Structural Basis for the Inhibition of Coronaviral Main Proteases by a Benzothiazole-Based Inhibitor

Abstract

The ongoing spread of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has caused hundreds of millions of cases and millions of victims worldwide with serious consequences to global health and economies. Although many vaccines protecting against SARS-CoV-2 are currently available, constantly emerging new variants necessitate the development of alternative strategies for prevention and treatment of COVID-19. Inhibitors that target the main protease (M^pro) of SARS-CoV-2, an essential enzyme that promotes viral maturation, represent a key class of antivirals. Here, we showed that a peptidomimetic compound with benzothiazolyl ketone as warhead, YH-53, is an effective inhibitor of SARS-CoV-2, SARS-CoV, and MERS-CoV M^pros. Crystal structures of M^pros from SARS-CoV-2, SARS-CoV, and MERS-CoV bound to the inhibitor YH-53 revealed a unique ligand-binding site, which provides new insights into the mechanism of inhibition of viral replication. A detailed analysis of these crystal structures defined the key molecular determinants required for inhibition and illustrate the binding mode of M^pros from other coronaviruses. In consideration of the important role of M^pro in developing antivirals against coronaviruses, insights derived from this study should add to the design of pan-coronaviral M^pro inhibitors that are safer and more effective.

Keywords: YH-53; coronavirus; inhibitor; main protease; warhead.

Figure 1

Enzymatic inhibition of YH-53 against main proteases of SARS-CoV-2 and MERS-CoV. (a) Inhibition of YH-53 against main protease of SARS-CoV-2. (b) Inhibition of YH-53 against main protease of MERS-CoV. Main proteases were preincubated in the reaction buffer with various concentrations of YH-53 at room temperature for 30 min before reacting with the FRET substrate. The IC₅₀ values were calculated using the GraphPad Prism software.

Figure 2

Crystal structure of SARS-CoV-2 M^pro in complex with YH-53. (a) Overall structure of SARS-CoV-2 M^pro in complex with YH-53. Three domains and two protomers of M^pro are labeled. The substrate binding pocket is situated within the black dotted box. YH-53 is shown as sticks. (b) An enlarged view of the substrate-binding pocket. M^pro is shown as surface and YH-53 is shown as sticks. (c) A C-S covalent bond forms between Cys145 and the benzothiazolyl ketone of YH-53. The 2Fo–Fc density map contoured at 1.0 σ is shown as blue mesh. (d) The detailed interaction in the SARS-CoV-2 M^pro-YH-53 structure is shown with the residues of SARS-CoV-2 involved in inhibitor binding (within 3.3 Å), shown as sticks. W represents the water molecule. Hydrogen-bonding interactions are indicated as black dashed lines. (e) Schematic interaction between YH-53 and M^pro. Hydrogen-bonding interactions are shown as orange dashed lines.

Figure 3

Structural comparison of M^pro-YH-53 complexes. (a) Overall comparison of SARS-CoV-2 M^pro-YH-53 (green), SARS-CoV M^pro-YH-53 (yellow), and MERS-CoV M^pro-YH-53 (orange) structures. YH-53 is shown as sticks. (b) A zoomed-in view of the substrate binding pocket of main protease. YH-53 in SARS-CoV-2 M^pro-YH-53, SARS-CoV M^pro-YH-53, and MERS-CoV M^pro-YH-53 structures are shown as green, yellow, and orange sticks, respectively.

Figure 4

Crystal structures of SARS-CoV and MERS-CoV M^pros in complex with YH-53. (a,b) A C-S covalent bond forms between Cys145 of SARS-CoV M^pro (a) or MERS-CoV M^pro (b) and the benzothiazole group of YH-53. The 2Fo-Fc density map contoured at 1.0 σ is shown as a blue mesh. (c,d) The detailed interaction in the complex structure is shown with the residues of SARS-CoV M^pro (c) or MERS-CoV M^pro (d) involved in inhibitor binding (within 3.3 Å), displayed as sticks. Hydrogen bond interactions are shown as black dashed lines.