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

However, enteroviruses are very different from coronaviruses. While both of them have a single-stranded RNA genome of positive polarity, that of enteroviruses is very small (just 7 -9 kb) whereas coronaviruses feature the largest RNA genome known to date (27 -34 kb) . Enteroviruses are small, naked particles, whereas coronaviruses are much larger and enveloped. Nevertheless, a related feature shared by these two groups of viruses is their type of major protease, 21 which in the enteroviruses is encoded by the 3C region of the genome (hence the protease is designated 3C pro ). In coronaviruses, non-structural protein 5 (Nsp5) is the main protease (M pro ). Similar to the enteroviral 3C pro , it is a cysteine protease in the vast majority of cases and has therefore also been called "3C-like protease" (3CL pro ). The first crystal structure of a CoV M pro or 3CL pro (ref. 22) revealed that two of the three domains of the enzyme together resemble the chymotrypsin-like fold of the enteroviral 3C pro , but there is an additional a-helical domain that is involved in the dimerization of the protease (Fig. 1A) . This dimerization is essential for the catalytic activity of the CoV M pro , whereas the enteroviral 3C pro (Fig. 1B) functions as a monomer. Further, the enteroviral 3C pro features a classical Cys...His...Glu/Asp catalytic triad, whereas the CoV M pro only has a Cys...His dyad. 22 Yet, there are a number of common features shared between the two types of proteases, in particular their almost absolute requirement for Gln in the P1 position of the substrate and space for only small amino-acid residues such as Gly, Ala, or Ser in the P1' position, encouraging us to explore the coronaviral M pro and the enteroviral 3C pro as a common target for the design of broad-spectrum antiviral compounds. The fact that there is no known human protease with a specificity for Gln at the cleavage site of the substrate increases the attractivity of this viral target, as there is hope that the inhibitors to be developed will not show toxicity versus the host cell. Indeed, neither the enterovirus 3C pro inhibitor rupintrivir, which was developed as a treatment of the common cold caused by HRV, nor the peptide aldehyde inhibitor of the coronavirus M pro that was recently demonstrated to lead to complete recovery of cats from the normally fatal infection with Feline Infectious Peritonitis Virus (FIPV), showed any toxic effects on humans or cats, respectively. 23, 24 Figure 1: Crystal structures of SARS-CoV main protease (M pro , ref. 26 ; PDB entry 2BX4) and Coxsackivirus B3 3C protease (3C pro ; Tan et al., unpublished; PDB entry 3ZYD). Catalytic residues are indicated by spheres (yellow, Cys; blue, His; red: Glu). A. The coronavirus M pro is a homodimer, with each monomer comprising three domains. B. The structure of the monomeric CVB3 3C pro resembles the Nterminal two domains of the SARS-CoV M pro . Structure is on the same scale as image A. C. Superimpostion of residues from the two structures involved in ligand binding. Superimposition was carried out by aligning the catalytic Cys-His pair of each protease. Residues of the SARS-CoV M pro are shown with carbon atoms in cyan, CVB3 3C pro residues have orange carbons and are labeled with an asterisk (*).