In conclusion, the SARS-CoV-2 specific 3CLpro enzyme was used as a target to screen the potential drugs that have high binding affinity for 3CLpro since it plays a major role during viral replication. We have identified that boceprevir, micafungin, ombitasvir, paritaprevir, and tipranavir exhibited partial inhibitory effect, whereas ivermectin was able to completely inhibit the SARS-COV-2 3CLpro enzymatic activity in vitro at the tested doses. The 100 ns MD simulation studies suggest that the ivermectin may require homodimeric form of 3CLpro enzyme for its inhibitory activity. This could be due to the interaction of the amino acid residue, Ser1, from the neighboring monomer. On the other hand, micafungin remained bound in the catalytic pocket of the monomeric from of 3CLpro throughout the period of simulation. The list of drugs that are reported in this study provides a rationale to prioritize these potential drugs to be tested pre-clinically followed by clinical studies to target the SARS-Cov-2 pathogenesis.
Reagents and drugs Molecular biology grade DMSO was purchased from Sigma Aldrich (St. Louis, MO, USA), Sterile PBS was from ThermoFisher Scientific (Waltham, MA, USA). 3CLpro inhibitor screening enzymatic assay kits (Catalog #79955-1) were from BPS Biosciences (San Diego, CA, USA). Amprenavir, Atazanavir sulfate, Candicin, Chloroquine Phosphate, Hydroychloroquine Sulfate, and Lopinavir were purchased from Sigma Aldrich (Saint Louis, MO). Beclabuvir, Temsavir were from Medchem Express (Monmouth Junction, NJ). Abacavir (sulfate), Arbidol hydrochloride, Asunaprevir, Atrovastatin, Boceprevir, Daclatasvir, Danoprevir, Darunavir, Delavirdine (mesylate), Edoxudine, Elbasvir, Elvitegravir, Etravirine, Favipiravir, Fumagillin, Glecaprevir, Grazoprevir, Indinavir sulfate, Itraconazole, Ivermectin, Ledipasvir (G-5885), Maraviroc, Methylprednisolone, micafungin sodium, ombitasvir, Oseltamivir phosphate, Paritaprevir, Peramivir, Pibrentasvir, Pimodivir, Pleconaril, Posaconazole, Quinine, Raltegravir (potassium salt), Remdesivir, Ribavirin 5’-monophosphate (lithium salt), Ribostamycin sulfate, Rilpivirine, Saquinavir, Telaprevir, Tenofovir diphosphate (sodium salt), and Velpatasvir were purchased from Cayman Chemicals (Ann Arbor, MI). Preparation of working solution of ligands One mg of drug was used to prepare 8 mM stock solution using either DMSO or PBS as solvent. This stock solution was used to prepare the working solution of 250 μM and 500 μM of drugs in PBS. 3CLpro protein preparation The crystal structure of COVID-19 main protease was retrieved from the protein data bank (www.rcsb.org) with PDB format (ID: 6LU7). Any structural issues if present in the protein were corrected using QuickPrep option in MOE. The QuickPrep option performs a protonation and calculates the minimum energy conformation of the protein. Default parameters of MOE software was used for QuickPrep function. Preparation of ligands The drug list of 3987 FDA approved drug molecules and active ingredients were downloaded from SUPERDRUGS2 database in sdf format. The database of these 3987 drugs were imported into Molecular Operating Environment MOE and were cleaned using wash function in MOE. This function rebalances protonation states and regenerate 3D coordinates to their minimum energy conformations. Default parameters of MOE software was used for wash function. Protein:drug docking studies Integrated Computer-Aided Molecular design computing method Molecular Operating Environment (MOE) software was used to dock the drugs with 3CLpro protein. Briefly, docking was performed on the ligand site of 6LU7 protease using washed dataset of 3987 drugs. 30 poses for London dG and 20 poses of GBVI/WSA dG were used for final docking. All docking results were sorted by the binding energy using S Score function and they were viewed for accuracy using the ligand interaction function in MOE. The drugs with a binding score (S-Score) of ≤−6.5 were considered for further studies. The list was further narrowed down based on the listed criteria (1) All peptidomimetic drugs were eliminated as they tend to elicit immune response in the body; (2) Drugs used as immunosuppressants or for the treatment of cardiovascular disorders were eliminated as they tend to be in high risk categories; (3) All prodrugs were eliminated unless the active form was readily available as our in vitro assay is specific for SARS-COV-2 3CLpro and does not provide an option to activate drugs in vitro; and (4) Drugs with a shorter half-life of <30 min were also eliminated. In addition, we also ensured that small molecules which are part of any current Phase I, II, or III clinical trials were included in this study irrespective of their S-Score. For e.g favipiravir has an S score of −4.23 and since it is under clinical trials for COVID-19, we have included favipiravir in the study. This screening led to the database of 56 drugs, which were selected to study their inhibitory effect on SARS-CoV-2 specific 3CLpro enzyme using an in vitro 3CLpro enzymatic assay. 