3clpro activity assay  (BPS Bioscience)

Journal: Biochemical and Biophysical Research Communications

Article Title: Highly polymerized proanthocyanidins (PAC) components from blueberry leaf and stem significantly inhibit SARS-CoV-2 infection via inhibition of ACE2 and viral 3CLpro enzymes

doi: 10.1016/j.bbrc.2022.04.072

Figure Lengend Snippet: Inhibitory activity of ACE2, BSS, and virus-derived 3CL protease enzymes by BB-PAC. A. ACE2 enzyme activity by BB-PAC fractions of Fr1(dotted line) as a control and Fr7 (solid line) at several different concentrations was measured by the ACE2 activity assay kit (BPS Bioscience). The right figure shows the results using the blueberry leaf fraction and the left figure shows the results using the blueberry stem fraction. B. TMPRSS2 enzyme activity by BB-PAC fractions of Fr1(dotted line) as a control and Fr7 (solid line) at several different concentrations was measured by the TMPRSS2 activity assay kit (BPS Bioscience). C. 3CLpro enzyme activity by BB-PAC fractions of Fr1(dotted line) as a control and Fr7 (solid line) at several different concentrations was measured by the 3CLpro activity assay kit (BPS Bioscience).

Article Snippet: C. 3CLpro enzyme activity by BB-PAC fractions of Fr1(dotted line) as a control and Fr7 (solid line) at several different concentrations was measured by the 3CLpro activity assay kit (BPS Bioscience).

Techniques: Activity Assay, Derivative Assay

Journal: Communications Biology

Article Title: Identification of 3-chymotrypsin like protease (3CLPro) inhibitors as potential anti-SARS-CoV-2 agents

doi: 10.1038/s42003-020-01577-x

Figure Lengend Snippet: Genetic material (RNA) of SARS-CoV-2 enters the host cell and borrows ribosome to translate into 16 nonstructural proteins upon auto-proteolytic cleavage by PLpro and 3CLpro enzymes. Structural proteins (S, E, M, and N) are translated by sg translation mechanism. All mature proteins assemble along with positive-sense single stranded RNA genome to form new virion. Arrows indicates the protease cleavage sites. ORF open reading frame, PLP papain like protease, 3CLP 3-chymotrypsin like protease, S spike protein, E envelop protein, M membrane protein, N nucleocaspid protein.

Article Snippet: SARS-CoV-2 specific 3CLpro assay kits were purchased from BPS Biosciences (CA) and enzymatic assay was carried out as per the manufacturer’s protocol using 96 well plates.

Techniques:

Journal: Communications Biology

Article Title: Identification of 3-chymotrypsin like protease (3CLPro) inhibitors as potential anti-SARS-CoV-2 agents

doi: 10.1038/s42003-020-01577-x

Figure Lengend Snippet: List of all the drugs with highest S score for 3CL protease calculated by computational studies (S score).

Article Snippet: SARS-CoV-2 specific 3CLpro assay kits were purchased from BPS Biosciences (CA) and enzymatic assay was carried out as per the manufacturer’s protocol using 96 well plates.

Techniques: Binding Assay

Journal: Communications Biology

Article Title: Identification of 3-chymotrypsin like protease (3CLPro) inhibitors as potential anti-SARS-CoV-2 agents

doi: 10.1038/s42003-020-01577-x

Figure Lengend Snippet: The selected FDA approved viral-protease inhibitors were screened for their inhibitory activity against SARS-CoV-2 3CLpro enzyme as described under Methods section. The fluorescence units were converted to percent enzymatic activity considering DMSO treated control as 100% activity. Blank values were subtracted from all the readings before calculating the percent activity. Representative of three individual experiments with triplicate values were presented graphically ( n = 3). P value < 0.001 considered as statistically significant. One-way ANOVA with Dunnett’s Multiple Comparison post-hoc test was used to calculate the statistical significance.

Article Snippet: SARS-CoV-2 specific 3CLpro assay kits were purchased from BPS Biosciences (CA) and enzymatic assay was carried out as per the manufacturer’s protocol using 96 well plates.

