Thiazolyl 4-carboxylate ketone as a new warhead for a highly potent SARS-CoV-2 main protease inhibitor

The SARS-CoV-2 main protease (M^pro), an enzyme essential for viral replication and lacking a human homologue, has emerged as a highly attractive target for the development of novel antiviral agents. Although several M^pro inhibitors have been developed - some receiving regulatory approval - their use is sometimes limited by drug-drug interactions. In this study, we designed and synthesized peptidomimetic SARS-CoV-2 M^pro inhibitors incorporating a novel thiazolyl 4-carboxylate ketone warhead, previously employed by our group in the development of cytosolic phospholipase A₂ inhibitors. The synthesized compounds were evaluated for their in vitro inhibitory potency against SARS-CoV-2 M^pro, and a highly potent M^pro inhibitor (GK730) was identified (IC₅₀ 5.75 nM). The melting temperature of the M^pro-GK730 complex revealed high stability, consistent with the high inhibitory potency. The X-ray crystal structures of inhibitors GK729 and GK730 bound to M^pro were determined, providing insights into the binding interactions and mechanism of action. Studies on the host cell proteases cathepsin B and L showed that GK730 did not inhibit cathepsin B, while exhibited weak inhibition of cathepsin L. Furthermore, GK730 demonstrated an EC₅₀ value of 5.70 μM against a wild-type SARS-CoV-2 strain in Vero E6 cells and minimal cytotoxicity (CC₅₀ value greater than 100 μM).