Journal: bioRxiv

Article Title: Drug repurposing screening identified tropifexor as a SARS-CoV-2 papain-like protease inhibitor

doi: 10.1101/2021.12.02.471030

Figure Lengend Snippet: Characterization of SARS-CoV-2 PL pro inhibitors identified from the high-throughput screening. (A) Chemical structures of the positive control GRL0617 and the three hits EACC, KY-226, and tropifexor. (B) IC 50 curves of the hits in inhibiting SARS CoV-2 PL pro with the FRET peptide substrate 1. (C) IC 50 curves of the hits in inhibiting SARS CoV PL pro with the FRET peptide substrate 1. (D) IC 50 curves of the hits in inhibiting MERS-CoV PL pro with the FRET peptide substrate 1. (E) IC 50 curves of the hits in inhibiting SARS CoV-2 M pro with the FRET peptide substrate 2. (F) IC 50 curves of the hits in inhibiting SARS CoV-2 PL pro with the Ub-AMC substrate. (G) IC 50 curves of the hits in inhibiting SARS CoV-2 PL pro with the ISG15-AMC substrate. Please refer to the methods and materials section for assay conditions. Values represent the average ± standard deviation of three replicates.

Article Snippet: Using the previously optimized FRET assay condition, we performed a high-throughput screening of SARS-CoV-2 PL pro against the MedChemExpress bioactive compound library which consists of 9,791 compounds including FDA-approved drugs, clinical candidates, and natural products.

Techniques: High Throughput Screening Assay, Positive Control, Standard Deviation

Journal: bioRxiv

Article Title: Drug repurposing screening identified tropifexor as a SARS-CoV-2 papain-like protease inhibitor

doi: 10.1101/2021.12.02.471030

Figure Lengend Snippet: Thermal shift binding assay of SARS-CoV-2 PL pro protease against identified inhibitors. All inhibitors display dose-dependent melting temperature (T m ) shift. Values represent the average ± standard deviation of three replicates.

Article Snippet: Using the previously optimized FRET assay condition, we performed a high-throughput screening of SARS-CoV-2 PL pro against the MedChemExpress bioactive compound library which consists of 9,791 compounds including FDA-approved drugs, clinical candidates, and natural products.

Techniques: Binding Assay, Standard Deviation

Journal: bioRxiv

Article Title: Drug repurposing screening identified tropifexor as a SARS-CoV-2 papain-like protease inhibitor

doi: 10.1101/2021.12.02.471030

Figure Lengend Snippet: FlipGFP SARS CoV-2 PL pro assay to determine cellular protease inhibitory activity of identified inhibitors. (A) Representative images of FlipGFP-PL pro assay with increasing concentrations of GRL0617 (positive control), GC376 (negative control), EACC, KY-226, and tropifexor. GRL0617 showed does-dependent decrease of GFP signal with the increasing drug concentration, while almost no GFP signal change was observed with the increasing concentration of negative control compound GC376. (B) Dose-response curves of the GFP/mCherry ratio with increasing drug concentrations. mCherry signal alone was used to calculate transfection efficiency and compound cytotoxicity. All three hits displayed significant cytotoxicity at high drug concentrations. Values represent the average ± standard deviation of three replicates.

Article Snippet: Using the previously optimized FRET assay condition, we performed a high-throughput screening of SARS-CoV-2 PL pro against the MedChemExpress bioactive compound library which consists of 9,791 compounds including FDA-approved drugs, clinical candidates, and natural products.

Techniques: Activity Assay, Positive Control, Negative Control, Concentration Assay, Transfection, Standard Deviation

Journal: bioRxiv

Article Title: Drug repurposing screening identified tropifexor as a SARS-CoV-2 papain-like protease inhibitor

doi: 10.1101/2021.12.02.471030

Figure Lengend Snippet: Antiviral activity of SARS-CoV-2 PL pro inhibitors GRL0617 (A), EACC(B), KY-226 (C), and tropifexor (D) against SARS-CoV-2 in Calu-3 cells. The results were quantified by immunofluorescence assay. Values represent the average ± standard deviation of three replicates.

Article Snippet: Using the previously optimized FRET assay condition, we performed a high-throughput screening of SARS-CoV-2 PL pro against the MedChemExpress bioactive compound library which consists of 9,791 compounds including FDA-approved drugs, clinical candidates, and natural products.

Techniques: Activity Assay, Immunofluorescence, Standard Deviation

Journal: bioRxiv

Article Title: Drug repurposing screening identified tropifexor as a SARS-CoV-2 papain-like protease inhibitor

doi: 10.1101/2021.12.02.471030

Figure Lengend Snippet: Molecular docking of SARS-CoV-2 PL pro inhibitors GRL0617 (A), EACC(B), KY-226 (C), and tropifexor (D) in PL pro (PDB: 7JRN).

