SARS-CoV-2 main protease inhibition through dimerization promotion by peptidomimetic inhibitors and disruption by ebselen
- Department of Life Sciences, Food Safety and Technology Research Centre, and Research Centre for Chinese Medicine Innovation, The Hong Kong Polytechnic University, China
- State Key Laboratory of Chinese Medicine and Molecular Pharmacology (Incubation), and Shenzhen Key Laboratory of Food Biological Safety Control, Hong Kong Polytechnic University Shenzhen Research Institute, China
Figures
Figure 1 with 4 supplements
The monomer-dimer equilibrium modulation of main protease (Mpro) induced by inhibitor binding.
(a) Crystal structure of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Mpro dimer, monomer, and active site pockets of SARS-CoV-2 Mpro with subsites and two catalytic dyad residues (H41 and C145) highlighted (PDB entry 7ALI). The upper structure represents the dimer, and the lower structure represents the monomer, respectively. Native mass spectra of (b) 2.5 μM unbound Mpro, (c) Mpro-PF-07321332 complex (2.5 μM Mpro with 3.75 μM PF-07321332), and (d) Mpro-ebselen complex (2.5 μM Mpro with 7.5 μM ebselen).
Figure 1—figure supplement 1
Surface views showing the key subpockets of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) main protease (Mpro) with: (a) PF-07321332 (PDB entry 8DZ2); (b) PF-00835231 (PDB entry 6XHM); (c) GC376 (PDB entry 7D1M); (d) boceprevir (PDB entry 7C6S); (e) carmofur (PDB entry 7BUY); (f) ebselen (PDB entry 7BFB; the orange part represents the Se atom); and (g) MR6-31-2 (PDB entry 7BAL; the orange sphere represents the Se atom).
The ligand structures of ebselen and MR6-31-2 are only partially resolved in the corresponding crystal structures.
Figure 1—figure supplement 2
Alignments of the free main protease (Mpro) (PDB entry 7ALI, gray) and various inhibitor-bound Mpro structures.
(a) PF-07321332-bound Mpro (PDB entry 8DZ2). (b) PF-00835231-bound Mpro (PDB entry 6XHM). (c) GC376-bound Mpro (PDB entry 7D1M). (d) Boceprevir-bound Mpro (PDB entry 7C6S). (e) Carmofur-bound Mpro (PDB entry 7BUY). (f) Ebselen-bound Mpro (PDB entry 7BFB). (g) MR6-31-2-bound Mpro (PDB entry 7BAL, the orange sphere is the Se atom). (h) AT7915-bound Mpro (PDB entry 7AGA) and (i) pelitinib-bound Mpro (PDB entry 7AXM). The gray structure is an unbound state of Mpro dimer, and the other colored structures are inhibitor-Mpro complexes. The residues H41 and C145, and various inhibitors, are highlighted in the enlarged figures.
Figure 1—figure supplement 3
Native mass spectra of main protease (Mpro) (2.5 µM) in the presence of (a) PF-00835231 (7.5 µM); (b) GC376 (7.5 µM); (c) boceprevir (7.5 µM); (d) carmofur (7.5 µM); (e) MR6-31-2 (7.5 µM); (f) AT7519 (37.5 µM);and (g) pelitinib (37.5 µM).
Figure 1—figure supplement 4
Hydrogen bond interactions between key residues of main protease (Mpro) dimerization.
(a) Residue S1 of one monomer with E166 of the other monomer from crystallography (Mpro alone, PDB ID 7ALI). (b) Residue R4 of one monomer with E290 of the other monomer from crystallography (Mpro alone, PDB ID 7ALI). (c) Residue S1 of one monomer with E166 of the other monomer from crystallography (Mpro with PF-07321332, PDB ID 8DZ2). (d) Residue R4 of one monomer with E290 of the other monomer from crystallography (Mpro with PF-07321332, PDB ID 8DZ2).
Figure 2 with 2 supplements
Main protease (Mpro) subunit exchange dynamics.
(a) The strategy for Mpro subunit exchange. (b) Monitoring of unbound Mpro subunit exchange by native mass spectrometry. Mpro subunit exchange is affected by (c) PF-07321332, (d) PF-00835231, (e) GC376, (f) boceprevir, and (g) ebselen. The red dashed lines indicate the heterodimers.
Figure 2—figure supplement 1
Characterization of main protease (Mpro) and 13C-labeled Mpro.
(a) Native mass spectrum of Mpro (2.5 μM). (b) Native mass spectrometry of 13C-labeled Mpro (2.5 μM).
Figure 2—figure supplement 2
Main protease (Mpro) subunit exchange was modulated by inhibitor binding.
Spectra were shown at the 15+ charge state of Mpro with subunit exchange of 3 min and 30 min, respectively. (a) 2.5 µM Mpro with 2.5 µM 13C-labeled Mpro, along with additional inhibitors: (b) carmofur (7.5 µM); (c) MR6-31-2 (7.5 µM); (d) AT7519 (7.5 µM); (e) pelitinib (7.5 µM). The red dashed lines indicate heterodimers.
