Exploiting high-energy hydration sites for the discovery of potent peptide aldehyde inhibitors of the SARS-CoV-2 main protease with cellular antiviral activity.

Small-molecule antivirals that prevent the replication of the SARS-CoV-2 virus by blocking the enzymatic activity of its main protease (Mpro) are and will be a tenet of pandemic preparedness. However, the peptidic nature of such compounds often precludes the design of compounds within favorable physical property ranges, limiting cellular activity. Here we describe the discovery of peptide aldehyde Mpro inhibitors with potent enzymatic and cellular antiviral activity. This structure-activity relationship (SAR) exploration was guided by the use of calculated hydration site thermodynamic maps (WaterMap) to drive potency via displacement of waters from high-energy sites. Thousands of diverse compounds were designed to target these high-energy hydration sites and then prioritized for synthesis by physics- and structure-based Free-Energy Perturbation (FEP+) simulations, which accurately predicted biochemical potencies. This approach ultimately led to the rapid discovery of lead compounds with unique SAR that exhibited potent enzymatic and cellular activity with excellent pan-coronavirus coverage.

Discovery of a Druggable, Cryptic Pocket in SARS-CoV-2 nsp16 Using Allosteric Inhibitors.

A collaborative, open-science team undertook discovery of novel small molecule inhibitors of the SARS-CoV-2 nsp16-nsp10 2'-O-methyltransferase using a high throughput screening approach with the potential to reveal new inhibition strategies. This screen yielded compound 5a, a ligand possessing an electron-deficient double bond, as an inhibitor of SARS-CoV-2 nsp16 activity. Surprisingly, X-ray crystal structures revealed that 5a covalently binds within a previously unrecognized cryptic pocket near the S-adenosylmethionine binding cleft in a manner that prevents occupation by S-adenosylmethionine. Using a multidisciplinary approach, we examined the mechanism of binding of compound 5a to the nsp16 cryptic pocket and developed 5a derivatives that inhibited nsp16 activity and murine hepatitis virus replication in rat lung epithelial cells but proved cytotoxic to cell lines canonically used to examine SARS-CoV-2 infection. Our study reveals the druggability of this newly discovered SARS-CoV-2 nsp16 cryptic pocket, provides novel tool compounds to explore the site, and suggests a new approach for discovery of nsp16 inhibition-based pan-coronavirus therapeutics through structure-guided drug design.