Article Snippet: Using the previously optimized FRET assay condition, we performed a high-throughput screening of SARS-CoV-2 PL pro against the MedChemExpress bioactive compound library which consists of 9,791 compounds including FDA-approved drugs, clinical candidates, and natural products.

Techniques:

Journal: PLoS ONE

Article Title: Biochemical characterization of protease activity of Nsp3 from SARS-CoV-2 and its inhibition by nanobodies

doi: 10.1371/journal.pone.0253364

Figure Lengend Snippet: A) Schematic of cleavage of Nsp1-3 by PLpro from the viral polypeptide and the domain structure of Nsp3: Ubl (ubiquitin-like domain), ADRP (ADP-ribose phosphatase, SUD (SARS-unique domain), PLpro (papain-like protease), NAB (nucleic acid binding domain), TM (Transmembrane domain), ZnF (zinc finger motif). B) Coomassie-stained gel of Nsp3 constructs used in this study with accompanying domain schematics (colour coding as in 1A; double slash indicates deletion). C) DUB assay directly comparing cleavage of Ub3 (upper) and ISG15 (lower) by PLpro and Nsp3 core . D) Assay directly comparing cleavage of Nsp1-2Δ by PLpro and Nsp3 core . E) Protease assay comparing the cleavage of Nsp1-2 FL by PLpro and Nsp3 core . F) DUB assay showing the polyubiquitin linkage preference of Nsp3 core . G) Cleavage assay comparing cleavage of Nsp1-2Δ and Nsp1-2Δ G180A mutant by Nsp3 core . H) UV-traces of analytical gel filtration analyses (Superdex 200 3.2/300) of Nsp3 core overlaid onto molecular weight standards. I) Mass photometry analysis measuring the molecular mass of Nsp3 core . Data shown are representative of two independent experiments.

Article Snippet: The cDNAs coding for SARS CoV-2 (MN908947.3) Nsp3 residues 743–1072 (PL-pro) and SARS CoV-2 Nsp3 residues 743–1072 (PL-pro) E912R E948R M953E Y1013K were synthesised by NBS Biologicals ( www.nbsbio.co.uk ) with 5’ BamHI and 3’ NotI sites.

Techniques: Ubiquitin Proteomics, Binding Assay, Staining, Construct, Protease Assay, Cleavage Assay, Mutagenesis, Filtration, Molecular Weight

Journal: PLoS ONE

Article Title: Biochemical characterization of protease activity of Nsp3 from SARS-CoV-2 and its inhibition by nanobodies

doi: 10.1371/journal.pone.0253364

Figure Lengend Snippet: Volcano plot showing significant interactors identified by pull down on Nsp3 DTM or PLpro from transiently transfected cells followed by quantitative mass spectrometry. Interactors enriched in PLpro pull downs are highlighted in blue and those enriched in Nsp3 pull downs are highlighted in red circles.

Article Snippet: The cDNAs coding for SARS CoV-2 (MN908947.3) Nsp3 residues 743–1072 (PL-pro) and SARS CoV-2 Nsp3 residues 743–1072 (PL-pro) E912R E948R M953E Y1013K were synthesised by NBS Biologicals ( www.nbsbio.co.uk ) with 5’ BamHI and 3’ NotI sites.

Techniques: Transfection, Mass Spectrometry

Journal: PLoS ONE

Article Title: Biochemical characterization of protease activity of Nsp3 from SARS-CoV-2 and its inhibition by nanobodies

doi: 10.1371/journal.pone.0253364

Figure Lengend Snippet: A) Schematic workflow of the MALDI-TOF DUB assay. K48 trimer is incubated with PLpro for 30 minutes at room temperature. The reaction is stopped by adding 2% TFA and 15 N ubiquitin (as internal standard). Enzymatic activity and inhibition were assessed by MALDI-TOF MS detection of ubiquitin and 15 N ubiquitin signals. B) FDA approved compound library screening by MALDI-TOF, Z’Prime score and HTS data distribution. C) IC50 calculation of compounds returning >50% inhibition values in the HTS: thioguanine, nordihydroguaiaretic acid, Disulfiram, Auranofin and Tideglusib. IC50s were calculated both against PLpro and Nsp3 core using the MALDI-TOF DUB Assay. D-E) Orthogonal, gel-based assays testing PLpro and Nsp3 protease activities against K48 trimer and pro-ISG15 as substrates. Nordihydroguaiaretic acid (NDA), Disulfiram (DSF), Auranofin (AUR) and Tideglusib (TID) were tested at the indicated concentrations against PLpro and Nsp3 core . Data shown are representative of two independent experiments. F) IC50 calculation of GRL0617 against PLpro and Nsp3 core . G) Selectivity assessment of GRL-0617 and HTS positive hits against a panel of 42 human DUBs and the viral ovarian tumor (vOTU). Compounds were tested at the final concentrations of 10 and 1 μM.

