Malleability of the SARS-CoV-2 3CL Mpro Active-Site Cavity Facilitates Binding of Clinical Antivirals

Abstract

The COVID-19 pandemic caused by SARS-CoV-2 requires rapid development of specific therapeutics and vaccines. The main protease of SARS-CoV-2, 3CL M^pro, is an established drug target for the design of inhibitors to stop the virus replication. Repurposing existing clinical drugs can offer a faster route to treatments. Here, we report on the binding mode and inhibition properties of several inhibitors using room temperature X-ray crystallography and in vitro enzyme kinetics. The enzyme active-site cavity reveals a high degree of malleability, allowing aldehyde leupeptin and hepatitis C clinical protease inhibitors (telaprevir, narlaprevir, and boceprevir) to bind and inhibit SARS-CoV-2 3CL M^pro. Narlaprevir, boceprevir, and telaprevir are low-micromolar inhibitors, whereas the binding affinity of leupeptin is substantially weaker. Repurposing hepatitis C clinical drugs as COVID-19 treatments may be a useful option to pursue. The observed malleability of the enzyme active-site cavity should be considered for the successful design of specific protease inhibitors.

Keywords: 3CL M(pro); 3CL main protease; SARS-CoV-2; drug design; enzyme kinetics; hepatitis C clinical drugs; protease inhibitor; repurposing clinical drugs; room temperature X-ray crystallography.

Graphical abstract

Scheme 1

Chemical Diagrams of the Inhibitors Used in This Study The reactive warheads to which the catalytic Cys145 covalently binds are colored red.

Figure 1

The Structure of the 3CL M^pro Homodimer from SARS-CoV-2 Protomers are drawn in cartoon representation showing leupeptin (in ball-and-stick) covalently bound in both active-site cavities (PDB: 6XCH). One of the protomers is shown with a transparent surface. The regions of the active-site cavity that demonstrate significant conformational changes after inhibitor binding are colored green and labeled as P2 helix, P4 β-hairpin flap, and P5 loop.

Figure 2

Binding Modes of Studied Inhibitors Leupeptin (A, PDB: 6XCH), telaprevir (B, PDB: 6XQS), narlaprevir (C, PDB: 6XQT), and boceprevir (D, PDB: 6XQU) are shown in ball-and-stick representation. The 2F_O-F_C electron density maps for the inhibitors are shown as violet meshes and are all contoured at 1.4 σ.

Figure 3

Hydrogen Bonding Interactions of the Studied Inhibitors (Blue Dashed Lines) with SARS-CoV-2 3CL M^pro Observed in the Room Temperature X-Ray Structures (A) Complex with leupeptin, (B) complex with telaprevir, (C) complex with narlaprevir, and (D) complex with boceprevir. The insets show the stereochemistry of the covalent conjugates with Cys145. Distances are in Å.

Figure 4

Superposition of the Four Inhibitor Complexes onto the Ligand-Free SARS-CoV-2 3CL M^pro (Black, PDB: 6WQF) in Cartoon Representation The complex with leupeptin is shown in yellow, the complex with telaprevir in dark pink, the complex with narlaprevir in blue, and the complex with boceprevir in gray. Narlaprevir and telaprevir are represented in ball-and-stick, whereas the other two inhibitors are omitted for clarity. The conformational changes for the P2 helix, the tip of the P4 β-hairpin flap, and the P5 loop due to inhibitor binding are shown as red arrows and maximal shifts are indicated with values colored red. The expansion of the active-site cavity is represented by black arrows and the maximal shifts are indicated with values colored black.