Gating modifier toxins (GMTs) are venom-derived peptides isolated from spiders and other venomous creatures and modulate activity of disease-relevant voltage-gated ion channels and are therefore being pursued as therapeutic leads. The amphipathic surface profile of GMTs has prompted the proposal that some GMTs simultaneously bind to the cell membrane and voltage-gated ion channels in a trimolecular complex. Here, we examined whether there is a relationship among spider GMT amphipathicity, membrane binding, and potency or selectivity for voltage-gated sodium (Na) channels. We used NMR spectroscopy and calculations to examine the structures and physicochemical properties of a panel of nine GMTs and deployed surface plasmon resonance to measure GMT affinity for lipids putatively found in proximity to Na channels. Electrophysiology was used to quantify GMT activity on Na1.7, an ion channel linked to chronic pain. Selectivity of the peptides was further examined against a panel of Na channel subtypes. We show that GMTs adsorb to the outer leaflet of anionic lipid bilayers through electrostatic interactions. We did not observe a direct correlation between GMT amphipathicity and affinity for lipid bilayers. Furthermore, GMT-lipid bilayer interactions did not correlate with potency or selectivity for Nas. We therefore propose that increased membrane binding is unlikely to improve subtype selectivity and that the conserved amphipathic GMT surface profile is an adaptation that facilitates simultaneous modulation of multiple Nas.
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.
IgG staining in human vagus nerves; H&E and Na v 1.7 expression in mouse sciatic nerves; tryptic digestion experiments for Hsp1a and Hsp1a-FL; epifluorescence images of sciatic nerves injected with Hsp1a-FL, block, or PBS, and the fluorescence quantification; fresh tissue confocal fluorescence microscopy (PDF) The authors declare the following competing financial interest(s): J.G., P.D.S.F., G.F.K. and T.R. are co-inventors on a Hsp1a-related patent application. S.K. and T.R. are shareholders of Summit Biomedical Imaging, LLC.
Compelling human genetic studies have identified the voltage-gated sodium channel Na V 1.7 as a promising therapeutic target for the treatment of pain. The analgesic spider-venom-derived peptide μ-theraphotoxin-Pn3a is an exceptionally potent and selective inhibitor of Na V 1.7; however, little is known about the structure−activity relationships or channel interactions that define this activity. We rationally designed 17 Pn3a analogues and determined their activity at hNa V 1.7 using patch−clamp electrophysiology. The positively charged amino acids K22 and K24 were identified as crucial for Pn3a activity, with molecular modeling identifying interactions of these residues with the S3−S4 loop of domain II of hNa V 1.7. Removal of hydrophobic residues Y4, Y27, and W30 led to a loss of potency (>250-fold), while replacement of negatively charged D1 and D8 residues with a positively charged lysine led to increased potencies (>13-fold), likely through alterations in membrane lipid interactions. Mutating D8 to an asparagine led to the greatest improvement in Pn3a potency at Na V 1.7 (20-fold), while maintaining >100-fold selectivity over the major off-targets Na V 1.4, Na V 1.5, and Na V 1.6. The Pn3a[D8N] mutant retained analgesic activity in vivo, significantly attenuating mechanical allodynia in a clinically relevant mouse model of postsurgical pain at doses 3-fold lower than those with wild-type Pn3a, without causing motor-adverse effects. Results from this study will facilitate future rational design of potent and selective peptidic Na V 1.7 inhibitors for the development of more efficacious and safer analgesics as well as to further investigate the involvement of Na V 1.7 in pain.
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