Computational design of peptides to target NaV1.7 channel with high potency and selectivity for the treatment of pain
- University of California, Davis, United States
- University of California Davis Medical Center, United States
- AnaBios, United States
- AmbioPharm Inc, United States
Abstract
The voltage-gated sodium NaV1.7 channel plays a key role as a mediator of action potential propagation in C-fiber nociceptors and is an established molecular target for pain therapy. ProTx-II is a potent and moderately selective peptide toxin from tarantula venom that inhibits human NaV1.7 activation. Here we used available structural and experimental data to guide Rosetta design of potent and selective ProTx-II-based peptide inhibitors of human NaV1.7 channels. Functional testing of designed peptides using electrophysiology identified the PTx2-3127 and PTx2-3258 peptides with IC50s of 7 nM and 4 nM for hNaV1.7 and more than 1,000-fold selectivity over human NaV1.1, NaV1.3, NaV1.4, NaV1.5, NaV1.8, and NaV1.9 channels. PTx2-3127 inhibits NaV1.7 currents in mouse and human sensory neurons and shows efficacy in rat models of chronic and thermal pain when administered intrathecally. Rationally-designed peptide inhibitors of human NaV1.7 channels have transformative potential to define a new class of biologics to treat pain.
Data availability
All data generated or analysed during this study are included in the manuscript.
Article and author information
Author details
Michael W Pennington
Theanne N Griffith
Vladimir Yarov-Yarovoy
Funding
National Institute of Neurological Disorders and Stroke (UG3NS114956)
- Phuong T Nguyen
- Hai M Nguyen
- Karen M Wagner
- Robert Stewart
- Vikrant Singh
- Parashar Thapa
- Yi-Je Chen
- Mark W Lillya
- Anh Tuan Ton
- Richard Kondo
- Andre Ghetti
- Michael W Pennington
- Bruce Hammock
- Theanne N Griffith
- Jon T Sack
- Heike Wulff
- Vladimir Yarov-Yarovoy
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Ethics
Animal experimentation: Research involving vertebrate animals was done at the University of California following protocols reviewed and approved by the UC Davis Institutional Animal Care and Use Committee (UCD IACUC) - Animal Welfare Assurance Number A3433-01. The animals were cared for by the Center for Laboratory Animal Science (CLAS) Veterinary Services under a currently AAALAC approved program under the direction of Dr. Laura Brignolo (Campus Veterinarian). The animals were housed in NIH-approved facilities in CLAS and are observed daily by technicians. Unusual events are reported to the on call veterinarian, as well as to the investigator according to posted protocols. Other maintenance veterinary care was conducted according to NIH guidelines on the Use and Care of Animals. Facilities were inspected regularly according to NIH and AAALAC guidelines.
Copyright
© 2022, Nguyen et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
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Computational design of peptides to target NaV1.7 channel with high potency and selectivity for the treatment of pain
eLife 11:e81727.
https://doi.org/10.7554/eLife.81727
Further reading
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Many voltage-gated potassium (Kv) channels display a time-dependent phenomenon called C-type inactivation, whereby prolonged activation by voltage leads to the inhibition of ionic conduction, a process that involves a conformational change at the selectivity filter toward a non-conductive state. Recently, a high-resolution structure of a strongly inactivated triple-mutant channel kv1.2-kv2.1-3m revealed a novel conformation of the selectivity filter that is dilated at its outer end, distinct from the well-characterized conductive state. While the experimental structure was interpreted as the elusive non-conductive state, our molecular dynamics simulations and electrophysiological measurements show that the dilated filter of kv1.2-kv2.1-3m is conductive and, as such, cannot completely account for the inactivation of the channel observed in the structural experiments. The simulation shows that an additional conformational change, implicating isoleucine residues at position 398 along the pore lining segment S6, is required to effectively block ion conduction. The I398 residues from the four subunits act as a state-dependent hydrophobic gate located immediately beneath the selectivity filter. By mutating I398 to Asparagine, ion permeation can be resumed in the kv1.2-kv2.1-3m channel, which was not a reversion C-type inactivation, since AgTxII fails to block the ionic permeation of kv1.2-kv2.1-3m_I398N. As a critical piece of the C-type inactivation machinery, this structural feature is the potential target of a broad class of QA blockers and negatively charged activators thus opening new research directions towards the development of drugs that specifically modulate gating-states of Kv channels.
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Kainate receptors are key modulators of synaptic transmission and plasticity in the central nervous system. Different kainate receptor isoforms with distinct spatiotemporal expressions have been identified in the brain. The GluK1-1 splice variant receptors, which are abundant in the adult brain, have an extra fifteen amino acids inserted in the amino-terminal domain (ATD) of the receptor resulting from alternative splicing of exon 9. However, the functional implications of this post-transcriptional modification are not yet clear. We employed a multi-pronged approach using cryogenic electron microscopy, electrophysiology, and other biophysical and biochemical tools to understand the structural and functional impact of this splice insert in the extracellular domain of GluK1 receptors. Our study reveals that the splice insert alters the key gating properties of GluK1 receptors and their modulation by the cognate auxiliary Neuropilin and tolloid-like (Neto) proteins 1 and 2. Mutational analysis identified the role of crucial splice residues that influence receptor properties and their modulation. Furthermore, the cryoEM structure of the variant shows that the presence of exon 9 in GluK1 does not affect the receptor architecture or domain arrangement in the desensitized state. Our study thus provides the first detailed structural and functional characterization of GluK1-1a receptors, highlighting the role of the splice insert in modulating receptor properties and their modulation.
