Scientists explore nanomolar inhibition of SARS-CoV-2 infection via unmodified peptide targeting spike protein pre-hairpin intermediate

In a recent study posted to the bioRxiv* preprint server, scientists identified an unmodified peptide addressing pre-hairpin spike (S) protein intermediate inhibiting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection.

Study: Nanomolar inhibition of SARS-CoV-2 infection by an unmodified peptide targeting the pre-hairpin intermediate of the spike protein. Image Credit: PHOTOCREO Michal Bednarek/Shutterstock


Fusion between the host and SARS-CoV-2 membranes, facilitated by the viral S glycoprotein, is necessary for SARS-CoV-2 infection. The significance of viral membrane fusion has made the S protein a well-liked target for the creation of therapeutics and vaccines. Nevertheless, through epitope alteration in the receptor binding domain (RBD) of S, SARS-CoV-2 variants pose a threat to presently available coronavirus disease 2019 (COVID-19) monoclonal antibody therapies and vaccines.

Hence, other antivirals that address processes unlikely to be impacted by the mutation, like the membrane fusion phase of viral entry into the host cell, are urgently needed. Peptide inhibitors are a subclass of the antivirals that prevent the SARS-CoV-2 S protein's so-called heptad repeat 1 heptad repeat 2 (HR1HR2) six-helix bundle from forming, preventing the viral membrane from fusing.

About the study

In the current research, the scientists conducted structural assessments of the HR1HR2 bundle using high-resolution cryo-electron microscopy (cryo-EM). Further, they developed an N-terminally extended HR2-stemmed peptide. In a cell-to-cell fusion analysis, the researchers looked at how the N-terminal elongation of HR2 affected the inhibition of the SARS-CoV-2 S membrane fusion ability.

The team investigated the possible antiviral impact of the longHR2_42 peptide. For this, they utilized a chimeric vesicular stomatitis virus (VSV) expressing a soluble enhanced green fluorescent protein (eGFP) infection reporter, where the VSV glycoprotein has been substituted by the S protein of SARS-CoV-2 Wuh strain, i.e., VSV-SARS-CoV-2-Wuh.

The investigators evaluated the impact of transmembrane serine protease 2 (TMPRSS2) on the S binding of the HR2 peptide. The results were validated using authentic SARS-CoV-2. The authors infected Caco-2 cells overexpressing the human angiotensin-converting enzyme 2 (ACE2) receptor, i.e., Caco-2+hACE2, with a SARS-CoV-2 Wuh strain patient isolate. Furthermore, they used the authentic and the VSV-SARS-CoV-2 infection tests to see whether the longHR2_42 could prevent infection by major SARS-CoV-2 variants.


Collectively, the researchers discovered a simple peptide that inhibited all significant SARS-CoV-2 variant infections in nanomolar ranges. They found an extended, well-folded N-terminal domain of HR2 between residues 1162 to 1203, interacting with the HR1 triple helix across the post-fusion HR1HR2 bundle. 

Based on this structure, the authors created an elongated HR2 peptide, longHR2_42. It inhibited SARS-CoV-2 at single-digit nanomolar concentrations without requiring modifications like chemical stapling or lipidation in authentic SARS-CoV-2 infection assays, cell-based fusion, and VSV-SARS-CoV-2 chimera.

Notably, the peptide robustly inhibited all relevant SARS-CoV-2 variants thus far. However, the activity was approximately 10-fold lower towards the Omicron variant. Since the D614G mutation, found in all significant SARS-CoV-2 variants, was not near the HR1HR2 interface, there was no discernible difference in the longHR2_42's effectiveness against this mutant. 

The presently developed peptide was around 100 times more effective than commonly utilized unmodified shorter HR2 peptides lacking a vital N-terminal extension. Moreover, it possesses a very extended inhibitory lifetime of more than three hours following washout in virus infection tests, implying that it addresses a pre-hairpin intermediary of the SARS-CoV-2 S protein. Since the transmembrane serine protease 2 (TMPRSS2) was required for the prolonged inhibitory lifetime following peptide washout indicates that TMPRSS2 (or possibly also cathepsin) cleavage of SARS-CoV-2 S creates a pre-hairpin intermediate that the peptide can easily bind.


Overall, in the present study, the team identified an unmodified monomeric N-terminally extended HR2 peptide lacking any hydrocarbon staples or modifications, longHR2_42, exhibiting around 100-fold higher SARS-CoV-2 inhibition than priorly documented peptides lacking chemical alterations. Specifically, they discovered nanomolar suppression by longHR2_42 in a cell-cell fusion test, a VSV-SARS-CoV-2 chimera infection evaluation, and an authentic SARS-CoV-2 infection analysis.

The research findings suggest that a simple peptide exhibiting the appropriate sequence could be a cost-effective and potent therapeutic against COVID-19. Besides, the study data reveal new information about the SARS-CoV-2 host entrance mechanism. These data add more evidence in favor of the pre-hairpin intermediate of the S protein and imply that sections beyond the HR2 helical region may present fresh prospects for robust peptide-stemmed therapeutics for SARS-CoV-2 and its variants as well as even more distantly linked viruses.

The authors suggested that a proper expansion of the peptide sequence might be able to produce more efficacy. Additional cycles of longHR2_42 sequence optimization might increase the peptide's activity and serve as a foundation for creating new variant-specific peptides. Such designs may be crucial for antiviral therapeutics to fight the present COVID-19 pandemic and other viruses of concern that are less closely related.

*Important notice

bioRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.

Journal reference:
  • Kailu Yang, Chuchu Wang, Alex J. B. Kreutzberger, Ravi Ojha, Suvi Kuivanen, Sergio Couoh-Cardel, Serena Muratcioglu, Timothy J. Eisen, K. Ian White, Richard G. Held, Subu Subramanian, Kendra Marcus, Richard A. Pfuetzner, Luis Esquivies, Catherine A. Doyle, John Kuriyan, Olli Vapalahti, Giuseppe Balistreri, Tomas Kirchhausen, Axel T. Brunger. (2022). Nanomolar inhibition of SARS-CoV-2 infection by an unmodified peptide targeting the pre-hairpin intermediate of the spike protein. bioRxivdoi:

Posted in: Medical Science News | Medical Research News | Disease/Infection News

Tags: ACE2, Angiotensin, Angiotensin-Converting Enzyme 2, Antibody, Cell, Coronavirus, Coronavirus Disease COVID-19, covid-19, Efficacy, Electron, Electron Microscopy, Enzyme, Fluorescent Protein, Glycoprotein, Helix, Membrane, Microscopy, Monoclonal Antibody, Mutation, Omicron, Pandemic, Peptides, Protein, Receptor, Research, Respiratory, SARS, SARS-CoV-2, Serine, Severe Acute Respiratory, Severe Acute Respiratory Syndrome, Stomatitis, Syndrome, Therapeutics, Virus

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Shanet Susan Alex

Shanet Susan Alex, a medical writer, based in Kerala, India, is a Doctor of Pharmacy graduate from Kerala University of Health Sciences. Her academic background is in clinical pharmacy and research, and she is passionate about medical writing. Shanet has published papers in the International Journal of Medical Science and Current Research (IJMSCR), the International Journal of Pharmacy (IJP), and the International Journal of Medical Science and Applied Research (IJMSAR). Apart from work, she enjoys listening to music and watching movies.

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