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

In our series of compounds, we used P1 = GlnLactam (g-lactam) throughout, because this substituent has proven to be an excellent surrogate for glutamine. 29, 32 While we made some efforts to optimize the P1' residue of the compounds as well as the N-cap (P3), we mainly focussed on optimization of the P2 substituent. In nearly all studies aiming at discovering peptidomimetic inhibitors of coronavirus M pro s, P2 is invariably isobutyl (leucine), and this residue has also been used in the efforts to design compounds that would inhibit enterovirus 3C pro s as well (see above). From crystal structures of our early lead compound, 11a (cinnamoyl-Phe-GlnLactam-CO-CO-NH-Bz), in complex with the M pro s of HCoV NL63 (as representative of the alphacoronavirus proteases) and SARS-CoV (beta-CoV) as well as the 3C pro of Coxsackievirus B3 (enterovirus proteases), we found that the S2 pocket has fundamentally different shapes in these enzymes. In the SARS-CoV M pro , the S2 subsite is a deep hydrophobic pocket that is truly three-dimensional in shape: the "walls" of the groove are formed by the polypeptide main chain around residues 186 -188 as well as by the side-chains of His41 (of the catalytic dyad) and Gln189, whereas the "floor" is formed by Met165 and the "lid" by residues 45 -51, in particular Met49. The two methionines provide important interaction points for the P2 substituents of inhibitors; while these interactions are mostly hydrophobic in character, we have previously described the surprising observation of the carboxylate of an aspartic residue in P2 that made polar interactions with the sulfur atoms of these methionines. 37 Because the pocket offers so many opportunities for interaction and features a pronounced plasticity, P2 substituents such as isobutyl (from Leu), which are too small to fill the pocket entirely, can still generate sufficient binding enthalpy. Accordingly, the S2 pocket of SARS-CoV M pro is the most tolerant among the three enzymes investigated here, in terms of versatility of the P2-substituents accepted.