Structural Basis of the Main Proteases of Coronavirus Bound to Drug Candidate PF-07321332

Abstract

The high mutation rate of COVID-19 and the prevalence of multiple variants strongly support the need for pharmacological options to complement vaccine strategies. One region that appears highly conserved among different genera of coronaviruses is the substrate-binding site of the main protease (M^pro or 3CL^pro), making it an attractive target for the development of broad-spectrum drugs for multiple coronaviruses. PF-07321332, developed by Pfizer, is the first orally administered inhibitor targeting the main protease of SARS-CoV-2, which also has shown potency against other coronaviruses. Here, we report three crystal structures of the main protease of SARS-CoV-2, SARS-CoV, and Middle East respiratory syndrome (MERS)-CoV bound to the inhibitor PF-07321332. The structures reveal a ligand-binding site that is conserved among SARS-CoV-2, SARS-CoV, and MERS-CoV, providing insights into the mechanism of inhibition of viral replication. The long and narrow cavity in the cleft between domains I and II of the main protease harbors multiple inhibitor-binding sites, where PF-07321332 occupies subsites S1, S2, and S4 and appears more restricted than other inhibitors. A detailed analysis of these structures illuminated key structural determinants essential for inhibition and elucidated the binding mode of action of the main proteases from different coronaviruses. Given the importance of the main protease for the treatment of SARS-CoV-2 infection, insights derived from this study should accelerate the design of safer and more effective antivirals. IMPORTANCE The current pandemic of multiple variants has created an urgent need for effective inhibitors of SARS-CoV-2 to complement vaccine strategies. PF-07321332, developed by Pfizer, is the first orally administered coronavirus-specific main protease inhibitor approved by the FDA. We solved the crystal structures of the main protease of SARS-CoV-2, SARS-CoV, and MERS-CoV that bound to the PF-07321332, suggesting PF-07321332 is a broad-spectrum inhibitor for coronaviruses. Structures of the main protease inhibitor complexes present an opportunity to discover safer and more effective inhibitors for COVID-19.

Keywords: COVID-19; PF-07321332; SARS-CoV-2; coronavirus; crystal structure; main protease.

FIG 1

Enzymatic inhibition of SARS-CoV-2 M^pro. (A) Inhibition of ebselen against SARS-CoV-2 M^pro. (B) Inhibition of PF-07321332 against SARS-CoV-2 M^pro. SARS-CoV-2 M^pro was preincubated in the reaction buffer with various concentrations of PF-07321332 at room temperature for 30 min before reacting with the FRET substrate. Ebselen was used as a control. The IC₅₀ was calculated using GraphPad Prism software.

FIG 2

Crystal structure of SARS-CoV-2 M^pro in complex with PF-07321332. (A) Overall structure of SARS-CoV-2 M^pro in complex with PF-07321332. The three domains and two protomers of M^pro are labeled. The substrate-binding pocket is located within the black dotted box. PF-07321332 is shown as sticks with the carbon atoms in magentas, oxygen atoms in bright red, nitrogen atoms in blue, and fluorine atom in pale cyan. (B) An enlarged view of the substrate-binding pocket. PF-07321332 forms a covalent bond with C145. The substrate-binding subsites (S1′, S1, S2, and S4) are labeled. (C) A C-S covalent bond forms between the Sγ atom of C145 and the nitrile carbon of PF-07321332. The 2Fo-Fc density map contoured at 1.0σ is shown as a blue mesh. (D) The detailed interaction in the complex structure is shown with the residues involved in inhibitor binding (within 3.5 Å) displayed as sticks. W1 and W2 represent the water molecules. Hydrogen bond interactions are shown as black dashed lines. (E) Schematic interaction between PF-07321332 and M^pro. Hydrogen bond interactions are shown as orange dashed lines.

FIG 3

Crystal structures of SARS-CoV and MERS-CoV M^pros in complex with PF-07321332. (A) Structural alignment of CoV M^pros complexed with PF-07321332 with SARS-CoV-2 M^pro-inhibitor complex in lime green, SARS-CoV M^pro-inhibitor complex in yellow, and MERS-CoV M^pro-inhibitor complex in orange. (B) An enlarged view of the substrate-binding pocket. (C and D) A C-S covalent bond forms between C145 of SARS-CoV M^pro (C) or MERS-CoV M^pro (D) and the nitrile group of PF-07321332. The 2Fo-Fc density map contoured at 1.0σ is shown as a blue mesh. (E and F) The detailed interaction in the complex structure is shown with the residues of SARS-CoV M^pro (E) or MERS-CoV M^pro (F) involved in inhibitor binding (within 3.5 Å) displayed as sticks. Hydrogen bond interactions are shown as black dashed lines. (G and H) Schematic interaction between PF-07321332 and SARS-CoV M^pro (G) or MERS-CoV M^pro (H). Hydrogen bonds interactions are shown as orange dashed lines.

FIG 4

Comparison of the binding modes of different inhibitors targeting SARS-CoV-2 M^pro. (A to I) The binding pockets of PF-07321332 (A) (PDB ID 7VLQ), N3 (B) (PDB ID 6LU7), GC376 (C) (PDB ID 7D1M), boceprevir (D) (PDB ID 7BRP), 13b (E) (PDB ID 6Y2F), 11a (F) (PDB ID 6LZE), MPI3 (G) (PDB ID 7JQO), 5h (H) (PDB ID 7JKV), and carmofur (I) (PDB ID 7BUY) bound to SARS-CoV-2 M^pro are shown. M^pros are shown as the gray surface, and the inhibitors are shown as sticks.