Alpha-ketoamides as broad-spectrum inhibitors of coronavirus and enterovirus replication

Linlin Zhang; Daizong Lin; Yuri Kusov; Yong Nian; Qingjun Ma; Jiang Wang; Albrecht von Brunn; Pieter Leyssen; Kristina Lanko; Johan Neyts; Adriaan de Wilde; Eric J. Snijder; Hong Liu; Rolf Hilgenfeld

We describe here the structure-based design, the synthesis, and the assessment of capped dipeptide a-ketoamides that target the main protease of alpha-or betacoronaviruses as well as the 3C protease of enteroviruses. Through crystallographic analyses of a total of six inhibitor complexes of three different proteases in this study, we found the a-ketoamide warhead (-CO-CO-NH-) to be sterically more versatile than other warheads such as Michael acceptors (-CH=CH-CO-) and aldehydes (-CH=O), because it features two acceptors for hydrogen bonds from the protein, namely the a-keto oxygen and the amide oxygen, whereas the other warheads have only one such acceptor. In the various complexes, the hydroxy group (or oxyanion) of the thiohemiketal that is formed by the nucleophilic attack of the activesite cysteine residue onto the a-keto carbon, can accept one or two hydrogen bonds from the main-chain amides of the oxyanion hole. In addition, the amide oxygen of the inhibitor accepts a hydrogen bond from the catalytic His side-chain. Alternatively, the thiohemiketal can interact with the catalytic His residue and the amide oxygen with the main-chain amides of the oxyanion hole. Depending on the exact interaction, the stereochemistry at the thiohemiketal C atom would be different. We have previously observed a similar difference in case of aldehyde inhibitors, where the single interaction point -the oxyanion of the thiohemiacetal -can accept a hydrogen bond either from the oxyanion hole or from the catalytic His side-chain, 37 resulting in different stereochemistry of the thiohemiacetal carbon. Both aketoamides and aldehydes react reversibly with the catalytic nucleophile of proteases, whereas Michael acceptors form irreversible adducts.