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 next analyzed the crystal structure of the complex between SARS-CoV M pro and compound 11s (Fig. 3f) . The cyclopropylmethyl substituent was found to be incorporated deeply into the S2 pocket, making hydrophobic interactions with Met49 (the lid), Met165 (the floor), and the Cb of Asp187 (the back-wall). In spite of the small size of the P2 substituent, this is possible because the S2 pocket of SARS-CoV M pro is flexible enough to contract and enclose the P2 moiety tightly. This plasticity is expressed in a conformational change of residue Gln189, both in the main chain and in the side-chain. The main-chain conformational change is connected with a flip of the peptide between Gln189 and Thr190. The c1 torsion angle of the Gln189 side-chain changes from roughly antiperiplanar (ap) to (-)synclinal (-sc) (Fig. 4) . The conformational variability of Gln189 has been noted before, both in Molecular Dynamics simulations 26 and in other crystal structures. 37 As a consequence of these changes, the sidechain oxygen of Gln189 can accept a 2.54-Å hydrogen bond from the main-chain NH of the P2 residue in the 11s complex (see Fig. 4 ). The affinity of 11s for the S2 pocket of HCoV-NL63 M pro is good because of an almost ideal match of size and not requiring conformational changes, which this enzyme would not be able to undergo because of the replacement of the flexible Gln189 by the more rigid Pro. On the other hand, docking of the same compound into the crystal structure of the CVB3 3C pro revealed that the cyclopropylmethyl moiety was probably unable to generate sufficient Free Energy of binding because of the missing lid and the large size of the S2 pocket in the enterovirus 3C pro , thereby explaining the poor inhibitory activity of 11s against these targets. (Table 1) . Experiments with the viral replicons confirmed this trend, although the EC50 value for SARS-CoV (6.8 µM) was surprisingly high (Table 2 ). In Huh7 cells infected with MERS-CoV, this compound exhibited EC50 = 0.1 µM (but 9.8 µM in Vero cells), whereas EC50 was 7.0 µM against SARS-CoV in Vero E6 cells. The compound was largely inactive against EV-A71 in RD cells and inhibited the replication of the two HRV subtypes tested (in HeLa Rh cells) with EC50 values of ~4 µM. The CC50 of 11t in HeLa cells was 65 µM, i.e. the therapeutic index was well above 15 (Table 3) .