Article Snippet: The cDNAs coding for SARS CoV-2 (MN908947.3) Nsp3 residues 743–1072 (PL-pro) and SARS CoV-2 Nsp3 residues 743–1072 (PL-pro) E912R E948R M953E Y1013K were synthesised by NBS Biologicals ( www.nbsbio.co.uk ) with 5’ BamHI and 3’ NotI sites.

Techniques: Incubation, Ubiquitin Proteomics, Activity Assay, Inhibition, Drug discovery

Journal: PLoS ONE

Article Title: Biochemical characterization of protease activity of Nsp3 from SARS-CoV-2 and its inhibition by nanobodies

doi: 10.1371/journal.pone.0253364

Figure Lengend Snippet: A) Schematic workflow of yeast-surface display selection to identify site-specific nanobodies as competitive Nsp3 core inhibitors. B) Flow cytometry analysis of yeast displaying anti-Nsp3 nanobodies NbSL17, NbSL18 or NbSL19, respectively. Yeast were stained with anti-HA-Alexa488 against HA-tagged Nb and Streptavidin-Alexa647 against biotinylated antigens. Top: Yeast incubated with PLpro wild-type, Bottom: Yeast incubated with PLpro S1*-mutant. Percentages indicate fraction of yeast positive for both fluorescent labels. C) UV-traces and coomassie-stained SDS-PAGE of analytical gel filtration analyses of PLpro-Nb complexes. PLpro alone (black), PLpro+NbSL17 (orange) and PLpro+NbSL18 (blue). D) Isothermal calorimetry titration measuring binding of PLpro to NbSL18. E) Silver stained gels of DUB assays of PLpro (left) and Nsp3 core (right) with K48-Ub3 (top) and proISG15 (middle) and Nsp1-2 FL (bottom) as substrates in the presence of increasing concentrations of NbSL18. Silver-stained SDS-PAGE gels shown are representative of two independent experiments. Also see .

Article Snippet: The cDNAs coding for SARS CoV-2 (MN908947.3) Nsp3 residues 743–1072 (PL-pro) and SARS CoV-2 Nsp3 residues 743–1072 (PL-pro) E912R E948R M953E Y1013K were synthesised by NBS Biologicals ( www.nbsbio.co.uk ) with 5’ BamHI and 3’ NotI sites.

Techniques: Selection, Flow Cytometry, Staining, Incubation, Mutagenesis, SDS Page, Filtration, Titration, Binding Assay

Journal: PLoS ONE

Article Title: Identification of repurposing therapeutics toward SARS-CoV-2 main protease by virtual screening

doi: 10.1371/journal.pone.0269563

Figure Lengend Snippet: The virtual screening scheme of molecular docking of SAR-CoV-2 3CL pro inhibitors from the crystal structures of potent peptidomimetic inhibitors (N3, 13b, and 11a) using the DrugBank database. The screened compounds were selected, and the common drugs were later included for testing the SAR-CoV-2 3CL pro inhibitory activity by enzyme-based assay.

Article Snippet: The MD trajectories at equilibrium state (2,000 frames) of lapatinib/SARS-CoV-2 3CL pro complex from the last 100 ns (401–500 ns) and the SARS-CoV-2 3CL pro complexes with three known inhibitors (N3, 11a, and 13b) from the last 20 ns (81–100 ns) in our previous work [ ] were used to create pharmacophore features using LigandScout 4.4.2 program combined with the KNIME analysis platform [ , ].

Techniques: Activity Assay, Enzymatic Assay

Journal: PLoS ONE

Article Title: Identification of repurposing therapeutics toward SARS-CoV-2 main protease by virtual screening

doi: 10.1371/journal.pone.0269563

Figure Lengend Snippet: Binding energy (kcal/mol) of approved drugs against three X-ray structures of SARS-CoV-2 3CL pro (PDB entry codes: 6LU7, 6LZE, and 6Y2F) derived from molecular docking.

