Journal: Journal of Medicinal Chemistry

Article Title: Discovery of the Clinical Candidate S‑892216 : A Second-Generation of SARS-CoV‑2 3CL Protease Inhibitor for Treating COVID-19

doi: 10.1021/acs.jmedchem.5c00754

Figure Lengend Snippet: X-ray crystal structures of SARS-CoV2 3CL pro complexed with (A) ensitrelvir (PDB: 7VU6 ), (B) nirmatrelvir (PDB: 7RFS ), (C) compound 1 (PDB: 9LVR) and (D) compound 4 (PDB: 9LVT), respectively. The inhibitors are shown as cyan sticks. The backbone of 3CL pro is shown as a white ribbon, the amino acids interacting to inhibitors are highlighted as gray sticks, and hydrogen bonds are indicated as yellow dashed lines. The structures of inhibitors are also depicted at the bottom.

Article Snippet: The reaction was initiated by adding 5 or 7.5 μL of 3CL pro (R&D Systems, Inc.) in assay buffer and incubated at room temperature for 4 to 5 h. The final concentration of enzyme was 0.3 nM in reaction mixture.

Techniques:

Journal: Journal of Medicinal Chemistry

Article Title: Discovery of the Clinical Candidate S‑892216 : A Second-Generation of SARS-CoV‑2 3CL Protease Inhibitor for Treating COVID-19

doi: 10.1021/acs.jmedchem.5c00754

Figure Lengend Snippet: X-ray crystal structure of S-892216 complexed with SARS-CoV-2 3CL pro (PDB: 9LVV). S-892216 is shown as a cyan stick, and the protein backbone is shown as a gray ribbon. Residues interacting with S-892216 are indicated as gray and orange sticks. Hydrogen bonds are indicated as yellow dashed lines, and π–π stacking is indicated as a cyan dashed line.

Article Snippet: The reaction was initiated by adding 5 or 7.5 μL of 3CL pro (R&D Systems, Inc.) in assay buffer and incubated at room temperature for 4 to 5 h. The final concentration of enzyme was 0.3 nM in reaction mixture.

Techniques:

Journal: Journal of Medicinal Chemistry

Article Title: Discovery of the Clinical Candidate S‑892216 : A Second-Generation of SARS-CoV‑2 3CL Protease Inhibitor for Treating COVID-19

doi: 10.1021/acs.jmedchem.5c00754

Figure Lengend Snippet: Dissociation kinetics of S-892216 from 3CL pro measured using a SPA-based assay. (A) Schematic illustration of S-892216 dissociation kinetics study. Sample 1: [ 14 C]- S-892216 alone; Sample 2: excess amount of nonlabeled S-892216 was added immediately before measurement; Sample 3 excess amount of nonlabeled S-892216 was added before preincubation. [ 14 C]- S-892216 : red circle, Nonlabeled S-892216 : blue circle, buffer: white circle. (B) Dissociation kinetics of [ 14 C]- S-892216 was monitored using scintillation signals. Data fitted in GraphPad Prism 9 using one-phase exponential decay.

Article Snippet: The reaction was initiated by adding 5 or 7.5 μL of 3CL pro (R&D Systems, Inc.) in assay buffer and incubated at room temperature for 4 to 5 h. The final concentration of enzyme was 0.3 nM in reaction mixture.

Techniques:

Journal: Journal of Medicinal Chemistry

Article Title: Discovery of the Clinical Candidate S‑892216 : A Second-Generation of SARS-CoV‑2 3CL Protease Inhibitor for Treating COVID-19

doi: 10.1021/acs.jmedchem.5c00754

Figure Lengend Snippet: X-ray crystal structures of SARS-CoV2 3CL pro complexed with (A) ensitrelvir (PDB: 7VU6 ), (B) nirmatrelvir (PDB: 7RFS ), (C) compound 1 (PDB: 9LVR) and (D) compound 4 (PDB: 9LVT), respectively. The inhibitors are shown as cyan sticks. The backbone of 3CL pro is shown as a white ribbon, the amino acids interacting to inhibitors are highlighted as gray sticks, and hydrogen bonds are indicated as yellow dashed lines. The structures of inhibitors are also depicted at the bottom.

Article Snippet: SARS-CoV-2 3CL protease (R&D systems) were attached to SPA beads by incubating 0.27 μM random biotinylated-SARS-CoV-2 3CL protease and 7 mg/mL streptavidin YSi SPA scintillation beads (Revvity) in assay buffer (10 mM Tris pH7.5, 5 mM DTT, 0.5 mM EDTA, 0.005% Tween20).

