A rational design strategy and validation for protease-resistant fusion-inhibitor antiviral peptides

Abstract

Peptide-based fusion inhibitors are promising pharmaceuticals in the fight against enveloped viruses relying on membrane fusion for host infection. However, peptide therapeutic applications have long been hindered by their poor stability in vivo. Here, we discovered that peptide inhibitors with the wildtype sequence of the heptad repeat 2 (HR2) domain of the SARS-CoV-2 spike protein are efficiently cleaved by Transmembrane Protease, Serine 2 (TMPRSS2), a key protease involved in the SARS-CoV-2 virus-cell fusion pathway. We then identified the corresponding cleavage sites and designed three protease-resistant peptides using ranking based on deep mutational scanning and natural occurrence. The three candidates all exhibit inhibitory activity in a cell-cell fusion assay. A high-resolution cryo-EM structure of the top candidate, HR2-NHN, bound to its HR1 target reveals the molecular basis for its potent activity. The top candidate of the cell-based screening assay significantly improved efficacy relative to the wildtype peptide when administered 12 h before infection in both an authentic virus-cell infection assay and a mouse assay. More broadly, our results suggest that the design strategies for protease-resistant peptides could be applied to a broad spectrum of other enveloped viruses and pave the way for the development of safe, prophylactic antivirals that can be administered before exposure.

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BibTeX RIS

BibTeX

@misc{yang2026rational,
  title = {{A rational design strategy and validation for protease-resistant fusion-inhibitor antiviral peptides}},
  author = {K. Yang and C. Wang and F. Topi and S. Muratcioglu and R. Kant and T. Strandin and Y. T. Bong and L. Kareinen and S. Mäki and S. H. Saber and T. Binder and T. Sironen and S. Subramanian and K. I. White and R. Pfuetzner and L. Esquivies and O. Vapalahti and M. Joensuu and J. Kuriyan and J. Hepojoki and G. Balistreri and A. T. Brunger},
  howpublished = {bioRxiv},
  year = {2026},
  month = sep,
  doi = {10.64898/2026.09.12.750390},
  url = {https://www.biorxiv.org/content/10.64898/2026.09.12.750390v1},
  note = {CC BY},
}

RIS

TY  - UNPB
TI  - A rational design strategy and validation for protease-resistant fusion-inhibitor antiviral peptides
AU  - K. Yang
AU  - C. Wang
AU  - F. Topi
AU  - S. Muratcioglu
AU  - R. Kant
AU  - T. Strandin
AU  - Y. T. Bong
AU  - L. Kareinen
AU  - S. Mäki
AU  - S. H. Saber
AU  - T. Binder
AU  - T. Sironen
AU  - S. Subramanian
AU  - K. I. White
AU  - R. Pfuetzner
AU  - L. Esquivies
AU  - O. Vapalahti
AU  - M. Joensuu
AU  - J. Kuriyan
AU  - J. Hepojoki
AU  - G. Balistreri
AU  - A. T. Brunger
PY  - 2026
DA  - 2026/09/12
PB  - bioRxiv
DO  - 10.64898/2026.09.12.750390
UR  - https://www.biorxiv.org/content/10.64898/2026.09.12.750390v1
AB  - Peptide-based fusion inhibitors are promising pharmaceuticals in the fight against enveloped viruses relying on membrane fusion for host infection. However, peptide therapeutic applications have long been hindered by their poor stability in vivo. Here, we discovered that peptide inhibitors with the wildtype sequence of the heptad repeat 2 (HR2) domain of the SARS-CoV-2 spike protein are efficiently cleaved by Transmembrane Protease, Serine 2 (TMPRSS2), a key protease involved in the SARS-CoV-2 virus-cell fusion pathway. We then identified the corresponding cleavage sites and designed three protease-resistant peptides using ranking based on deep mutational scanning and natural occurrence. The three candidates all exhibit inhibitory activity in a cell-cell fusion assay. A high-resolution cryo-EM structure of the top candidate, HR2-NHN, bound to its HR1 target reveals the molecular basis for its potent activity. The top candidate of the cell-based screening assay significantly improved efficacy relative to the wildtype peptide when administered 12 h before infection in both an authentic virus-cell infection assay and a mouse assay. More broadly, our results suggest that the design strategies for protease-resistant peptides could be applied to a broad spectrum of other enveloped viruses and pave the way for the development of safe, prophylactic antivirals that can be administered before exposure.
ER  -

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