Discovery of multiple new SARS-CoV-2 MPro inhibitors


In a recent study posted to the bioRxiv* preprint server, researchers have performed extensive library docking for novel covalent and non-covalent inhibitors of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) major protease (Mpro).

Study: Docking of large library for novel SARS-CoV-2 master protease non-covalent and covalent inhibitors. ​​​​​​​Image Credit: creativeneko/Shutterstock

Background

Antivirals against SARS-CoV-2 are urgently needed to fight the ongoing CoV disease 2019 (COVID-19) pandemic. The MPro, also called 3-chymotrypsin-like protease (3CLPro), is a well-known SARS-CoV-2 enzyme. An approved drug, Paxlovid, and several experimental new drugs, such as S-217622, ​​target the SARS-CoV-2 Mpro. Nevertheless, there is still a need for novel chemical scaffolds targeting MPro, given the inherent drawbacks of current novel drugs and the threat of viral resistance.

About the study

In the current study, the researchers focused on the SARS-CoV-2 MPo structure for mass library docking in search of new starting points for the discovery of non-covalent or covalent inhibitors. They linked nearly 1.2 billion non-covalent and a new pool of 6.5 million electrophilic compounds against the Mpro enzyme structure from the easily accessible (REAL) space of Enamine.

The team simulated an aggregate of SARS-CoV-2 MPro coupled with a non-covalent SARS-CoV MPro inhibitor to characterize hotspots for ligand binding across the active site. Three of the crystal structures of the refined non-covalent inhibitors, with resolutions from 2.12 A to 2.59 A, were established to study how the docked positions of the new inhibitors corresponded to their actual binding modes and to guide further refinement.

The authors performed a second docking experiment because other researchers had discovered efficient inhibitors with extensive structure-activity-relationship (SAR) information and scaffolds similar to those used in the current work during the study period. They sought to incorporate the conclusions drawn from the current findings and those of other studies highlighting the identification of new chemotypes. The non-covalent ligand from the COVID-19 Moonshot consortium MAT-POS-b3e365b9-1 (MPro-x11612.pdb), in combination with the SARS-CoV-2 MPro crystal structure, was the target of the new docking screen.

The scientists searched the 1.4 billion compounds in the ZINC15/ZINC20 libraries for three Cys-reactive covalent warheads, nitriles, aldehydes and alpha-ketoamides to find electrophiles that can covalently alter the catalytic Cys145. DOCKovalent was used to make linkable three-dimensional (3D) molecules for covalent bonding.

The crystal structures of five aldehyde inhibitors bound with MPro were developed to investigate how the docked positions of the covalent inhibitors correlated with the actual binding modes and to support further optimization. As the optimization of non-covalent and covalent inhibitor progressed, the team evaluated several compounds in a reverse transcription quantitative polymerase chain reaction (RT-qPCR) viral infectivity assay in Henrietta Lacks angiotensin-converting enzyme 2 (HeLa-ACE2) cells.

Substrate design and test development make it possible to discover structure-based inhibitors.  (A) The chemical structure of the optimized NSP7 substrate shown as a schematic (top) of the substrate sequence highlights the role of each residue (bottom).  The substrate contains the P4-P4′ NSP7 extended substrate sequence (blue), the fluorophore (yellow), the fluorescent quencher (purple), and the solubility enhancer residues (green).  (B) A list of the viral polypeptide NSP sequences (P4-P4′) cleaved by MPro (left).  The sequence LOGO emphasizes the substrate specificity of MPro, yielding a P4-P4′ consensus sequence: ATLQ(S/A)XXA (right).  (C) The Michaelis-Menten kinetics for the NSP7 substrate with MPro yield parameters indicative of an optimized, efficient substrate.  (D) SARS-CoV-2 MPro active site (PDB 6Y2G)26 (green; subboxes S1′, S1, S2, S3, S4), shown here with substrate preferences (pink; P1′, P1, P2, P3, P4) (modelled after PDB 3SNE)27, was used to link 1.2 billion non-covalent molecules and 6.5 million electrophilic molecules.  The best ranked molecules were filtered and 395 were synthesized for in vitro testing.  Some docking hits were prioritized for linkage optimization, crystallography, pan-viral enzymatic activity, and cell-based antiviral activity.  For C, experiments were performed in triplicate.Substrate design and test development make it possible to discover structure-based inhibitors. (A) The chemical structure of the optimized NSP7 substrate shown as a schematic (top) of the substrate sequence highlights the role of each residue (bottom). The substrate contains the P4-P4′ NSP7 extended substrate sequence (blue), the fluorophore (yellow), the fluorescent quencher (purple), and the solubility enhancer residues (green). (B) A list of the viral polypeptide NSP sequences (P4-P4′) cleaved by MPro (left). The sequence LOGO emphasizes the substrate specificity of MPro, yielding a P4-P4′ consensus sequence: ATLQ(S/A)XXA (right). (C) The Michaelis-Menten kinetics for the NSP7 substrate with MPro yield parameters indicative of an optimized, efficient substrate. (D) SARS-CoV-2 MPro active site (PDB 6Y2G)26 (green; subboxes S1′, S1, S2, S3, S4), shown here with substrate preferences (pink; P1′, P1, P2, P3, P4) (modelled after PDB 3SNE)27, was used to link 1.2 billion non-covalent molecules and 6.5 million electrophilic molecules. The best ranked molecules were filtered and 395 were synthesized for in vitro testing. Some docking hits were prioritized for linkage optimization, crystallography, pan-viral enzymatic activity, and cell-based antiviral activity. For C, experiments were performed in triplicate.

