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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- Neuroscience
- Structural Biology and Molecular Biophysics
We present near-atomic-resolution cryoEM structures of the mammalian voltage-gated potassium channel Kv1.2 in open, C-type inactivated, toxin-blocked and sodium-bound states at 3.2 Å, 2.5 Å, 3.2 Å, and 2.9 Å. These structures, all obtained at nominally zero membrane potential in detergent micelles, reveal distinct ion-occupancy patterns in the selectivity filter. The first two structures are very similar to those reported in the related Shaker channel and the much-studied Kv1.2–2.1 chimeric channel. On the other hand, two new structures show unexpected patterns of ion occupancy. First, the toxin α-Dendrotoxin, like Charybdotoxin, is seen to attach to the negatively-charged channel outer mouth, and a lysine residue penetrates into the selectivity filter, with the terminal amine coordinated by carbonyls, partially disrupting the outermost ion-binding site. In the remainder of the filter two densities of bound ions are observed, rather than three as observed with other toxin-blocked Kv channels. Second, a structure of Kv1.2 in Na+ solution does not show collapse or destabilization of the selectivity filter, but instead shows an intact selectivity filter with ion density in each binding site. We also attempted to image the C-type inactivated Kv1.2 W366F channel in Na+ solution, but the protein conformation was seen to be highly variable and only a low-resolution structure could be obtained. These findings present new insights into the stability of the selectivity filter and the mechanism of toxin block of this intensively studied, voltage-gated potassium channel.
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- Structural Biology and Molecular Biophysics
xCas9 is an evolved variant of the CRISPR-Cas9 genome editing system, engineered to improve specificity and reduce undesired off-target effects. How xCas9 expands the DNA targeting capability of Cas9 by recognising a series of alternative protospacer adjacent motif (PAM) sequences while ignoring others is unknown. Here, we elucidate the molecular mechanism underlying xCas9’s expanded PAM recognition and provide critical insights for expanding DNA targeting. We demonstrate that while wild-type Cas9 enforces stringent guanine selection through the rigidity of its interacting arginine dyad, xCas9 introduces flexibility in R1335, enabling selective recognition of specific PAM sequences. This increased flexibility confers a pronounced entropic preference, which also improves recognition of the canonical TGG PAM. Furthermore, xCas9 enhances DNA binding to alternative PAM sequences during the early evolution cycles, while favouring binding to the canonical PAM in the final evolution cycle. This dual functionality highlights how xCas9 broadens PAM recognition and underscores the importance of fine-tuning the flexibility of the PAM-interacting cleft as a key strategy for expanding the DNA targeting potential of CRISPR-Cas systems. These findings deepen our understanding of DNA recognition in xCas9 and may apply to other CRISPR-Cas systems with similar PAM recognition requirements.