Article Snippet: The MD trajectories at equilibrium state (2,000 frames) of lapatinib/SARS-CoV-2 3CL pro complex from the last 100 ns (401–500 ns) and the SARS-CoV-2 3CL pro complexes with three known inhibitors (N3, 11a, and 13b) from the last 20 ns (81–100 ns) in our previous work [ ] were used to create pharmacophore features using LigandScout 4.4.2 program combined with the KNIME analysis platform [ , ].

Techniques: Binding Assay, Derivative Assay

Journal: PLoS ONE

Article Title: Identification of repurposing therapeutics toward SARS-CoV-2 main protease by virtual screening

doi: 10.1371/journal.pone.0269563

Figure Lengend Snippet: Heat map of the top compounds interacting with SARS-CoV-2 3CL pro derived from relative to the known inhibitors derived from LigPlot version 2.2.

Article Snippet: The MD trajectories at equilibrium state (2,000 frames) of lapatinib/SARS-CoV-2 3CL pro complex from the last 100 ns (401–500 ns) and the SARS-CoV-2 3CL pro complexes with three known inhibitors (N3, 11a, and 13b) from the last 20 ns (81–100 ns) in our previous work [ ] were used to create pharmacophore features using LigandScout 4.4.2 program combined with the KNIME analysis platform [ , ].

Techniques: Derivative Assay

Journal: PLoS ONE

Article Title: Identification of repurposing therapeutics toward SARS-CoV-2 main protease by virtual screening

doi: 10.1371/journal.pone.0269563

Figure Lengend Snippet: The selected compounds and common drugs for SAR-CoV-2 3CL pro inhibitory activity assay.

Article Snippet: The MD trajectories at equilibrium state (2,000 frames) of lapatinib/SARS-CoV-2 3CL pro complex from the last 100 ns (401–500 ns) and the SARS-CoV-2 3CL pro complexes with three known inhibitors (N3, 11a, and 13b) from the last 20 ns (81–100 ns) in our previous work [ ] were used to create pharmacophore features using LigandScout 4.4.2 program combined with the KNIME analysis platform [ , ].

Techniques: Activity Assay, Protease Inhibitor, Infection

Journal: PLoS ONE

Article Title: Identification of repurposing therapeutics toward SARS-CoV-2 main protease by virtual screening

doi: 10.1371/journal.pone.0269563

Figure Lengend Snippet: (A) Relative activity of 3CL pro in the presence of 100 μM of compounds and (B) inhibition of 3CL pro by lapatinib at various concentrations.

Article Snippet: The MD trajectories at equilibrium state (2,000 frames) of lapatinib/SARS-CoV-2 3CL pro complex from the last 100 ns (401–500 ns) and the SARS-CoV-2 3CL pro complexes with three known inhibitors (N3, 11a, and 13b) from the last 20 ns (81–100 ns) in our previous work [ ] were used to create pharmacophore features using LigandScout 4.4.2 program combined with the KNIME analysis platform [ , ].

Techniques: Activity Assay, Inhibition

Journal: PLoS ONE

Article Title: Identification of repurposing therapeutics toward SARS-CoV-2 main protease by virtual screening

doi: 10.1371/journal.pone.0269563

Figure Lengend Snippet: (A) Distance between the C m of lapatinib and the C m of SARS-CoV-2 3CL pro active site residues, (B) # H-bonds, (C) # atom contacts, (D) RMSD plot for protein backbone (CA, C, O, and N atoms) and lapatinib, (E) interaction energy, and (F) radius of gyration (R g ) of 3CL pro in each chain plotted along with the 500-ns MD simulation.

Article Snippet: The MD trajectories at equilibrium state (2,000 frames) of lapatinib/SARS-CoV-2 3CL pro complex from the last 100 ns (401–500 ns) and the SARS-CoV-2 3CL pro complexes with three known inhibitors (N3, 11a, and 13b) from the last 20 ns (81–100 ns) in our previous work [ ] were used to create pharmacophore features using LigandScout 4.4.2 program combined with the KNIME analysis platform [ , ].

Techniques:

Journal: PLoS ONE

Article Title: Identification of repurposing therapeutics toward SARS-CoV-2 main protease by virtual screening

doi: 10.1371/journal.pone.0269563

Figure Lengend Snippet: (A) Binding free energy contribution per residue ( Δ G b i n d r e s i d u e ) for lapatinib binding derived from the last 100 ns, colored from dark red to green according to the highest to lowest free energies. The residues with Δ G b i n d r e s i d u e ≤ −0.5 kcal/mol and ≥ 0.5 kcal/mol are labeled. The key residues are colored according to their Δ G b i n d r e s i d u e values. The representative structure was taken from the last MD snapshot. Noted that the quinazoline scaffold of lapatinib highly interacted with L141 at the S1 site ( Δ G b i n d r e s i d u e of -3.33 kcal/mol). Their percentages of hydrogen bond occupation are shown in (B). (C) 2D projection of MD trajectories on the first two PC modes and PCA scree plot, (D) PC1 porcupine plot of the holo and apo forms, where the arrowhead and length represent the direction and amplitude of motion, respectively, and (E) RMSF plot of 3CL pro in each chain.

