Alkyne as a Latent Warhead to Covalently Target SARS-CoV-2 Main Protease

[Image: see text] There is an urgent need for improved therapy to better control the ongoing COVID-19 pandemic. The main protease M(pro) plays a pivotal role in SARS-CoV-2 replications, thereby representing an attractive target for antiviral development. We seek to identify novel electrophilic warheads for efficient, covalent inhibition of M(pro). By comparing the efficacy of a panel of warheads installed on a common scaffold against M(pro), we discovered that the terminal alkyne could covalently modify M(pro) as a latent warhead. Our biochemical and X-ray structural analyses revealed the irreversible formation of the vinyl-sulfide linkage between the alkyne and the catalytic cysteine of M(pro). Clickable probes based on the alkyne inhibitors were developed to measure target engagement, drug residence time, and off-target effects. The best alkyne-containing inhibitors potently inhibited SARS-CoV-2 infection in cell infection models. Our findings highlight great potentials of alkyne as a latent warhead to target cystine proteases in viruses and beyond.