Introduction:

Bats are recognized as natural reservoirs of coronaviruses. Major human disease outbreaks caused by SARS-CoV, SARS-CoV-2, and MERS-CoV are presumed to have originated from zoonotic spillover of bat coronaviruses. Multiple bat species harbor SARS-CoV-like (Sarbecovirus) and MERS-CoV-like (Merbecovirus) viruses that use angiotensin-converting enzyme-2 (ACE2) or dipeptidyl peptidase-4 (DPP4) as a host cell receptor, similar to SARS-CoV, SARS-CoV-2, and MERS-CoV. This shared receptor tropism highlights the zoonotic and pandemic potential of bat coronaviruses and underscores the importance of evaluating the antiviral activity of inhibitors against them. Coronavirus 3CLpro cleaves the viral polyprotein into functional nonstructural proteins, essential for the viral replication cycle, and is conserved among coronaviruses, making it an attractive broad-spectrum antiviral target. This study aims to evaluate the antiviral activity of 3CLpro inhibitors against ACE2 or DPP4 utilizing bat coronaviruses.

Methodology:

Ten bat coronaviruses from the Betacoronavirus (Sarbecovirus and Merbecovirus) and Alphacoronavirus genera, along with SARS-CoV, SARS-CoV-2, MERS-CoV, and FIPV (Feline Infectious Peritonitis Virus), were included in this study. Their full-length 3CLpro genes were cloned and expressed. Nine 3CLpro inhibitors, including GC376, its analogs, and Nirmatrelvir, were tested against the expressed 3CLpros in the Fluorescence Resonance Energy Transfer (FRET) assay to determine 50% inhibitory concentrations (IC₅₀). X‑ray crystallography of bat coronavirus 3CLpro in complex with GC376 was performed. Additionally, we analyzed approximately 220 bat coronavirus 3CLpro sequences for amino acid homology and phylogeny.

Result:

Sequence analysis of these bat coronavirus 3CLpros revealed high homology within the Sarbecovirus (87-100%) and Merbecovirus (78-100%) subgenera and 41-100% homology within the Alphacoronavirus genus. Fifty percent or less homology was observed among the different coronavirus genera. In the FRET assay, some compounds demonstrated potent broad-spectrum inhibition across all tested coronaviruses, including those utilizing ACE2 or DPP4 receptors.

Conclusion:

These preliminary results highlight the potential of 3CLpro inhibitors as antiviral candidates against bat coronaviruses with zoonotic potential.