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Structure and inhibition of the SARS-CoV-2 main protease reveal strategy for developing dual inhibitors against Mpro and cathepsin L

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Science Advances  09 Dec 2020:
Vol. 6, no. 50, eabe0751
DOI: 10.1126/sciadv.abe0751
  • Fig. 1 X-ray crystal structures of SARS-CoV-2 Mpro in complex with calpain inhibitors II (PDB: 6XA4) and XII (PDB: 6XFN).

    Unbiased Fo-Fc electron density map, shown in gray, is contoured at 2 σ. Hydrogen bonds are shown as red dashed lines. SARS-CoV-2 Mpro in complex with (A) calpain inhibitor II (orange) and (B) calpain inhibitor XII (cyan). (C) Binding mode and interactions of a previously reported α-ketoamide inhibitor 13b (purple, PDB ID: 6Y2F) with SARS-CoV-2 Mpro. (D) Close-up comparison of calpain inhibitor XII (blue) and 13b (purple) in the S1 and S1′ sites. The P1 pyrrolidinone ring and P1′ benzene of 13b occupy the S1 and S1′ sites, respectively. Conversely, the P1 norvaline and P1′ pyridine of calpain inhibitor XII adopt the S1′ and S1 sites, respectively. Accompanying the noncanonical positioning of the P1 and P1′ moieties, a stereochemical inversion of the thiohemiketal adduct with Cys145 is observed for calpain inhibitor XII. Like all other known α-ketoamide inhibitors, 13b adopts a (S) configuration. However, calpain inhibitor XII forms a covalent adduct in the (R) configuration.

  • Fig. 2 Biochemical and biophysical characterizations of calpain inhibitors II and XII and their analogs as SARS-CoV-2 Mpro inhibitors.

    (A) Chemical structures of calpain inhibitors II, I, and XII and UAWJ257. (B to E) IC50 plots from in vitro FRET-based enzymatic assay against SARS-CoV-2 Mpro of calpain inhibitor II (B), calpain inhibitor I (C), calpain inhibitor XII (D), and UAWJ257 (E). (F) Thermal shift binding assay of calpain inhibitors and their analogs with 3 μM SARS-CoV-2 Mpro protein. Forty micromolar compounds were preincubated with Mpro protein for 30 min at 30°C. Tm was calculated as described in Materials and Methods. (G and H) IC50 plots from in vitro FRET-based enzymatic assay against human cathepsin L of calpain inhibitor II (G) and calpain inhibitor XII (H). (I) Counter screening of calpain inhibitors II, I, and XII and UAWJ257 against SARS-CoV-2 PLpro in the FRET-based enzymatic assay. GRL0617 was added as a positive control. The calculated enzymatic activity with each compound was normalized to dimethyl sulfoxide (DMSO) control. The results are average ± SD of two or more repeats.

  • Fig. 3 Pharmacological characterization of the mechanism of action of GC-376 analogs UAWJ246, UAWJ247, and UAWJ248 in inhibiting SARS-CoV-2 Mpro.

    (A) Chemical structures of GC-376, UAWJ246, UAWJ247, and UAWJ248. (B to E) Morrison plot of the proteolytic reaction progression curves of SARS-CoV-2 Mpro in the presence or the absence of compounds. Original proteolytic reaction progression curves are in fig. S4. Detailed methods are described in Materials and Methods. UAWJ246 (B), UAWJ247 (C), UAWJ248 (D), and GC-376 (E). Kobs, observed pseudo first order rate constant. (F) Counter screening of GC-376, UAWJ246, UAWJ247, and UAWJ248 against SARS-CoV-2 PLpro in the FRET-based enzymatic assay. (G) Thermal shift binding assay of GC-376 analogs with different SARS-CoV-2 Mpro constructs. (H to K) Binding of inhibitors to SARS-CoV-2 Mpro using native mass spectrometry. Native mass spectra with the inset deconvolved spectra revealing ligand binding with (H) 10 μM GC-376 added, (I) 10 μM UAWJ246, (J) 10 μM UAWJ247 added, and (K) 10 μM UAWJ248 with 4 mM DTT added. The peaks are annotated with the blue circle as the dimer, green down triangle as the dimer with one ligand bound, and the purple up triangle as the dimer with two ligands bound. VO, initial velocity.

