α-Ketoamides as Broad-Spectrum Inhibitors of Coronavirus and Enterovirus Replication: Structure-Based Design, Synthesis, and Activity Assessment

Abstract

The main protease of coronaviruses and the 3C protease of enteroviruses share a similar active-site architecture and a unique requirement for glutamine in the P1 position of the substrate. Because of their unique specificity and essential role in viral polyprotein processing, these proteases are suitable targets for the development of antiviral drugs. In order to obtain near-equipotent, broad-spectrum antivirals against alphacoronaviruses, betacoronaviruses, and enteroviruses, we pursued a structure-based design of peptidomimetic α-ketoamides as inhibitors of main and 3C proteases. Six crystal structures of protease−inhibitor complexes were determined as part of this study. Compounds synthesized were tested against the recombinant proteases as well as in viral replicons and virus-infected cell cultures; most of them were not cell-toxic. Optimization of the P2 substituent of the αketoamides proved crucial for achieving near-equipotency against the three virus genera. The best near-equipotent inhibitors, 11u (P2 = cyclopentylmethyl) and 11r (P2 = cyclohexylmethyl), display low-micromolar EC 50 values against enteroviruses, alphacoronaviruses, and betacoronaviruses in cell cultures. In Huh7 cells, 11r exhibits three-digit picomolar activity against the Middle East Respiratory Syndrome coronavirus.

Substrate specificity profiling of SARS-CoV-2 main protease enables design of activity-based probes for patient-sample imaging

“…The Gln is also involved in proteases, which explains less stringent specificity. [11] The S2 pocket can form hydrophobic interactions with P2 residues that are not only limited to leucine. The S3 pocket of SARS M pro is not very well defined which is also reflected in our P3 substrate specificity profile.…”

“…This observation, along with further studies on the M pro , can potentially lead to new broad-spectrum anti-coronaviral inhibitors with minimum side effects. [11] In the present study, we applied the HyCoSuL (Hybrid Combinatorial Substrate…”

Structure-based design of antiviral drug candidates targeting the SARS-CoV-2 main protease

“…In our design of new inhibitors, an aldehyde was selected as a new warhead in P1 in order to form a covalent bond with cysteine. The reported SARS-CoV M pro inhibitors often have an (S)-γlactam ring that occupies the S1 site of M pro , and this ring was expected to be a good choice in P1 (23). Furthermore, the S2 site of coronavirus M pro is usually large enough to accommodate the bigger P2 fragment.…”

Structure-Based Design, Synthesis and Biological Evaluation of Peptidomimetic Aldehydes as a Novel Series of Antiviral Drug Candidates Targeting the SARS-CoV-2 Main Protease

“…The substrates of coronaviruses 3CL pro (M pro ) show some similarity, and most 3CL protease inhibitors are peptidomimetic covalent inhibitors derived from the natural substrates. The active sites are highly conserved among all CoV M pro and are usually composed of four pockets (S1', S1, S2 and S4) 22,23 . The thiol of a cysteine residue in the S1' pocket can anchor inhibitors by a covalent linkage, which is important for the inhibitors to maintain anti-viral activity.…”