SARS-COV-2 3CL-PROTEASE INHIBITORS AS ANTIVIRAL AGENT AGAINST COVID-19

The SARS-CoV-2 virus causes coronavirus, and the pandemic has led to efforts to develop appropriate drugs for treatment. Understanding the structure and function of SARS-CoV-2 3CL is crucial in unlocking ways of developing effective drugs. Some studies have described the structure of the protease at the DNA and protein levels. Notably, two important proteases help in the drug development process: PLpro and 3CLpro. The 3CLpro, for instance, is helpful in viral replication alongside transcription. The PL is associated with NsP3, a multi-domain protein part of the viral replication and transcription complex which cleaves peptide bonds at specific sites. In vitro studies have shown that SARS-CoV-2 3CL-protease inhibitors can contribute to antiviral drug development, especially MG-132, boceprevir, telaprevir, and calpain, which are protein inhibitors with lethal dose values appropriate for drug development. In contrast, there are very limited studies in vivo reporting the appropriateness of protease inhibitors in antiviral drug development

Inhibition of SARS-CoV 3C-like Protease Activity by Theaflavin-3,3′-digallate (TF3)

SARS-CoV is the causative agent of severe acute respiratory syndrome (SARS). The virally encoded 3C-like protease (3CL(Pro)) has been presumed critical for the viral replication of SARS-CoV in infected host cells. In this study, we screened a natural product library consisting of 720 compounds for inhibitory activity against 3CL(Pro). Two compounds in the library were found to be inhibitive: tannic acid (IC(50) = 3 µM) and 3-isotheaflavin-3-gallate (TF2B) (IC(50) = 7 µM). These two compounds belong to a group of natural polyphenols found in tea. We further investigated the 3CL(Pro)-inhibitory activity of extracts from several different types of teas, including green tea, oolong tea, Puer tea and black tea. Our results indicated that extracts from Puer and black tea were more potent than that from green or oolong teas in their inhibitory activities against 3CL(Pro). Several other known compositions in teas were also evaluated for their activities in inhibiting 3CL(Pro). We found that caffeine, (—)-epigallocatechin gallte (EGCg), epicatechin (EC), theophylline (TP), catechin (C), epicatechin gallate (ECg) and epigallocatechin (EGC) did not inhibit 3CL(Pro) activity. Only theaflavin-3,3′-digallate (TF3) was found to be a 3CL(Pro) inhibitor. This study has resulted in the identification of new compounds that are effective 3CL(Pro) inhibitors

HIGH-THROUGHPUT SCREENING AND DYNAMIC STUDIES OF SELECTED COMPOUNDS AGAINST SARS-COV-2

Objective: This study was aimed to analyze the inhibitory effect of the drugs used in nanocarrier as well as nanoparticles formulation based drug delivery system selected from PubChem database literature against 3CLpro (3C-like protease) receptor of SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) by implementing several in silico analysis techniques. Methods: This paper detailed a molecular docking-based virtual screening of 5240 compounds previously utilized in nanoparticle and nanocarrier drug delivery systems utilizing AutoDock Vina software on 3CL protease to discover potential inhibitors using a molecular docking technique. Results: According to the results of the screening, the top two compounds, PubChem Id 58823276 and PubChem Id 60838 exhibited a high affinity for the 3CL protease binding region. Their binding affinities were-9.6 and-8.5 kJ/mol, indicating that they were tightly bound to the target receptor, respectively. These results outperformed those obtained using the co-crystallized native ligand, which exhibited a binding affinity of-7.4 kJ/mol. PubChem Id 60838, the main hit compound in terms of both binding affinity and ADMET analysis, displayed substantial deformability after MD simulation. As a result of the VS and molecular docking techniques, novel 3CL protease inhibitors from the PubChem database were discovered using the Lipinski rule of five and functional molecular contacts with the target protein, as evidenced by the findings of this work. Conclusion: The findings suggest that the compounds discovered may represent attractive opportunities for the development of COVID-19 3CLpro inhibitors and that they need further evaluation and investigation

The Potential of <i>Clerodendrum paniculatum</i> Leaves Fraction as a 3-Chymotrypsin-Like (3CL) Protease Inhibitor of SARS-CoV-2

We described the biological activity of the Clerodendrum paniculatum leaf fraction against the SARS-CoV-2 3-Chymotrypsin-like 3CL protease at the molecular level. This study applied LC-MS/MS to identify bioactive compounds from fractions, computational studies, and fluorescence resonance energy transfer (FRET) assays to ascertain their inhibitory activity. LC-MS/MS analysis of the three samples revealed that sample 1 contained 18 compound peaks. In samples 2 and 3, there were 23 and 25 compounds with different molecular weights, respectively. Docking's study identified that the alkaloids (komarovicine and roemerine) have lower binding energies than other metabolites and standard compounds, with values of -33.47 and -32.63 kJ/mol, respectively. Roemerine demonstrated excellent stability based on dynamic simulation results and confirmed its affinity for 3CL protease predicted by the MM-PBSA approach of -89.44 kJ/mol. The FRET method for testing 3CL protease activity revealed that sample 2 had an enzyme inhibitory activity of 94.3%, which was close to that of GC376 (98.19%). Meanwhile, samples 1 and 3 yielded satisfactory inhibition activity by 89.64% and 85.24%, respectively. The antiviral activity of C. paniculatum leaves was discovered for the first time by inhibiting the 3CL protease SARS-CoV-2, providing an excellent opportunity for its development as an anti-SARS-CoV-2