  • Fig. 4 SARS-CoV-2 Mpro in complex with GC-376 analogs.

    Unbiased Fo-Fc electron density map, shown in gray, is contoured at 2 σ. Hydrogen bonds are shown as red dashed lines. Solved as a dimer in the P21 spacegroup, two different conformations of the Cbz group of UAWJ246 were observed in the (A) protomer A and (B) protomer B. (C) The complex structure of UAWJ247, revealing that the P2 position can accommodate a Phe side chain. (D) Comparison of the binding poses of UAWJ247 (dark green/salmon) and GC-376 (light green/gray, PDB ID: 7BRR). (E) The complex structure of UAWJ248, solved as a dimer in the P1 space-group. Protomer A is shown here, and the inhibitor binding pose is identical in protomer B (fig. S8). (F) Comparison of the binding poses of UAWJ248 (purple) and UAWJ246 (yellow) in protomer A.

  • Fig. 5 MD simulations of SARS-CoV-2 Mpro with calpain inhibitors II and XII and UAWJ248.

    In (A), (C), (E), and (G), hydrogen bonding interactions bar is depicted in light blue, van der Waals in orange, and water bridges in blue. Interactions are plotted from 100-ns MD simulations for the complexes of the covalently bound calpain inhibitor II, calpain inhibitor XII, UAWJ248 (pose 1: first protomer), and UAWJ248 (pose 2: second protomer) in the active site of SARS-CoV-2 Mpro. They are considered important when the frequency bar is ≥0.2. In (B), (D), (F), and (H), the last snapshots of the abovementioned 100-ns MD-simulated complexes overlaid with experimental structures with PDB IDs 6XA4, 6XFN, 6XBI, respectively, are shown. In (I) to (L), the RMSD plots of Cα carbons (blue diagram, left axis) and of ligand (red diagram, right axis) of the abovementioned 100-ns MD-simulated complexes are shown.

  • Fig. 6 Antiviral activity of GC-376 analogs.

    (A to D) Antiviral activity of GC-376 analogs against SARS-CoV-2 in the immunofluorescence assay. (A) GC-376; (B) UAWJ246; (C) UAWJ247; (D) UAWJ248. Vero E6 cells in a 96-well plate were infected with SARS-CoV-2 (USA-WA1/2020 isolate) at an MOI of 0.05 in the presence of the indicated concentrations of the tested compounds. At 48 hours post infection (hpi), the cells were fixed and stained with a rabbit monoclonal antibody against the SARS-CoV-2 NP and a secondary antibody conjugated with Alexa 488 (green). The nuclei were counterstained with Hoechst dye (blue). For each well, fluorescence images of approximately 10,000 cells were acquired and shown. The images are representatives of three repeats. (E to H) Antiviral activity of GC-376 analogs against SARS-CoV-2 in the plaque assay. (E) GC-376; (F) UAWJ246; (G) UAWJ247; (H) UAWJ248. Vero E6 cells in six-well plates were infected with approximately 40 plaque-forming units per well of SARS-CoV-2 (USA-WA1/2020 isolate). After 1 hour, the inoculum was removed, and the cells were overlaid with medium containing the indicated concentrations of the tested compounds and 1.2% Avicel RC-591. At 3 days post infection, the overlay was removed, and the cells were stained with 0.2% crystal violet. The images are representatives of two repeats. Data fitting of the antiviral activity of GC-376 analogs against SARS-CoV-2 in the immunofluorescence assay (I) and the plaque assay (J).

Supplementary Materials

  • Supplementary Materials

    Structure and inhibition of the SARS-CoV-2 main protease reveals strategy for developing dual inhibitors against Mpro and cathepsin L

    Michael Dominic Sacco, Chunlong Ma, Panagiotis Lagarias, Ang Gao, Julia Alma Townsend, Xiangzhi Meng, Peter Dube, Xiujun Zhang, Yanmei Hu, Naoya Kitamura, Brett Hurst, Bart Tarbet, Michael Thomas Marty, Antonios Kolocouris, Yan Xiang, Yu Chen, Jun Wang

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