Breakthrough peptide inhibitor effective against multiple SARS-CoV-2 variants

SARS-CoV-2, the virus responsible for COVID-19, infects cells by binding its spike protein to angiotensin-converting enzyme 2 (ACE2) receptors. Blocking this interaction with inhibitors could prevent infection. Since these inhibitors act directly on the virus without affecting human cells, they may be safer than some existing treatments. However, mutations in the spike protein can alter its structure, reducing the effectiveness of these inhibitors.

In a significant breakthrough, a research team led by Professor Yoshinori Fujiyoshi and Project Assistant Professor Shun Nakamura from the Cellular and Structural Physiology Laboratory, Advanced Research Initiative. Institute of Science Tokyo, in collaboration with the Department of Microbiology and Infection Control, Faculty of Medicine, Osaka Medical and Pharmaceutical University, has developed COVID-19 eliminative Short Peptide Inhibiting ACE2 binding (CeSPIACE), a mutation-tolerant spike protein inhibitor that remains effective against SARS-CoV-2 variants, including Omicron XBB.1.5. The study was published in the Proceedings of the National Academy of Sciences on January 24, 2025.

All pathogen proteins, like the SARS-CoV-2 spike, have invariant structures critical for their functions, making them good targets for mutation-tolerant drugs, as seen in our peptide engineering." Dr. Yoshinori Fujiyoshi, Institute of Science Tokyo

The team targeted the receptor-binding domain (RBD), a critical region of the spike protein responsible for binding to ACE2 receptors. Because this region is essential for viral function, it is less likely to mutate, making it an ideal target. Using cryo-electron microscopy and X-ray crystallography, the researchers analyzed the RBD structure to identify target sites. Starting with LCB1, a mutation-sensitive RBD-binding molecule, they developed a 39-amino acid peptide, enhancing its stability, mutation tolerance, and binding affinity to create CeSPIACE.

CeSPIACE is a short peptide made up of natural amino acids. It forms a two-helix bundle, which then self-assembles into a four-helix bundle with its RBD-binding site exposed, maximizing its ability to block the spike protein from binding to ACE2 receptors. While CeSPIACE primarily targets the ACE2-binding site, ensuring that mutations outside this region do not weaken its effectiveness, it was further engineered to recognize the stable backbone of the RBD, which remains unchanged even when side chains mutate. To ensure broad effectiveness across variants, the researchers adjusted its binding surface to accommodate specific mutations, such as Y501 in many strains after alpha one and N501 in the wild-type, making it effective against multiple SARS-CoV-2 variants.

CeSPIACE demonstrated strong binding to the RBDs of major SARS-CoV-2 variants, with a picomolar (pM) affinity ranging from 44 pM to 928 pM. In vivo tests with Syrian hamsters showed that a three-day intranasal treatment against the Delta variant led to a 1,000-fold drop in the amount of virus compared to untreated controls. In vitro experiments with human lung-derived Calu-3 cells showed clear efficacy against multiple variants (WT, Alpha, Delta, and Omicron BA.5) , blocking viral entry into pre-treated cells and preventing reinfection of cells already exposed to the virus.

These findings suggest that CeSPIACE can be used both as a prophylactic (preventive measure) to block infection and as a therapeutic in treating infection after exposure to the virus. Unlike biological antibodies, which are complex and costly to produce, peptides like CeSPIACE are simpler, cheaper, and easier to manufacture, allowing for rapid large-scale production during outbreaks. Additionally, peptides are chemically stable and do not require cold storage, making them easier to distribute globally.

Such an approach could also be used to develop potential treatments for other viruses, such as influenza or human immunodeficiency virus. "Unknown infectious diseases will continue to emerge. Our strategy of engineering mutation-tolerant inhibitors can be applied to developing therapeutics against other existing infections or future pandemics," says Dr. Fujiyoshi.

Nakamura, S., et al. (2025). Structure-guided engineering of a mutation-tolerant inhibitor peptide against variable SARS-CoV-2 spikes. Proceedings of the National Academy of Sciences. doi.org/10.1073/pnas.2413465122.