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 <i><b>5c</b></i> and <i><b>11c</b></i> inhibited SARS-CoV-2 replication with EC₅₀ values of 12 and 11 nM, respectively. Furthermore, the corresponding aldehyde bisulfite adducts <i><b>5d</b></i> and <i><b>11d</b></i> were equipotent with EC₅₀ values of 13 and 12 nM, respectively. The safety index (SI) values for compounds <i><b>5c</b></i> / <i><b>11c</b></i> and <i><b>5d</b></i> / <i><b>11d</b></i> ranged between 7692 and 9090. Importantly, aldehydes <i><b>5c</b></i> / <i><b>11c</b></i> and bisulfite adducts <i><b>5d</b></i> / <i><b>11d</b></i> potently inhibited MERS-CoV 3CL^pro with IC₅₀ values of 80 and 120 nM, and 70 and 70 nM, respectively. Likewise, compounds <i><b>5c</b></i> / <i><b>11c</b></i> and <i><b>5d</b></i> / <i><b>11d</b></i> inhibited SARS-CoV-1 with IC₅₀ 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.