Techniques:

Journal: Journal of Medicinal Chemistry

Article Title: Discovery of the Clinical Candidate S‑892216 : A Second-Generation of SARS-CoV‑2 3CL Protease Inhibitor for Treating COVID-19

doi: 10.1021/acs.jmedchem.5c00754

Figure Lengend Snippet: X-ray crystal structure of S-892216 complexed with SARS-CoV-2 3CL pro (PDB: 9LVV). S-892216 is shown as a cyan stick, and the protein backbone is shown as a gray ribbon. Residues interacting with S-892216 are indicated as gray and orange sticks. Hydrogen bonds are indicated as yellow dashed lines, and π–π stacking is indicated as a cyan dashed line.

Article Snippet: SARS-CoV-2 3CL protease (R&D systems) were attached to SPA beads by incubating 0.27 μM random biotinylated-SARS-CoV-2 3CL protease and 7 mg/mL streptavidin YSi SPA scintillation beads (Revvity) in assay buffer (10 mM Tris pH7.5, 5 mM DTT, 0.5 mM EDTA, 0.005% Tween20).

Techniques:

Journal: Journal of Medicinal Chemistry

Article Title: Discovery of the Clinical Candidate S‑892216 : A Second-Generation of SARS-CoV‑2 3CL Protease Inhibitor for Treating COVID-19

doi: 10.1021/acs.jmedchem.5c00754

Figure Lengend Snippet: Dissociation kinetics of S-892216 from 3CL pro measured using a SPA-based assay. (A) Schematic illustration of S-892216 dissociation kinetics study. Sample 1: [ 14 C]- S-892216 alone; Sample 2: excess amount of nonlabeled S-892216 was added immediately before measurement; Sample 3 excess amount of nonlabeled S-892216 was added before preincubation. [ 14 C]- S-892216 : red circle, Nonlabeled S-892216 : blue circle, buffer: white circle. (B) Dissociation kinetics of [ 14 C]- S-892216 was monitored using scintillation signals. Data fitted in GraphPad Prism 9 using one-phase exponential decay.

Article Snippet: SARS-CoV-2 3CL protease (R&D systems) were attached to SPA beads by incubating 0.27 μM random biotinylated-SARS-CoV-2 3CL protease and 7 mg/mL streptavidin YSi SPA scintillation beads (Revvity) in assay buffer (10 mM Tris pH7.5, 5 mM DTT, 0.5 mM EDTA, 0.005% Tween20).

Techniques:

Journal: Viruses

Article Title: Development of a Cell-Based Luciferase Complementation Assay for Identification of SARS-CoV-2 3CL pro Inhibitors

doi: 10.3390/v13020173

Figure Lengend Snippet: Development of cell-based luciferase complementation reporters to detect inhibition of SARS-CoV-2 3CL pro activity. ( A ) Strategy for detection of 3CL pro inhibition using NanoBiT, a luciferase complementation reporter comprised of Large BiT (L) and Small BiT (S). When connected by a linker containing a 3CL pro cleavage site, 3CL pro cleavage should result in a loss of complementation and low luciferase activity. Conversely, 3CL pro inhibitors should prevent cleavage, resulting in high luciferase activity. ( B ) Lentiviral vectors that express 3CL pro and reporters. P2A, a self-cleaving peptide, was inserted between 3CL pro and reporters. Reporters consisted of GFP linked to L and S in various orders. L and S were separated by the SARS-CoV-2 nsp4-nsp5 cleavage site. LTR, long terminal repeat; P EF1α , EF1α promoter; IRES, internal ribosome entry site; puro, puromycin resistance gene; WPRE, woodchuck hepatitis virus post-transcriptional regulatory element. ( C ) Western blotting to examine reporter expression and cleavage. Lentiviral vectors expressing 3CL pro WT or C145A (a catalytically inactive mutant) were transfected into 293T cells, and DMSO (control) or the 3CL pro inhibitor GC376 (100 µM) was added. Cell lysates were collected 30 h post-transfection. 3CL pro was detected using anti-SARS-CoV 3CL pro or anti-P2A antibodies, whereas reporters were detected using anti-GFP or anti-L antibodies. Arrows denote cleavage products of expected sizes. For 3CL pro C145A, two different plasmid amounts were transfected: 2 or 0.4 µg. HSP90 was included as a loading control. ( D ) NanoBiT luciferase activity. 293T cells were transfected (same sample order as in C ), and NanoBiT activity (expressed as relative luciferase units, or RLU) was measured 30 h post-transfection. The data represent the mean ± standard deviation of four independent experiments; ns: not significant; * p < 0.05, ** p < 0.01, *** p < 0.001 (relative to 3CL pro WT without drug; mixed effects analysis with Dunnett’s post-test).