Techniques: Activity Assay, Fluorescence

Journal: Communications Biology

Article Title: Identification of 3-chymotrypsin like protease (3CLPro) inhibitors as potential anti-SARS-CoV-2 agents

doi: 10.1038/s42003-020-01577-x

Figure Lengend Snippet: The non-protease anti-viral drugs selected by computational studies were screened for their inhibitory activity against SARS-CoV-2 3CLpro enzyme as described under Methods section. The percent enzymatic activity was calculated as described in Fig. legend. Blank values were subtracted from all the readings before calculating the percent activity. Representative of three individual experiments with triplicate values were presented graphically ( n = 3). P value < 0.001 considered as statistically significant. One-way ANOVA with Dunnett’s Multiple Comparison post-hoc test used to calculate the statistical significance.

Article Snippet: SARS-CoV-2 specific 3CLpro assay kits were purchased from BPS Biosciences (CA) and enzymatic assay was carried out as per the manufacturer’s protocol using 96 well plates.

Techniques: Activity Assay

Journal: Communications Biology

Article Title: Identification of 3-chymotrypsin like protease (3CLPro) inhibitors as potential anti-SARS-CoV-2 agents

doi: 10.1038/s42003-020-01577-x

Figure Lengend Snippet: The off-target drugs that are being used to treat non-viral ailments selected by in silico studies were screened for their inhibitory activity against SARS-CoV-2 3CLpro enzyme as described under Methods section. The percent enzymatic activity was calculated as described in Fig. legend. Blank values were subtracted from all the readings before calculating the percent activity. Representative of three individual experiments with triplicate values were presented graphically ( n = 3). P value < 0.001 considered as statistically significant. One-way ANOVA with Dunnett’s Multiple Comparison post-hoc test used to calculate the statistical significance.

Article Snippet: SARS-CoV-2 specific 3CLpro assay kits were purchased from BPS Biosciences (CA) and enzymatic assay was carried out as per the manufacturer’s protocol using 96 well plates.

Techniques: In Silico, Activity Assay

Journal: Communications Biology

Article Title: Identification of 3-chymotrypsin like protease (3CLPro) inhibitors as potential anti-SARS-CoV-2 agents

doi: 10.1038/s42003-020-01577-x

Figure Lengend Snippet: Inhibitors were docked with active site of 3CLpro protein. Panel-I: ligand interaction map. Panel-II: interaction of inhibitors specific amino acids of 3CLpro at active site. Panel-III: lipophilic cavity of active site with drugs interacting with specific amino acids. Boceprevir ( a ), partapravir ( b ), tipranavir ( c ), ombitasvir ( d ), ivermectin ( e ), and micafungin ( f ) are arranged in columns for comparison. Drugs are represented in green color with ball and stick model. Arrows indicate the C–H, N–H, and C–O bonds between drugs and with Cys 145 , His 41 , and Glu 166 residues since they are essential for the enzymatic activity of 3CLpro enzyme. However, we also observed the drugs interacting with neighboring amino acid residues.

Article Snippet: SARS-CoV-2 specific 3CLpro assay kits were purchased from BPS Biosciences (CA) and enzymatic assay was carried out as per the manufacturer’s protocol using 96 well plates.

Techniques: Activity Assay

Journal: Communications Biology

Article Title: Identification of 3-chymotrypsin like protease (3CLPro) inhibitors as potential anti-SARS-CoV-2 agents

doi: 10.1038/s42003-020-01577-x

Figure Lengend Snippet: MD simulation studies were carried as described under Methods section. Interaction of micafungin ( a ), and ivermectin ( b ) with monomeric form of 3 CLpro enzyme. c Interaction of ivermectin with active site of 3CLpro homodimer. Panel I–III represents the interaction of ligand at different time points in nano second (ns). Panel-IV represents the ligand-binding fingerprint of micafungin and ivermectin with specific amino acids of 3CLpro enzymes.

Article Snippet: SARS-CoV-2 specific 3CLpro assay kits were purchased from BPS Biosciences (CA) and enzymatic assay was carried out as per the manufacturer’s protocol using 96 well plates.

Techniques: Ligand Binding Assay

Journal: Communications Biology

Article Title: Identification of 3-chymotrypsin like protease (3CLPro) inhibitors as potential anti-SARS-CoV-2 agents

doi: 10.1038/s42003-020-01577-x

Figure Lengend Snippet: Hypothetical model illustrating the inhibition of SARS-CoV-2 replication by ivermectin mediated through the blocking of α/β1-importin (imp) as well as 3CLpro enzymatic activity.

Article Snippet: SARS-CoV-2 specific 3CLpro assay kits were purchased from BPS Biosciences (CA) and enzymatic assay was carried out as per the manufacturer’s protocol using 96 well plates.

Techniques: Inhibition, Blocking Assay, Activity Assay