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

Crystal structures of compound 11a in complex with SARS-CoV M pro , HCoV-NL63 M pro , and CVB3 3C pro demonstrated that the aketo-carbon is covalently linked to the active-site Cys (no. 145, 144, and 147, resp.) of the protease (Fig. 2, 3a-c) . The resulting thiohemiketal is in the R configuration in the SARS-CoV and HCoV-NL63 M pro but in the S configuration in the CVB3 3C pro complex. The reason for this difference is that the oxygen atom of the thiohemiketal accepts a hydrogen bond from the catalytic His40 in the CVB3 protease, rather than from the main-chain amides of the oxyanion hole as in the SARS-CoV and HCoV-NL63 enzymes (Fig. 3a ,b,c insets). It is remarkable that we succeeded in obtaining a crystal structure of compound 11a in complex with the HCoV-NL63 M pro , even though it has no inhibitory effect on the activity of the enzyme (IC50 > 50 µM), (Fig. 2c) . Apparently, the compound is able to bind to this M pro in the absence of peptide substrate, but cannot compete with substrate for the binding site due to low affinity. A similar observation has been made in one of our previous studies, where we were able to determine the crystal structure of a complex between the inactive Michaelacceptor compound SG74 and the EV-D68 3C pro (ref. 29 ; PDB entry 3ZV9). Figure 2 : Fit of compound 11a (pink carbon atoms) to the target proteases (wheat surfaces) as revealed by X-ray crystallography of the complexes. A. Fo -Fc difference density (contoured at 3s) for 11a in the substrate-binding site of the SARS-CoV M pro (transparent surface). Selected side-chains of the protease are shown with green carbon atoms. B. Another view of 11a in the substrate binding site of the SARS-CoV M pro . Note the "lid" formed by residue Met49 and its neighbors above the S2 pocket. C. 11a in the substrate-binding site of HCoV-NL63 M pro . Because of the restricted size of the S2 pocket, the P2 benzyl group of the compound cannot enter deeply into this site. Note that the S2 pocket is also covered by a "lid" centred around Thr47. D. 11a in the substrate-binding site of the CVB3 3C pro . The S2 site is large and not covered by a "lid".