Figure 3 with 5 supplements
Conformational dynamics analysis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) main protease (Mpro) with PF-07321332.
(a) The deuterium uptake plot for all identified peptides from Mpro at 1, 10, and 60 min. (b) Deuterium uptake of PF-07321332-bound Mpro by global hydrogen/deuterium exchange mass spectrometry (HDX-MS). (c) The heat map of Mpro upon binding with PF-07321332 at 10 min was overlaid onto the crystal model of Mpro (PDB entry 7ALI). (d) The uptake line plots in the PF-07321332-bound Mpro at 10 min HDX labeling. Residues 1–17 (***), 35–47 (***), 104–111 (**), 116–125 (***), 135–150 (***), 160–177 (***), 182–199 (***), and 296–306 (***). ** for p<0.01, and *** for p<0.001.
Figure 3—figure supplement 1
Coverage map of main protease (Mpro) showing 94.4% sequence coverage.
Figure 3—figure supplement 2
The deuterium uptake difference plots of main protease (Mpro) upon binding with (a) PF-00835231; (b) GC376; (c) boceprevir; (d) carmofur; and (e) MR6-31-2, respectively, at 10 min.
Figure 3—figure supplement 3
Hydrogen/deuterium exchange (HDX) profile of main protease (Mpro) with peptidomimetic inhibitors: (a) PF-07321332; (b) PF-00835231; (c) GC376; and (d) boceprevir.
Deuterium uptake differences are presented in a Woods plot, which provides a breakdown of the peptide’s ensemble for 1-, 10-, and 60 min time points. It displays peptide length, global coverage, and deuterium uptake. A 99% confidence limit was applied to the dataset to identify peptides with significant deuterium uptake. Deprotected, protected, and non-significantly different peptides are in red, blue, and gray, respectively. The presented plots have been created in Deuteros 2.0.
Figure 3—figure supplement 4
Hydrogen/deuterium exchange (HDX) profiles of main protease (Mpro) with inhibitors: (a) carmofur; (b) ebselen; (c) MR6-31-2; (d) AT7519; and (e) pelitinib.
Deuterium uptake differences are presented in a Woods plot, which provides a breakdown of the peptide ensemble for 1-, 10-, and 60 min time points. It displays peptide length, global coverage, and deuterium uptake. A 99% confidence limit was applied to the dataset to identify peptides with significant deuterium uptake. Deprotected, protected, and non-significantly different peptides are in red, blue, and gray, respectively. The presented plots have been created in Deuteros 2.0.
Figure 3—figure supplement 5
The deuterium uptake difference plots for all identified peptides from main protease (Mpro) upon binding with inhibitors at 10 min were labeled onto the crystal model of Mpro (PDB ID 7ALI).
(a) PF-00835231; (b) GC376; (c) boceprevir; (d) carmofur; and (e) MR6-31-2.
Figure 4 with 5 supplements
A new allosteric binding mode of ebselen on main protease (Mpro).
(a) Deuterium uptake plot of ebselen-bound Mpro by global hydrogen/deuterium exchange mass spectrometry (HDX-MS). (b) The heat map of Mpro upon binding with ebselen at 10 min was overlaid onto the crystal model of Mpro (PDB entry 7ALI). (c) The uptake line plots in the ebselen-bound Mpro at 10 min HDX labelling. Residues 1–17 (***), 104–111 (**), 160–177 (**), 182–202 (**), and 296–306 (*). * for p<0.05, ** for p<0.01, and *** for p<0.001. (d) MD simulation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Mpro in the presence of ebselen on C300. Shifts in key intra-dimer interactions before and after covalent binding of ebselen at C300 on Mpro. Left panel: key amino acids involved in intra-dimer interactions are depicted on the crystal model of Mpro (PDB entry 7ALI). Right panel: the distances between R4 of one monomer and E290 of the other monomer Cα atoms, and residues S1 of one monomer and the other monomer E166 Cα atoms were measured, respectively.
Figure 4—figure supplement 1
Tandem mass spectrometry analysis reveals that ebselen covalently binds to C44 and C300 of main protease (Mpro).
(a) At m/z 868.8633 of the Mpro peptide FDVVRQCSGVTFQ, containing a modification (-C13H9NOSe) induced by ebselen on C300 for Mpro bound with ebselen at a molar ratio of 2:1. (b) At m/z 868.3600 of the Mpro-modified peptide FDVVRQCSGVTFQ containing a modification (-C13H9NOSe) induced by ebselen on C300 for Mpro bound with ebselen at a molar ratio of 1:3. (c) At m/z 830.6810 of the Mpro-modified peptide VICTSEDMLNPNYEDLLIR containing a modification (-C13H9NOSe) induced by ebselen on C44 for Mpro bound with ebselen at a molar ratio of 2:1. (d) At m/z 889.6963 of the Mpro-modified peptide hVICTSEDMLNPNYEDLLIR containing a modification (-C13H9NOSe) induced by ebselen on C44 for Mpro bound with ebselen at a molar ratio of 1:3.
