The main protease of coronaviruses and the 3C protease of enteroviruses share a similar active-site architecture and a unique requirement for glutamine in the P1 position of the substrate. Because of their unique specificity and essential role in viral polyprotein processing, these proteases are suitable targets for the development of antiviral drugs. In order to obtain near-equipotent, broad-spectrum antivirals against alphacoronaviruses, betacoronaviruses, and enteroviruses, we pursued a structure-based design of peptidomimetic α-ketoamides as inhibitors of main and 3C proteases. Six crystal structures of protease−inhibitor complexes were determined as part of this study. Compounds synthesized were tested against the recombinant proteases as well as in viral replicons and virus-infected cell cultures; most of them were not cell-toxic. Optimization of the P2 substituent of the αketoamides proved crucial for achieving near-equipotency against the three virus genera. The best near-equipotent inhibitors, 11u (P2 = cyclopentylmethyl) and 11r (P2 = cyclohexylmethyl), display low-micromolar EC 50 values against enteroviruses, alphacoronaviruses, and betacoronaviruses in cell cultures. In Huh7 cells, 11r exhibits three-digit picomolar activity against the Middle East Respiratory Syndrome coronavirus.

A novel series of peptidomimetic aldehydes was designed and synthesized to target 3C protease (3Cpro) of enterovirus 71 (EV71). Most of the compounds exhibited high antiviral activity, and among them, compound 18p demonstrated potent enzyme inhibitory activity and broad-spectrum antiviral activity on a panel of enteroviruses and rhinoviruses. The crystal structure of EV71 3Cpro in complex with 18p determined at a resolution of 1.2 Å revealed that 18p covalently linked to the catalytic Cys147 with an aldehyde group. In addition, these compounds also exhibited good inhibitory activity against the 3CLpro and the replication of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), especially compound 18p (IC50 = 0.034 μM, EC50 = 0.29 μM). According to our previous work, these compounds have no reasons for concern regarding acute toxicity. Compared with AG7088, compound 18p also exhibited good pharmacokinetic properties and more potent anticoronavirus activity, making it an excellent lead for further development.

The main protease of coronaviruses and the 3C protease of enteroviruses share a similar active-site architecture and a unique requirement for glutamine in the P1 position of the substrate. Because of their unique specificity and essential role in viral polyprotein processing, these proteases are suitable targets for the development of antiviral drugs. In order to obtain nearequipotent, broad-spectrum antivirals against alphacoronaviruses, betacoronaviruses, and enteroviruses, we pursued structure-based design of peptidomimetic a-ketoamides as inhibitors of main and 3C proteases. Six crystal structures of protease:inhibitor complexes were determined as part of this study. Compounds synthesized were tested against the recombinant proteases as well as in viral replicons and virus-infected cell cultures; most of them were not cell-toxic. Optimization of the P2 substituent of the aketoamides proved crucial for achieving near-equipotency against the three virus genera. The best near-equipotent inhibitors, 11u (P2 = cyclopentylmethyl) and 11r (P2 = cyclohexylmethyl), display low-micromolar EC50 values against enteroviruses, alphacoronaviruses, and betacoronaviruses in cell cultures. In Huh7 cells, 11r exhibits three-digit picomolar activity against Middle East Respiratory Syndrome coronavirus.We chose the chemical class of peptidomimetic a-ketoamides to assess the feasibility of achieving antiviral drugs targeting coronaviruses and enteroviruses with near-equipotency. Here we describe the structure-based design, synthesis, and evaluation of inhibitory activity of a series of compounds with broad-spectrum activities afforded by studying the structure-activity relationships mainly with respect to the P2 position of the peptidomimetics. One of the compounds designed and synthesized exhibits excellent activity against MERS-CoV. RESULTS Structure-based design of a-ketoamidesOur efforts to design novel a-ketoamides as broad-spectrum inhibitors of coronavirus M pro s and enterovirus 3C pro s started with a detailed analysis of the following crystal structures of unliganded target enzymes: SARS-CoV M pro (ref. 25-27; PDB entries 1UJ1, 2BX3, 2BX4); bat coronavirus HKU4 M pro as a surrogate for the closely related MERS-CoV protease (our unpublished work (Ma, Xiao et al.; PDB entry 2YNA; see also ref. 27); HCoV-229E M pro (ref. 27,28; PDB entry: 1P9S); Coxsackievirus B3 3C pro (our unpublished work; Tan et al., PDB entry 3ZYD); enterovirus D68 3C pro (ref. 29; PDB entry: 3ZV8); and enterovirus A71 3C pro (ref. 30; PDB entry: 3SJK).During the course of the present study, we determined crystal structures of a number of lead a-ketoamide compounds in complex with SARS-CoV M pro , HCoV-NL63 M pro , and CVB3 3C pro , in support of the design of improvements in the next round of lead optimization. Notably, unexpected differences between alpha-and betacoronavirus M pro were found in this study. The structural foundation of these was elucidated in detail in a subproject involving the M pro of HCoV NL63; because of its volume, this work wil...