Article Snippet: After transfer to PVDF membranes, blots were probed with 1:5000 rabbit anti-SARS-CoV 3CL pro (Rockland Immunochemicals; Limerick, PA, USA; catalog #200-401-A51), mouse anti-P2A (Novus Biologicals; Littleton, CO, USA; catalog #NBP2-59627), mouse anti-L (Promega; catalog #N710A), or rabbit anti-GFP antibodies (Invitrogen; Carlsbad, CA, USA; catalog #A6455).

Techniques: Luciferase, Inhibition, Activity Assay, Virus, Western Blot, Expressing, Mutagenesis, Transfection, Control, Plasmid Preparation, Standard Deviation

Journal: Viruses

Article Title: Development of a Cell-Based Luciferase Complementation Assay for Identification of SARS-CoV-2 3CL pro Inhibitors

doi: 10.3390/v13020173

Figure Lengend Snippet: The luciferase complementation assay accurately reflects concentration-dependent inhibition of SARS-CoV-2 3CL pro -mediated cleavage. ( A ) Western blotting to examine reporter cleavage. 293T cells were transfected with the lentiviral vector containing 3CL pro WT and the S-L-GFP reporter cassette, and DMSO (control) or GC376 (0.1, 0.4, 1.6, 6.3, 25, 100, or 200 µM) was added. Mock transfection (MT) and 3CL pro C145A S-L-GFP were included as controls. On the GFP and LgBiT (L) panels, the bottom band corresponds to cleaved reporter (L-GFP, 46 kDa, indicated by arrows), while the top band corresponds to uncleaved reporter (S-L-GFP, 49 kDa). ( B ) Quantification of the percentage of uncleaved reporter from three independent western blots based on GFP (circle) or L (triangle) detection. ( C ) NanoBiT luciferase activity and cell viability. 293T cells were transfected with the S-L-GFP lentiviral vector, and DMSO (control) or GC376 (0.1, 0.4, 1.6, 6.3, 25, or 100 µM) was added. NanoBiT activity (circle, left axis) and cell viability (measured with the CellTiter-Glo 2.0 assay; triangle, right axis) were analyzed 30 h post-transfection and are expressed as relative luciferase units (RLU). Cell viability data were normalized to the DMSO sample, which was set to 100%. NanoBiT data were normalized to the DMSO and 100 µM GC376 samples, which were set to 0 and 100%, respectively. The data represent the mean ± standard deviation of four independent experiments; ns: not significant; * p < 0.05, ** p < 0.01, *** p < 0.001 (relative to no-drug; one-way ANOVA with Dunnett’s post-test).

Article Snippet: After transfer to PVDF membranes, blots were probed with 1:5000 rabbit anti-SARS-CoV 3CL pro (Rockland Immunochemicals; Limerick, PA, USA; catalog #200-401-A51), mouse anti-P2A (Novus Biologicals; Littleton, CO, USA; catalog #NBP2-59627), mouse anti-L (Promega; catalog #N710A), or rabbit anti-GFP antibodies (Invitrogen; Carlsbad, CA, USA; catalog #A6455).

Techniques: Luciferase, Concentration Assay, Inhibition, Western Blot, Transfection, Plasmid Preparation, Control, Activity Assay, Standard Deviation

Journal: Viruses

Article Title: Development of a Cell-Based Luciferase Complementation Assay for Identification of SARS-CoV-2 3CL pro Inhibitors

doi: 10.3390/v13020173

Figure Lengend Snippet: The luciferase complementation assay can easily distinguish 3CL pro inhibition from cytotoxicity. To determine if the luciferase complementation reporter assay can differentiate 3CL pro inhibition from cytotoxicity, 293T cells were transfected with the S-L-GFP lentiviral vector, and DMSO (control) or the anti-cancer drug doxorubicin (0.1–100 µM) was added. NanoBiT activity (circle, left axis) and cell viability (measured with the CellTiter-Glo 2.0 assay; triangle, right axis) were analyzed 30 h post-transfection and are expressed as relative luciferase units (RLU). Cell viability data were normalized to the DMSO sample, which was set to 100%. NanoBiT data were normalized to the DMSO and 100 µM GC376 samples, which were set to 0 and 100%, respectively. The data represent the mean ± standard deviation of three independent experiments.

Article Snippet: After transfer to PVDF membranes, blots were probed with 1:5000 rabbit anti-SARS-CoV 3CL pro (Rockland Immunochemicals; Limerick, PA, USA; catalog #200-401-A51), mouse anti-P2A (Novus Biologicals; Littleton, CO, USA; catalog #NBP2-59627), mouse anti-L (Promega; catalog #N710A), or rabbit anti-GFP antibodies (Invitrogen; Carlsbad, CA, USA; catalog #A6455).