Figure 4—figure supplement 2
Structural stability and flexibility analysis of unbound Mpro and Mpro-ebselen complex.
(a) Root mean square deviation (RMSD) results of unbound main protease (Mpro) and Mpro-2ebselen complex. Three colors represent the three replicates. (b) Root mean square fluctuation (RMSF) results of the unbound Mpro protein and Mpro-ebselen complex. (c) The RMSF comparison between the N-terminal of the unbound Mpro protein and the N-terminal of the Mpro-ebselen complex. (d) The RMSF comparison between the C-terminal of the unbound Mpro protein and the C-terminal of the Mpro-ebselen complex.
Figure 4—figure supplement 3
SDS-PAGE and native PAGE analyses of WT, C300S, and C300F Mpro.
SDS-PAGE was used to assess protein integrity, whereas native PAGE was used to compare oligomeric states under non-denaturing conditions.
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Figure 4—figure supplement 3—source data 1
Raw, uncropped images for SDS-PAGE and native PAGE analyses shown in Figure 4—figure supplement 3.
- https://cdn.elifesciences.org/articles/109956/elife-109956-fig4-figsupp3-data1-v1.zip
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Figure 4—figure supplement 3—source data 2
Uncropped, labelled images for SDS-PAGE and native PAGE analyses shown in Figure 4—figure supplement 3.
- https://cdn.elifesciences.org/articles/109956/elife-109956-fig4-figsupp3-data2-v1.tif
Figure 4—figure supplement 4
Native mass spectra of C300S main protease (Mpro) in the (a) absence of ebselen and (b) presence of ebselen at a 1:3 Mpro-to-ebselen molar ratio.
Ebselen-bound monomeric and dimeric species were detected, whereas no marked redistribution toward the monomeric state was observed.
Figure 4—figure supplement 5
Effects of C300 mutations on main protease (Mpro) proteolytic activity and ebselen sensitivity.
(a) Proteolytic activities of WT, C300S, and C300F Mpro variants. Activities were normalized to WT Mpro (100%). Data are presented as mean ± SD from three independent experiments. (b) Differential sensitivities of WT and C300S Mpro to ebselen inhibition. Activities in the presence of ebselen were normalized to the corresponding untreated enzyme. Data are presented as mean ± SD from three independent experiments.
Tables
Table 1
Inhibitors against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Mpro and their inhibitory properties.
| Inhibitor | Binding site | Structure | EC50 (μM) | Hydrogen bonds | PDB ID | Reference |
|---|---|---|---|---|---|---|
| PF-07321332 | Active site | Peptidomimetic | 0.075 | H41, G143, H164, E166, Q192 | 7VH8 | Owen et al., 2021 |
| PF-00835231 | Active site | Peptidomimetic | 0.23 | F140, H164, E166, Q189 | 6XHM | Deshmukh et al., 2021 |
| GC376 | Active site | Peptidomimetic | 0.92 | F140, G143, C145, H163, H164, E166 | 7D1M | Vuong et al., 2020 |
| boceprevir | Active site | Peptidomimetic | 15.57 | H41, G143, H164, E166 | 7C6S | Fu et al., 2020 |
| carmofur | Active site | Non-peptidomimetic | 24.3 | G143, C145 | 7BUY | Jin et al., 2020b |
| ebselen | Active site | Non-peptidomimetic | 4.67 | - | 7BFB, 7BAK | Amporndanai et al., 2021 |
| MR6-31-2 | Active site | Non-peptidomimetic | 1.78 | - | 7BAL | Amporndanai et al., 2021 |
| AT7519 | Allosteric site | Non-peptidomimetic | 25.16 | Q110, D153 | 7AGA | Günther et al., 2021 |
| pelitinib | Allosteric site | Non-peptidomimetic | 1.25 | - | 7AXM | Günther et al., 2021 |
Additional files
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Supplementary file 1
Summaries of HDX experimental details.
- https://cdn.elifesciences.org/articles/109956/elife-109956-supp1-v1.xlsx
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Supplementary file 2
The file contains peptide-level deuterium uptake data for the HDX-MS experiments described in the main text.
- https://cdn.elifesciences.org/articles/109956/elife-109956-supp2-v1.xlsx
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MDAR checklist
- https://cdn.elifesciences.org/articles/109956/elife-109956-mdarchecklist1-v1.docx
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SARS-CoV-2 main protease inhibition through dimerization promotion by peptidomimetic inhibitors and disruption by ebselen
eLife 15:RP109956.
https://doi.org/10.7554/eLife.109956.4
