Alpha-ketoamides as broad-spectrum inhibitors of coronavirus and enterovirus replication

Abstract

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PDB entries 1UJ1, 2BX3, 2BX4); bat coronavirus HKU4 M pro as a surrogate for the closely related MERS-CoV protease (our unpublished work (Ma, Xiao et al.; PDB entry 2YNA; see also ref. 27); HCoV-229E M pro (ref. 27,28; PDB entry: 1P9S); Coxsackievirus B3 3C pro (our unpublished work; Tan et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...

Drug Development and Medicinal Chemistry Efforts toward SARS‐Coronavirus and Covid‐19 Therapeutics

“…Starting from the information provided from complex of lead compound 13 (Figures and , IC 50 SARS‐CoV 3CLpro=1.95 μM) with the Mpro of HCoV NL63 (an α‐CoV protease) and SARS‐CoV (β‐CoV) as well as the 3Cpro of Coxsackievirus B3 (enterovirus proteases), a systematic study was embarked in order to modify size and flexibility of P2 ligands, in order to obtain compounds able to adapt to target proteases’ S2 pockets. The compounds which better performed were derivatives 14 and 15 , bearing a P2 cyclohexylmethyl and cyclopentylmethyl moiety, respectively …”

“…Inhibitor 14 showed potent antiviral activity against MERS‐CoV when tested in Huh7 cells (EC 50 =400 pM), while the activity strongly lowered when Vero cells were used as the host system (EC 50 =5 μM). The authors speculated that, due to the strong similarity between SARS‐CoV and SARS‐CoV‐2, the new compound is likely to inhibit this new virus as well . Based on inhibitor 14 , Zhang and co‐workers very recently developed optimized compounds as SARS‐CoV‐2 3CLpro inhibitors …”

AI-aided design of novel targeted covalent inhibitors against SARS-CoV-2

“…Although compound #46 has the best covalent docking score, it has the alerting group aldehyde. Considering there is still much space for compound #46 to fill in the S1' subsite and α-ketoamides may be good to fit the oxyanion hole ( Figure 4A) of 3CL pro , 3 we replaced the aldehyde by formamide and also replaced the 1,4 Michael acceptors by alpha-ketoamides. Thus, we optimized compound #46 to compound 46-14-1 (Figure 4 A and B).…”

“…Luckily, there are serval reported privileged core structures targeting SARS M pro . 3,52 Here, we chose 4-aminopent-2-enal and 3-amino-2-oxobutanal as the starting cores as Figure 8 displays, because both cores have been validated to generate covalent bonds with the Cys145 of SARS or SARS-CoV-2 3CL pro .…”

“…To face the considerable threat of SARS-CoV-2, it is urgent to develop new inhibitors or drugs against this deadly virus. Unfortunately, since the outbreak of severe acute respiratory syndrome (SARS) eighteen years ago, there has been no approved treatment against the SARS coronavirus (SARS-CoV), 3 which is similar to SARS-CoV-2. Repurposing potential drugs such as lopinavir and ritonavir also failed to SARS-CoV-2 injected patients.…”