Techniques: Luciferase, Inhibition, Reporter Assay, Transfection, Plasmid Preparation, Control, Activity Assay, Standard Deviation

Journal: Viruses

Article Title: Development of a Cell-Based Luciferase Complementation Assay for Identification of SARS-CoV-2 3CL pro Inhibitors

doi: 10.3390/v13020173

Figure Lengend Snippet: HIV protease inhibitors do not block SARS-CoV-2 3CL pro activity in a cell-based luciferase complementation assay. To determine if HIV protease inhibitors are active against SARS-CoV-2 3CL pro in cells, 293T cells were transfected with the S-L-GFP lentiviral vector, and DMSO (control) or inhibitors (0.1, 0.4, 1.6, 6.3, 25, or 100 µM) were added. NanoBiT activity (circle, left axis) and cell viability (measured with the CellTiter-Glo 2.0 assay; triangle, right axis) were analyzed 30 h post-transfection and are expressed as relative luciferase units (RLU). Cell viability data were normalized to the DMSO sample, which was set to 100%. NanoBiT data were normalized to the DMSO and 100 µM GC376 samples, which were set to 0 and 100%, respectively. The data represent the mean ± standard deviation of three independent experiments; * p < 0.05, ** p < 0.01, *** p < 0.001 (relative to no-drug; one-way ANOVA with Dunnett’s post-test).

Article Snippet: After transfer to PVDF membranes, blots were probed with 1:5000 rabbit anti-SARS-CoV 3CL pro (Rockland Immunochemicals; Limerick, PA, USA; catalog #200-401-A51), mouse anti-P2A (Novus Biologicals; Littleton, CO, USA; catalog #NBP2-59627), mouse anti-L (Promega; catalog #N710A), or rabbit anti-GFP antibodies (Invitrogen; Carlsbad, CA, USA; catalog #A6455).

Techniques: Blocking Assay, Activity Assay, Luciferase, Transfection, Plasmid Preparation, Control, Standard Deviation

Journal: Viruses

Article Title: Development of a Cell-Based Luciferase Complementation Assay for Identification of SARS-CoV-2 3CL pro Inhibitors

doi: 10.3390/v13020173

Figure Lengend Snippet: Compounds screened for inhibition of SARS-CoV-2 3CL pro in cells.

Article Snippet: After transfer to PVDF membranes, blots were probed with 1:5000 rabbit anti-SARS-CoV 3CL pro (Rockland Immunochemicals; Limerick, PA, USA; catalog #200-401-A51), mouse anti-P2A (Novus Biologicals; Littleton, CO, USA; catalog #NBP2-59627), mouse anti-L (Promega; catalog #N710A), or rabbit anti-GFP antibodies (Invitrogen; Carlsbad, CA, USA; catalog #A6455).

Techniques: Inhibition

Journal: Viruses

Article Title: Development of a Cell-Based Luciferase Complementation Assay for Identification of SARS-CoV-2 3CL pro Inhibitors

doi: 10.3390/v13020173

Figure Lengend Snippet: Boceprevir, Z-FA-FMK, calpain inhibitor XII, and GRL-0496, inhibit SARS-CoV-2 3CL pro in a cell-based luciferase complementation assay. To determine if other compounds are active against SARS-CoV-2 3CL pro in cells, 293T cells were transfected with the S-L-GFP lentiviral vector, and DMSO (control) or compounds (0.1, 0.4, 1.6, 6.3, 25, or 100 µM) were added. NanoBiT activity (circle, left axis) and cell viability (measured with the CellTiter-Glo 2.0 assay; triangle, right axis) were analyzed 30 h post-transfection and are expressed as relative luciferase units (RLU). Cell viability data were normalized to the DMSO sample, which was set to 100%. NanoBiT data were normalized to the DMSO and 100 µM GC376 samples, which were set to 0 and 100%, respectively. The data represent the mean ± standard deviation of three independent experiments; ** p < 0.01, *** p < 0.001 (relative to no-drug; one-way ANOVA with Dunnett’s post-test).

Article Snippet: After transfer to PVDF membranes, blots were probed with 1:5000 rabbit anti-SARS-CoV 3CL pro (Rockland Immunochemicals; Limerick, PA, USA; catalog #200-401-A51), mouse anti-P2A (Novus Biologicals; Littleton, CO, USA; catalog #NBP2-59627), mouse anti-L (Promega; catalog #N710A), or rabbit anti-GFP antibodies (Invitrogen; Carlsbad, CA, USA; catalog #A6455).

Techniques: Luciferase, Transfection, Plasmid Preparation, Control, Activity Assay, Standard Deviation