Computational design of peptides to target NaV1.7 channel with high potency and selectivity for the treatment of pain
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The authors of this manuscript set out to improve on a peptide, ProTxII, which had been previously put forward as a promising blocker of Nav1.7 channels and thus offers a possible non-opiate way to block pain. They develop a useful computational workflow that is based on in silico manipulations of the interaction of ProTxII with a Na channel structure determined previously and evaluation of the predicted mutations with electrophysiology. The authors succeed in producing two peptides with improved selectivity for Nav1.7 over other subtypes and capable of producing ion channel block at low concentrations and the experimental evidence presented is solid, and the general applicability of the method is compelling.
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Abstract
The voltage-gated sodium Na V 1.7 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 Na V 1.7 activation. Here we used available structural and experimental data to guide Rosetta design of potent and selective ProTx-II-based peptide inhibitors of human Na V 1.7 channels. Functional testing of designed peptides using electrophysiology identified the PTx2-3127 and PTx2-3258 peptides with IC 50 s of 7 nM and 4 nM for hNa V 1.7 and more than 1000-fold selectivity over human Na V 1.1, Na V 1.3, Na V 1.4, Na V 1.5, Na V 1.8, and Na V 1.9 channels. PTx2-3127 inhibits Na V 1.7 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 Na V 1.7 channels have transformative potential to define a new class of biologics to treat pain.
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eLife assessment
The authors of this manuscript set out to improve on a peptide, ProTxII, which had been previously put forward as a promising blocker of Nav1.7 channels and thus offers a possible non-opiate way to block pain. They develop a useful computational workflow that is based on in silico manipulations of the interaction of ProTxII with a Na channel structure determined previously and evaluation of the predicted mutations with electrophysiology. The authors succeed in producing two peptides with improved selectivity for Nav1.7 over other subtypes and capable of producing ion channel block at low concentrations and the experimental evidence presented is solid, and the general applicability of the method is compelling.
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Reviewer #1 (Public Review):
Voltage-gated sodium channels are fundamental in the generation and transmission of painful signals. For this reason their inhibition has been proposed as a potential way to treat the worst forms of chronic pain. Since the main subtype of sodium channels involved in pain signaling is Nav1.7, its important that potential inhibitors target this subtype with high efficacy and in a selective manner.
In this manuscript, the authors set out to improve on a peptide, ProTxII, which had been previously put forward as a promising blocker of Nav1.7 channels. For this task, they develop a computational workflow that is based on in silico manipulations of the interaction of ProTxII with a Na channel structure determined previously and evaluation of the predicted mutations with electrophysiology. The method employs …
Reviewer #1 (Public Review):
Voltage-gated sodium channels are fundamental in the generation and transmission of painful signals. For this reason their inhibition has been proposed as a potential way to treat the worst forms of chronic pain. Since the main subtype of sodium channels involved in pain signaling is Nav1.7, its important that potential inhibitors target this subtype with high efficacy and in a selective manner.
In this manuscript, the authors set out to improve on a peptide, ProTxII, which had been previously put forward as a promising blocker of Nav1.7 channels. For this task, they develop a computational workflow that is based on in silico manipulations of the interaction of ProTxII with a Na channel structure determined previously and evaluation of the predicted mutations with electrophysiology. The method employs previously validated algorithms implemented in Rossetta.
The authors succeed in producing two peptides with improved selectivity for Nav1.7 over other subtypes and capable of blocking at low nanomolar concentrations.
The method seems to be robust enough to be implemented for similar tasks in other protein-protein interaction scenarios, although this remains to be proven.
The results and methods presented here should be useful in several ways. First, the developed peptides can be further evaluated in a clinical setting or at least serve as a scaffold to develop further. Second, the methods should be useful to other groups working on biologicals as clinical pharmacological agents and in pure biophysics to probe surfaces of interactions.
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Reviewer #2 (Public Review):
In this manuscript, Nguyen et al. make use of recently determined cryo-EM structures of Nav1.7 channels in complex with ProTX-II, a peptide spider toxin that binds to VSD2 and stabilizes the deactivated state of the channel in addition to reducing peak currents. Previous work on making modified spider toxin peptides as potent and selective Nav1.7 inhibitors by Merck, Amgen, and others was conducted in a structure-blind manner. This manuscript demonstrates that it is possible to use structure data and computational tools to identify modified spider toxin peptides that show even better potency and selectivity properties.
The authors did a very nice job presenting their detailed results. This detailed material should be very helpful to researchers wanting to expand on this work toward the development of …
Reviewer #2 (Public Review):
In this manuscript, Nguyen et al. make use of recently determined cryo-EM structures of Nav1.7 channels in complex with ProTX-II, a peptide spider toxin that binds to VSD2 and stabilizes the deactivated state of the channel in addition to reducing peak currents. Previous work on making modified spider toxin peptides as potent and selective Nav1.7 inhibitors by Merck, Amgen, and others was conducted in a structure-blind manner. This manuscript demonstrates that it is possible to use structure data and computational tools to identify modified spider toxin peptides that show even better potency and selectivity properties.
The authors did a very nice job presenting their detailed results. This detailed material should be very helpful to researchers wanting to expand on this work toward the development of peptide-based pain drugs that selectively target Nav1.7. Their in-vitro electrophysiological analysis is excellent, showing full selectivity profiles (including difficult to work with channels such as hNav1.8 and hNav1.9) from HEK293 cells and also showing inhibition of the TTX-S current with both mouse and human cultured DRG neurons. The in-vivo work shows very strong analgesia in the hotplate model as well as in a model of oxaliplatin-induced peripheral neuroparthy, showing that PTx2-3127 is a powerful analgesic in rats.
Overall, this is an excellent investigation into the feasibility of using structural information and computational tools to design potent and selective Nav1.7 inhibitors. Such peptide-based inhibitors might be developed in the future as novel pain drugs.
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Reviewer #3 (Public Review):
This communication describes the molecular design of antinociceptive peptides with the aim to improve the peptide affinity and blocking activity towards Nav1.7. The authors performed in vivo experimental assays of such molecular designed peptides to validate them. The methods incorporate state-of-the-art techniques, and the results are clear and of great quality.
Strengths: Many synthetic variants were generated to accomplish the best antinociceptive peptides including its in vitro and in vivo assays.
Weaknesses: Some of the reasonings for creating some of the in-silico peptide variants were not clear at all even though they were designed based on ligand-receptor models.
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