) are specialised ion channels that have a voltage dependent mode of action, where ion conduction, or gating, is controlled by a voltage-sensing mechanism. VGICs are critical for electrical signalling and are therefore important pharmacological targets. Among these, voltage-gated sodium channels (Nas) have attracted particular attention as potential analgesic targets. Nas, however, comprise several structurally similar subtypes with unique localisations and distinct functions, ranging from amplification of action potentials in nociception (e.g. Na1.7) to controlling electrical signalling in cardiac function (Na1.5). Understanding the structural basis of Na function is therefore of great significance, both to our knowledge of electrical signalling and in development of subtype and state selective drugs. An important tool in this pursuit has been the use of peptides from animal venoms as selective Na modulators. In this review, we look at peptides, particularly from spider venoms, that inhibit Nas by binding to the voltage sensing domain (VSD) of this channel, known as gating modifier toxins (GMT). In the first part of the review, we look at the structural determinants of voltage sensing in VGICs, the gating cycle and the conformational changes that accompany VSD movement. Next, the modulation of the analgesic target Na1.7 by GMTs is reviewed to develop bioinformatic tools that, based on sequence information alone, can identify toxins that are likely to inhibit this channel. The same approach is also used to define VSD sequences, other than that from Na1.7, which are likely to be sensitive to this class of toxins. The final section of the review focuses on the important role of the cellular membrane in channel modulation and also how the lipid composition affects measurements of peptide-channel interactions both in binding kinetics measurements in solution and in cell-based functional assays.

The lipid composition of the cellular membrane plays an important role in a number of biological processes including the binding of membrane-active peptides. Characterization of membrane binding remains challenging, due to the technical limitations associated with the use of standard biophysical techniques and available membrane models. Here, we investigate the lipid binding properties of two membrane-active peptides, VSTx1, a well characterized ion-channel inhibitor, identified from spider venom, that preferentially binds to anionic lipid mixtures, and AA139 an antimicrobial β-hairpin peptide with uncharacterised lipid binding properties, currently in pre-clinical development. The lipid binding properties of these peptides are elucidated using nanodiscs formed by both linear and circularized (sortase-mediated) forms of a membrane scaffold protein (MSP1D1ΔH5). We find that nanodiscs formed by circularized MSPs—in contrast to those formed by linear MSPs—are sufficiently stable under sample conditions typically used for biophysical measurements (including lipid composition, a range of buffers, temperatures and concentrations). Using these circularized nanodiscs, we are able to extract detailed thermodynamic data using isothermal titration calorimetry (ITC) as well as atomic resolution mapping of the lipid binding interfaces of our isotope labeled peptides using solution-state, heteronuclear, nuclear magnetic resonance (NMR) spectroscopy. This represents a novel and general approach for elucidating the thermodynamics and molecular interface of membrane-active peptides toward flat lipid bilayers of variable composition. Our approach is validated by first determining the thermodynamic parameters and binding interface of VSTx1 toward the lipid bilayer, which shows good agreement with previous studies using lipid micelles and liposomes. The method is then applied to AA139, where the membrane binding properties are unknown. This characterization, involved solving the high-resolution structure of AA139 in solution using NMR spectroscopy and the development of a suitable expression system for isotope labeling. AA139 was found to bind exclusively to anionic membranes with moderate affinity ( K d ~low μM), and was found to have a lipid binding interface involving the termini of the β-hairpin structure. The preference of AA139 for anionic lipids supports a role for membrane binding in the mode-of-action of this peptide, which is also consistent with its higher inhibitory activity against bacterial cells compared to mammalian cells. The described approach is a powerful method for investigation of the membrane binding properties of this important class of molecules.