Production of a functionally active recombinant SARS-CoV-2 (COVID-19) 3C-Like protease and a soluble inactive 3C-like protease-RBD chimeric in a prokaryotic expression system

During the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) intracellular life-cycle, two large polyproteins, pp1a and pp1ab, are produced. Processing of these by viral cysteine proteases, the papain-like protease (PLpro) and the chymotrypsin-like 3C-like protease (3CL-pro) release non-structural proteins necessary for the establishment of the viral replication and transcription complex (RTC), crucial for viral replication. Hence, these proteases are considered prime targets against which anti-coronavirus disease 2019 (COVID-19) drugs could be developed. Here, we describe the expression of a highly soluble and functionally active recombinant 3CL-pro using Escherichia coli BL21 cells. We show that the enzyme functions in a dimeric form and exhibits an unexpected inhibitory profile because its activity is potently blocked by serine rather than cysteine protease inhibitors. In addition, we assessed the ability of our 3CL-pro to function as a carrier for the receptor binding domain (RBD) of the Spike protein. The co-expressed chimeric protein, 3CLpro-RBD, did not exhibit 3CL-pro activity, but its enhanced solubility made purification easier and improved RBD antigenicity when tested against serum from vaccinated individuals in ELISAs. Chimeric proteins containing the 3CL-pro could represent an innovative approach to developing new COVID-19 vaccines

Profiling of Substrate Specificities of 3C-Like Proteases from Group 1, 2a, 2b, and 3 Coronaviruses

BACKGROUND: Coronaviruses (CoVs) can be classified into alphacoronavirus (group 1), betacoronavirus (group 2), and gammacoronavirus (group 3) based on diversity of the protein sequences. Their 3C-like protease (3CL(pro)), which catalyzes the proteolytic processing of the polyproteins for viral replication, is a potential target for anti-coronaviral infection. METHODOLOGY/PRINCIPAL FINDINGS: Here, we profiled the substrate specificities of 3CL(pro) from human CoV NL63 (group 1), human CoV OC43 (group 2a), severe acute respiratory syndrome coronavirus (SARS-CoV) (group 2b) and infectious bronchitis virus (IBV) (group 3), by measuring their activity against a substrate library of 19 × 8 of variants with single substitutions at P5 to P3' positions. The results were correlated with structural properties like side chain volume, hydrophobicity, and secondary structure propensities of substituting residues. All 3CL(pro) prefer Gln at P1 position, Leu at P2 position, basic residues at P3 position, small hydrophobic residues at P4 position, and small residues at P1' and P2' positions. Despite 3CL(pro) from different groups of CoVs share many similarities in substrate specificities, differences in substrate specificities were observed at P4 positions, with IBV 3CL(pro) prefers P4-Pro and SARS-CoV 3CL(pro) prefers P4-Val. By combining the most favorable residues at P3 to P5 positions, we identified super-active substrate sequences 'VARLQ↓SGF' that can be cleaved efficiently by all 3CL(pro) with relative activity of 1.7 to 3.2, and 'VPRLQ↓SGF' that can be cleaved specifically by IBV 3CL(pro) with relative activity of 4.3. CONCLUSIONS/SIGNIFICANCE: The comprehensive substrate specificities of 3CL(pro) from each of the group 1, 2a, 2b, and 3 CoVs have been profiled in this study, which may provide insights into a rational design of broad-spectrum peptidomimetic inhibitors targeting the proteases

Profiling of Substrate Specificity of SARS-CoV 3CLpro

BACKGROUND: The 3C-like protease (3CL(pro)) of severe acute respiratory syndrome-coronavirus is required for autoprocessing of the polyprotein, and is a potential target for treating coronaviral infection. METHODOLOGY/PRINCIPAL FINDINGS: To obtain a thorough understanding of substrate specificity of the protease, a substrate library of 198 variants was created by performing saturation mutagenesis on the autocleavage sequence at P5 to P3' positions. The substrate sequences were inserted between cyan and yellow fluorescent proteins so that the cleavage rates were monitored by in vitro fluorescence resonance energy transfer. The relative cleavage rate for different substrate sequences was correlated with various structural properties. P5 and P3 positions prefer residues with high β-sheet propensity; P4 prefers small hydrophobic residues; P2 prefers hydrophobic residues without β-branch. Gln is the best residue at P1 position, but observable cleavage can be detected with His and Met substitutions. P1' position prefers small residues, while P2' and P3' positions have no strong preference on residue substitutions. Noteworthy, solvent exposed sites such as P5, P3 and P3' positions favour positively charged residues over negatively charged one, suggesting that electrostatic interactions may play a role in catalysis. A super-active substrate, which combined the preferred residues at P5 to P1 positions, was found to have 2.8 fold higher activity than the wild-type sequence. CONCLUSIONS/SIGNIFICANCE: Our results demonstrated a strong structure-activity relationship between the 3CL(pro) and its substrate. The substrate specificity profiled in this study may provide insights into a rational design of peptidomimetic inhibitors