X-ray screening identifies active site and allosteric inhibitors of SARS-CoV-2 main protease

Abstract

The coronavirus disease (COVID-19) caused by SARS-CoV-2 is creating tremendous human suffering. To date, no effective drug is available to directly treat the disease. In a search for a drug against COVID-19, we have performed a high-throughput x-ray crystallographic screen of two repurposing drug libraries against the SARS-CoV-2 main protease (M^pro), which is essential for viral replication. In contrast to commonly applied x-ray fragment screening experiments with molecules of low complexity, our screen tested already-approved drugs and drugs in clinical trials. From the three-dimensional protein structures, we identified 37 compounds that bind to M^pro In subsequent cell-based viral reduction assays, one peptidomimetic and six nonpeptidic compounds showed antiviral activity at nontoxic concentrations. We identified two allosteric binding sites representing attractive targets for drug development against SARS-CoV-2.

Fig. 1. The x-ray screening of drug-repurposing libraries reveals compound binding sites distributed across the complete M^pro surface.

(A) Schematic drawing of M^pro dimer structure. Protomer A is shown in white, and protomer B is in red. For clarity, the 29 binding compounds (yellow sticks) are only depicted on one of the two protomers. Catalytic residues His⁴¹ (H41) and Cys¹⁴⁵(C145), the active site, and two allosteric drug binding sites are highlighted. (B) Close-up view of the active site with peptide substrate bound (blue sticks), modeled after SARS-CoV M^pro (PDB 2Q6G). The scissile bond is indicated in yellow and with the green arrowhead. Substrate binding pockets S1ʹ, S1, S2, and S4 are indicated by colored regions.

Fig. 2. Effect of selected compounds on SARS-CoV-2 replication in Vero E6 cells.

The vRNA yield (solid circles), viral titers (half-solid circles), and cell viability (empty circles) were determined by reverse transcription–quantitative polymerase chain reaction, immunofocus assays, and the CCK-8 method, respectively. EC₅₀ for the viral titer reduction is shown. Individual data points represent means ± SD from three independent replicates in one experiment.

Fig. 3. Covalent and noncovalent binders in the active site of M^pro.

Bound compounds are depicted as colored sticks, and the surface of M^pro is shown in gray with selected interacting residues shown as sticks. Substrate binding pockets are colored as in Fig. 1. Hydrogen bonds are depicted by dashed lines. (A) Tolperisone. (B) HEAT. (C) Isofloxythepin. (D) Triglycidyl isocyanurate. (E) Calpeptin. (F) MUT056399.

Fig. 4. Screening hits at allosteric sites of M^pro.

(A) Close-up view of the binding site in the dimerization domain (protomer A, gray cartoon representation), close to the active site of the second protomer (protomer B, surface representation) in the native dimer. Residues forming the hydrophobic pocket are indicated. Pelitinib (dark green) binds to the C-terminal α-helix at Ser³⁰¹ and pushes against Asn¹⁴² and the β-turn of the pocket S1 of protomer B (residues marked with an asterisk). The inset shows the conformational change of Gln²⁵⁶ (gray sticks) compared with the M^pro apo structure (white sticks). (B) RS-102895 (purple), ifenprodil (cyan), PD-168568 (orange), and tofogliflozin (blue) occupy the same binding pocket as pelitinib. (C) AT7519 occupies a deep cleft between the catalytic and dimerization domain of M^pro. (D) Conformational changes in the AT7519-bound M^pro structure (gray) compared with those in the apo structure (white).