Inhibition of SARS-CoV-2 3CLpro enzymatic activity by selected drug candidates

To further validate the SARS-CoV-2 3CLpro inhibitory activity of the selected drugs from computational studies, we performed an in vitro enzymatic inhibitory assay using commercially available assay kits. The background autofluorescence of the selected 56 compounds was measured by excitation/emission wavelength of 360/460 nm. Nine out of 56 drugs exhibited extremely high autofluorescence (Supplementary Fig. 2) and were therefore eliminated from the in vitro protease inhibitor enzymatic assay. The remaining 47 drugs were sorted as PI, VNIs, and OTDs. The specific mechanism of action and their clinical uses are listed in Supplementary Table 1. The 3CLpro inhibitory activity was screened at 50 µM concentration of the drugs. Among the 17 PIs screened, boceprevir, paritaprevir and tipranavir significantly inhibited the 3CLpro enzymatic activity by 45, 70, and 64%, respectively (Fig. 2). Out of the 17 VNIs, only ombitasvir was able to partially block (65%) the 3CLpro enzymatic activity (Fig. 3). Interestingly, as shown in Fig. 4, out of 13 OTDs only ivermectin completely blocked (>80%) the 3CLpro activity at 50 µM concentration. Additionally, micafungin exhibited partial inhibitory activity (57%) against 3CLpro of SARS-CoV-2.


Inhibition of SARS-CoV-2 3CLpro enzymatic activity by selected drug candidates

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.


Inhibition of SARS-CoV-2 3CLpro enzymatic activity by selected drug candidates

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. 1 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.


Inhibition of SARS-CoV-2 3CLpro enzymatic activity by selected drug candidates

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. 1 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.

The compounds that exhibited more than 50% inhibitory activity were subjected to subsequent dose-dependent studies to calculate the concentration required to inhibit 50% of the 3CLpro enzymatic activity (IC50). Boceprevir, ivermectin, micafungin, ombitasvir, paritaprevir, and tipranavir were subjected to dose-dependent inhibitory activity studies. As shown in the Fig. 5, ivermectin inhibited more than 85% of the enzymatic activity at 50 µM concentration, whereas micafungin and paritaprevir inhibited around 80% of the enzymatic activity at 100 µM concentration. Both tipranavir and ombitasvir were able to inhibit only 50% of the enzymatic activity even at 100 µM concentration (Fig. 5). The percent enzymatic activity versus the log concentration of the inhibitors was used to calculate the IC50 values using non-linear curve fit model as described under Methods section. The calculated IC50 values for ivermectin, tipranavir, boceprevir, micafungin, paritaprevir, and ombitasvir were found to be 21.5, 27.7, 31.4, 47.6, 73.4, and 75.5 µM, respectively (Table 2). Taken together, these studies suggest that the molecules listed above exhibited inhibitory activity against 3CLpro enzyme of SARS-CoV-2.

Results

All the 3987 FDA approved drugs (downloaded from SuperDrugs2) were sorted as PIs, VNIs, and OTDs and docked with monomeric form of 3CLpro protein using the Molecular Operating Environment (MOE) software. Drugs were ranked according to the highest binding energy (S score). S score was calculated using the London dG score for placement and GBVI/WSA dG score for refinement of poses. A binding energy with ≤−6.5 kcal/mol (S score) was selected as a cutoff to rank the drugs with highest binding affinity16. The list was further narrowed down based on the criteria listed in the Methods section. We found that 56 drugs have a binding affinity of <−6.50 kcal/mol for the 3CLpro enzyme (Table 1). The computational study suggested that the list of drugs identified might inhibit the SARS-CoV-2 viral replication by targeting the viral 3CL protease. However, the potential for false positives with the predicted binding score is one of the most common limitation of docking studies24,25. Hence, to rule out any ambiguity in false prediction, we carried out the in vitro enzymatic assay to identify potential therapeutics and investigate correlation between the binding score and the in vitro activity. In the current study, we did not find any correlation between the in vitro results of selected drugs and their computational inhibition constants. Even though, computational studies are being widely used to predict the initial protein-drug interactions, in vitro screening of the drugs is necessary to confirm the inhibitory activities of the drugs.


Credited to communication biology


 


2 views0 comments