Single Residue Substitutions That Confer Voltage-Gated Sodium Ion Channel Subtype Selectivity in the Na_V1.7 Inhibitory Peptide GpTx-1

Abstract

There is interest in the identification and optimization of new molecular entities selectively targeting ion channels of therapeutic relevance. Peptide toxins represent a rich source of pharmacology for ion channels, and we recently reported GpTx-1 analogs that inhibit NaV1.7, a voltage-gated sodium ion channel that is a compelling target for improved treatment of pain. Here we utilize multi-attribute positional scan (MAPS) analoging, combining high-throughput synthesis and electrophysiology, to interrogate the interaction of GpTx-1 with NaV1.7 and related NaV subtypes. After one round of MAPS analoging, we found novel substitutions at multiple residue positions not previously identified, specifically glutamic acid at positions 10 or 11 or lysine at position 18, that produce peptides with single digit nanomolar potency on NaV1.7 and 500-fold selectivity against off-target sodium channels. Docking studies with a NaV1.7 homology model and peptide NMR structure generated a model consistent with the key potency and selectivity modifications mapped in this work.

Breaking barriers to novel analgesic drug development

“…This approach exploits amino-acid differences among different sodium channel types in extracellular loops of the channel proteins and has yielded peptide inhibitors with very high affinity for blocking Nav1.7 channels and a high degree of subtype selectivity 158 . However, delivery of these large peptide compounds may present a significant challenge for clinical translation.…”

Spider peptide toxin HwTx-IV engineered to bind to lipid membranes has an increased inhibitory potency at human voltage-gated sodium channel hNa V 1.7

“…Loss of function mutations on SCN9A, the gene encoding hNa V 1.7, result in unresponsiveness to pain, whereas gain of function mutations result in increased chronic and acute pain sensations [3,4]. Because of this link to pain, efforts to identify potent and selective inhibitors of hNa V 1.7 are underway [5][6][7][8].…”

The tarantula toxin β/δ-TRTX-Pre1a highlights the importance of the S1-S2 voltage-sensor region for sodium channel subtype selectivity

“…From extensive structure-activity studies carried out by several pharmaceutical companies on spider venom peptides closely related to Pre1a (i.e. GpTx1, CcoTx1 and HwTxIV 26–28, 34 ), the aromatic residues at the tip of Loop 1 are key residues for the interaction of these peptides with Na V channels, in particular for their inhibitory effects on channel activation. Whether the flexibility of loop 1 in Pre1a has any role in its ability to potently interact with both DII and/or DIV VSDs (i.e., resulting in two conformations of the same pharmacophore residues) remains to be elucidated.…”

“…A deeper understanding of the molecular basis of the interactions between spider venom peptides with Na V channels is helping efforts to design a new generation of pharmacological tools and potential therapeutic lead molecules 26–28 . To this end, we identified β/δ-TRTX-Pre1a (Pre1a) from the venom of the tarantula Psalmopoeus reduncus in a screen for Na V 1.7 inhibitors.…”