Broad-Spectrum Cyclopropane-Based Inhibitors of Coronavirus 3C-like Proteases: Biochemical, Structural, and Virological Studies

Abstract

The advent of SARS-CoV-2, the causative agent of COVID-19, and its worldwide impact on global health, have provided the impetus for the development of effective countermeasures that can be deployed against the virus, including vaccines, monoclonal antibodies, and direct-acting antivirals (DAAs). Despite these efforts, the current paucity of DAAs has created an urgent need for the creation of an enhanced and diversified portfolio of broadly acting agents with different mechanisms of action that can effectively abrogate viral infection. SARS-CoV-2 3C-like protease (3CL pro ), an enzyme essential for viral replication, is a validated target for the discovery of SARS-CoV-2 therapeutics. In this report, we describe the structure-guided utilization of the cyclopropane moiety in the design of highly potent inhibitors of SARS-CoV-2 3CL pro , SARS-CoV-1 3CL pro , and MERS-CoV 3CL pro . High-resolution cocrystal structures were used to identify the structural determinants associated with the binding of the inhibitors to the active site of the enzyme and unravel the mechanism of action. Aldehydes 5c and 11c inhibited SARS-CoV-2 replication with EC 50 values of 12 and 11 nM, respectively. Furthermore, the corresponding aldehyde bisulfite adducts 5d and 11d were equipotent with EC 50 values of 13 and 12 nM, respectively. The safety index (SI) values for compounds 5c/11c and 5d/11d ranged between 7692 and 9090. Importantly, aldehydes 5c/11c and bisulfite adducts 5d/11d potently inhibited MERS-CoV 3CL pro with IC 50 values of 80 and 120 nM, and 70 and 70 nM, respectively. Likewise, compounds 5c/11c and 5d/11d inhibited SARS-CoV-1 with IC 50 values of 960 and 350 nM and 790 and 240 nM, respectively. Taken together, these studies suggest that the inhibitors described herein have low cytotoxicity and high potency and are promising candidates for further development as broad-spectrum direct-acting antivirals against highly pathogenic coronaviruses.

Structure-guided design of direct-acting antivirals that exploit the gem-dimethyl effect and potently inhibit 3CL proteases of severe acute respiratory syndrome Coronavirus-2 (SARS-CoV-2) and middle east respiratory syndrome coronavirus (MERS-CoV)

“…The rest of the alcohols ( 10–16 ) were subsequently treated with disuccinimidyl carbonate [ 42 ] and the activated mixed esters were reacted with a Leu-Gln surrogate amino alcohol to yield the dipeptidyl alcohols ( 10b-16b ), which were then oxidized to the corresponding aldehydes ( 10c-16c ) with Dess-Martin periodinane reagent ( Scheme 2 ). Compounds 1-17c and 1e were transformed into the bisulfite salts ( 1-17d and 1f ) by treatment with sodium bisulfite as described previously ( Scheme 1 , Scheme 2 ) [ 28 , 43 ]. α-Ketoamide 1e was prepared by reacting aldehyde 1c with cyclopropyl isonitrile followed by oxidation with Dess-Martin periodinane reagent, while nitrile inhibitor 1g was obtained by converting dipeptidyl ester 1b to the primary amide, followed by dehydration of the amide ( Scheme 1 ) [ 24 ].…”

“…Importantly, the substrate binding cleft is fairly conserved among coronaviruses, suggesting that the design of broad-spectrum DAAs may be feasible [ 23 ]. The protease is a validated target for the development of SARS-CoV-2 therapeutics [ [24] , [25] , [26] , [27] ] and has been the subject of intense investigations by us [ 28 ] and others [ 29 ]. We report herein the structure-guided design, synthesis, structure-activity relationships, and biochemical, structural, and antiviral activity studies of potent, broad-spectrum inhibitors of SARS-CoV-2 3CL pro that exploit the conformational control and other beneficent attributes of the gem -dimethyl moiety to access new chemical space and enhance enzyme-inhibitor interactions in the S3-S4 active site domains of the protease.…”

On the origins of SARS-CoV-2 main protease inhibitors

“…Many analogues of GC376 (53) were reported, 185,260,261 such as compounds resulting from a "fluorine walk" on the benzyl moiety and/or its replacement by a substituted cyclohexyl, a bulkier adamentyl and even more elaborated substituents. 195,199,201,262,263 As an illustration of the "leeway" on this position, the difluorocyclohexyl-bearing analogue 57 was found to be effective against a mouse model of MERS coronavirus infection. 188 Moreover, a deuterated derivative of 50 was evaluated on a mouse model of SARS-CoV-2 infection but this analogue showed no real advantage.…”

Potent 3CLpro inhibitors effective against SARS-CoV-2 and MERS-CoV in animal models by therapeutic treatment

“…Some inhibitors of SARS-CoV-2 3CLpro, including those from our lab ( 28 , 35 – 38 ), have dual inhibitory effects against cathepsins ( 28 , 33 , 39 , 40 ). In this report, we further examined selected compounds ( 5c/d and 11c/d ) from our previous report ( 41 ) for the potential dual roles of the inhibitors against 3CLpro and cathepsins in virus replication using lentivirus-based pseudotyped viruses expressing coronavirus S ( 42 ). We then examined the in vivo efficacy of compounds 5d and 11d using animal models of SARS-CoV-2 and MERS-CoV using BALB/c and human dipeptidyl peptidase 4 (hDDP4) knock-in (KI) mice, respectively.…”