3CLpro enzymatic assay 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. Briefly, 4 ng 3CLpro-MBP tagged enzyme in 30 µl of assay buffer was pre-incubated with 10 µl of (250 µM) drugs for 1 h. The enzymatic reaction was initiated by adding 10 µl (250 µM) fluorescent substrate. The final volume of the assay samples was 50 µl. The final concentration of drugs and substrate in the reaction mixture was 50 µM. Incubation was continued at room temperature for 16–18 h. Fluorescence reading was taken at 360/40 excitation and 460/40 nm emission using Synergy HT fluorescent plate reader. For IC50 calculation, drugs were screened from 0 to 100 µM dose range. Wells with 1% DMSO with 4 ng of enzyme and 50 µM of substrate served as positive control with no enzyme inhibition. Wells with 50 µM of GC367 compound (provided by the BPS Biosciences) served as standard inhibitor and negative control. Wells with 1% DMSO with 50 µM of substrate without enzyme served as blank. All the values were subtracted from blank values. Molecular dynamic simulation studies The structure of COVID-19 main protease 6LU7 (PDB ID: 6LU7) was prepared in its monomeric and functional dimeric form using the QuickPrep application of MOE2019 with default parameters. This atomistic model was used for generating the input files for all MD simulations. The best dock pose of micafungin was placed into the binding pocket of the prepared monomeric 3CLprotease structure. The best pose of ivermectin was placed into the monomeric and dimeric protease structure to test which form can stabilize the inhibitor. The simulation cell and NAMD 2.1439 input files were generated using MOE2019. The crystallographic water molecules were removed prior to solvation. Next, the protein/ligand complexes were embedded in a TIP3P water box with cubic periodic boundary conditions, keeping a distance of 10 Å between the boundaries and the protein. The net charge of the protein was neutralized with 100 mM NaCl. For energy minimization and MD simulations, the AMBER10:EHT force field was used and the electrostatic interactions were evaluated by the particle-mesh Ewald method. Each system was energy-minimized for 5000 steps using the Steepest Descent and Conjugate Gradient method. For equilibration the system was subjected to a 100-ps simulation to gradually heating the system from 10 K to 300 K. Next, a 100-ps NVT ensemble was generated at 300 K followed by an NPT ensemble for 200 ps at 300 K and 1 bar. Then, for each complex, a 100-ns production trajectory was generated for further analysis. The trajectory analysis was done using scripts shared by the CCG support group. Identification of ligand-binding mode The protein ligand interaction fingerprint application in MOE2019 was used to study the average binding mode of each inhibitor bound to the monomer or dimer of 6LU7. The calculation used the default parameters on the recorded MD trajectories. Statistics and reproducibility One-way analysis of variance (ANOVA) with Dunnett’s Multiple Comparison post-hoc test was performed with 99.9% confidence intervals to compare the statistical significance and represented as the mean ± SEM. P values < 0.001 considered statistically significant. Non-linear regression (curve fit) with four variable dose vs inhibition was used to calculate the IC50 values. Statistical analysis was performed using GraphPad Prism (version 6.07; La Jolla, CA, USA). All the experiments were carried out minimum three times with triplicates for reproducibility and the representative of three individual experiments is presented in this report. The data generated at different time points were combined to make the final graphs. Investigators performing the assay were blinded for the drugs being tested in the assay.
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