Results

In the present work, the authors identified 132 SARS-CoV-2 MPro inhibitors belonging to 37 different scaffold classes and exhibiting half-maximal inhibitory concentrations (IC50) of less than 150 M. Of these, 15 inhibitors across three scaffolds had IC50 values ​​below 10 µM and inhibited the enzyme. The most effective covalent inhibitor, ‘7021, was shown to function reversibly, probably reflecting the fast-on/fast-off kinetics of covalent aldehyde inhibitors.

Furthermore, the team provided an improved MPro substrate for the upcoming evaluation of inhibitors. The scientists initially established an electrophilic library using the growing collection of tangible compounds, including aldehydes, nitriles and alpha-ketoamides. The community can openly access this library of more than 6.5 million new electrophiles at https://covalent2022.docking.org.

Eight of the crystal structures of the new inhibitors closely matched the docking estimates. The antiviral activities of two of the new aldehyde inhibitors were comparable in their enzymatic IC50 values, indicating that further optimization of this class for onenzyme efficacy may predict antiviral activity.

Optimization of non-covalent compounds towards low μM potencies.  (A) Progress of the '0273 jetty.  (B) Predicted binding pose of '0273.  (C) Comparison of crystal structure (gray protein, red compound) and docked complex (green protein, blue compound) of SG-0001 (PDB 8DII).  (D) Predicted binding pose of '0541.  (E), (F) Comparison of crystal structures and coupled complexes of '5548 (VOB 8DIG) and '6111 (VOB 8DIH), respectively.  (G) Additional '0541 analogs with improved affinities.  The 2fo-fc ligand density maps (blue contour) are displayed at 1 s.  Hungarian quadratic deviations (RMSD) were calculated using DOCK6.Optimization of non-covalent compounds towards low μM potencies. (A) Progress of the ‘0273 jetty. (B) Predicted binding pose of ‘0273. (C) Comparison of crystal structure (gray protein, red compound) and docked complex (green protein, blue compound) of SG-0001 (PDB 8DII). (D) Predicted binding pose of ‘0541. (E), (F) Comparison of crystal structures and coupled complexes of ‘5548 (VOB 8DIG) and ‘6111 (VOB 8DIH), respectively. (G) Additional ‘0541 analogs with improved affinities. The 2fo-fc ligand density maps (blue contour) are displayed at 1 s. Hungarian quadratic deviations (RMSD) were calculated using DOCK6.

conclusions

In total, the team found 37 different scaffolds of SARS-CoV-2 Mpro inhibitors, with the most effective covalent and non-covalent compounds having an IC50 of 20 and 29 M, respectively. Inhibitors with low activity in the micromolar range were identified after the optimization of different series. The projected linkage binding modes were later validated by crystallography, which can serve as a template for future optimization. The compounds identified in the current work reveal new chemotypes that aid in the future search for MPro inhibitors for SARS-CoV-2 and other potential CoVs.

*Important announcement

bioRxiv publishes preliminary scientific reports that are not peer-reviewed and therefore should not be considered conclusive, should guide clinical practice/health-related behavior or be treated as established information.

Reference magazine:

  • Large library docking for novel SARS-CoV-2 major protease non-covalent and covalent inhibitors; Elissa A Fink, Conner Bardine, Stefan Gahbauer, Isha Singh, Kris White, Shuo Gu, Xiaobo Wan, Beatrice Ary, Isabelle Glenn, Joseph O’Connell, Henry O’Donnell, Pavla Fajtova, Jiankun Lyu, Seth Vigneron, Nicholas J Young , Ivan S Kondratov , Anthony J O’Donoghue , Yurii Moroz , Jack Taunton , Adam R Renslow , John J Irwin , Adolfo Garcia-Tailor , Brian K Shoichet , Charles S Craik . bioRxiv preprint 2022, DOI: https://doi.org/10.1101/2022.07.05.498881, https://www.biorxiv.org/content/10.1101/2022.07.05.498881v1
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