Article Snippet: The MD trajectories at equilibrium state (2,000 frames) of lapatinib/SARS-CoV-2 3CL pro complex from the last 100 ns (401–500 ns) and the SARS-CoV-2 3CL pro complexes with three known inhibitors (N3, 11a, and 13b) from the last 20 ns (81–100 ns) in our previous work [ ] were used to create pharmacophore features using LigandScout 4.4.2 program combined with the KNIME analysis platform [ , ].

Techniques: Binding Assay, Residue, Derivative Assay, Labeling

Journal: PLoS ONE

Article Title: Identification of repurposing therapeutics toward SARS-CoV-2 main protease by virtual screening

doi: 10.1371/journal.pone.0269563

Figure Lengend Snippet: The 2D and 3D pharmacophore models of the first representative frame of lapatinib at 401 ns, and peptidomimetic inhibitors N3, 13b, and 11a at 81 ns, where RPMs derived from MD trajectories of lapatinib-3CL pro complex (401–500 ns), and peptidomimetic inhibitor(s)-3CL pro complex (81–100 ns) are illustrated on the right column. The green arrow, red arrow, purple arrow, and yellow color sphere (or circle in 2D) are pharmacophore features of hydrogen bond donor (HBD) and acceptor (HBA), halogen bond donor (XBD), and hydrophobic interaction properties, respectively.

Article Snippet: The MD trajectories at equilibrium state (2,000 frames) of lapatinib/SARS-CoV-2 3CL pro complex from the last 100 ns (401–500 ns) and the SARS-CoV-2 3CL pro complexes with three known inhibitors (N3, 11a, and 13b) from the last 20 ns (81–100 ns) in our previous work [ ] were used to create pharmacophore features using LigandScout 4.4.2 program combined with the KNIME analysis platform [ , ].

Techniques: Derivative Assay

Journal: PLoS ONE

Article Title: Identification of repurposing therapeutics toward SARS-CoV-2 main protease by virtual screening

doi: 10.1371/journal.pone.0269563

Figure Lengend Snippet: Interaction map of four inhibitors against SAR-CoV-2 3CL pro derived from 401–500 ns MD trajectories for lapatinib 81–100 ns, and MD trajectories for peptidomimetic inhibitors N3, 13b, and 11a. The abbreviations of H, HBA, and HBD represent the pharmacophore features of hydrophobic interaction, hydrogen bond acceptor, and hydrogen bond donor properties. The numbers in the blue box are the percentage of appearance in each interaction per residue.

Article Snippet: The MD trajectories at equilibrium state (2,000 frames) of lapatinib/SARS-CoV-2 3CL pro complex from the last 100 ns (401–500 ns) and the SARS-CoV-2 3CL pro complexes with three known inhibitors (N3, 11a, and 13b) from the last 20 ns (81–100 ns) in our previous work [ ] were used to create pharmacophore features using LigandScout 4.4.2 program combined with the KNIME analysis platform [ , ].

Techniques: Derivative Assay, Residue

Journal: PLoS ONE

Article Title: Identification of repurposing therapeutics toward SARS-CoV-2 main protease by virtual screening

doi: 10.1371/journal.pone.0269563

Figure Lengend Snippet: (A) 2D structure of lapatinib and designed lapatinibs, (B) predicted binding affinity of the designed compounds A-H against SARS-CoV-2 3CL pro in comparison with lapatinib using LigandScout 4.4.2 program, (C) the binding free energy per residue of modified lapatinib, compound F/SARS-CoV-2 3CL pro complex, and its hydrogen bond interactions (D). The results were obtained from one snapshot of the complex after system minimization.

Article Snippet: The MD trajectories at equilibrium state (2,000 frames) of lapatinib/SARS-CoV-2 3CL pro complex from the last 100 ns (401–500 ns) and the SARS-CoV-2 3CL pro complexes with three known inhibitors (N3, 11a, and 13b) from the last 20 ns (81–100 ns) in our previous work [ ] were used to create pharmacophore features using LigandScout 4.4.2 program combined with the KNIME analysis platform [ , ].

Techniques: Binding Assay, Comparison, Residue, Modification