Targeting allosteric pockets of SARS-CoV-2 main protease Mpro

Repurposing of antivirals is an attractive therapeutic option for the treatment of COVID-19. Mpro (also called 3CLpro) is a key protease of SARS-CoV-2 involved in viral replication, and is a promising drug target for testing the existing antivirals. A major challenge to test the efficacy of antivirals is the conformational plasticity of Mpro and its future mutation prone flexibility. To address this, we hereby propose combination therapy by drugging two specific additional pockets of Mpro probed in our studies. Long scale Molecular Dynamics (MD) simulations provide evidence of these additional sites being allosteric. Suitable choice of drugs in catalytic and allosteric pockets appear to be essential for combination therapy. Current study, based on docking and extensive set of MD simulations, finds the combination of Elbasvir, Glecaprevir, Ritonavir to be a viable candidate for further experimental drug testing/pharmacophore design for Mpro.

The severe acute respiratory syndrome (SARS) coronavirus (CoV) main protease represents an attractive target for the development of novel anti-SARS agents. The tertiary structure of the protease consists of two distinct folds. One is the N-terminal chymotrypsin-like fold that consists of two structural domains and constitutes the catalytic machinery; the other is the C-terminal helical domain, which has an unclear function and is not found in other RNA virus main proteases. To understand the functional roles of the two structural parts of the SARS-CoV main protease, we generated the full-length of this enzyme as well as several terminally truncated forms, different from each other only by the number of amino acid residues at the C- or N-terminal regions. The quaternary structure and Kd value of the protease were analyzed by analytical ultracentrifugation. The results showed that the N-terminal 1–3 amino acid-truncated protease maintains 76% of enzyme activity and that the major form is a dimer, as in the wild type. However, the amino acids 1–4-truncated protease showed the major form to be a monomer and had little enzyme activity. As a result, the fourth amino acid seemed to have a powerful effect on the quaternary structure and activity of this protease. The last C-terminal helically truncated protease also exhibited a greater tendency to form monomer and showed little activity. We concluded that both the C- and the N-terminal regions influence the dimerization and enzyme activity of the SARS-CoV main protease.

Severe acute respiratory syndrome (SARS) is an emerging infectious disease caused by a novel human coronavirus (CoV), which infected more than 8000 people during the 2003 outbreak. The viral maturation requires a main protease (3CLpro) to cleave the virus-encoded polyproteins. Accordingly, in human picornavirus (PV) family which consists of over 200 medically relevant viruses, a chymotrypsin-like protease (3Cpro) is required for viral replication through the processing the polyproteins. As a result, the 3CLpro and 3Cpro are regarded as anti-viral drug targets. However, known inhibitors (AG7088) against PV 3Cpro failed to inhibit SARS-CoV 3CLpro, indicating differences in their active-site structures. In this study, we have prepared the recombinant SARS 3CLpro without redundant residues at both N- and C-termini and characterized its kinetic property using a fluorogenic substrate. Combined with our crystallography data, we proposed a model to illustrate the maturation process of SARS 3CLpro. In addition, we evaluated several types of inhibitors and investigated their inhibitory mechanisms. Additionally, the recombinant 3Cpro from PV (including entervirus, coxsackievirus, and rhinovirus, abbreviated as EV, CV, and RV, respectively) were purified and characterized as well. We have identified several inhibitors which show their potencies against viral replication. Furthermore, we tested 6800 small molecules by high-throughput screening for anti-SARS agents and found one hit and its analogues could serve as the common inhibitors against CoV 3CLpro and PV 3Cpro. By computer modeling, the structural features of these compounds were elucidated to enhance our knowledge for developing anti-viral agents against PV and CoV. In order to determine the amino acid residues essential for the substrate specificity and engineer 3CLpro as a tool for tag removal of the recombinant fusion proteins, we developed a mutant 3CLpro (T25G) which has altered substrate specificity to cleave Gln↓Met. We have also constructed E. coli and yeast vectors to express recombinant fusion proteins with the T25G 3CLpro recognition site (Ala-Val-Leu-Gln↓Met) between the tags and the target proteins for tag removal.

Aim. To provide a brief literature review regarding the structure of the human coronavirus SARS-CoV-2, the mechanism of its replication and the role of viral proteases in this process; to analyze the ability of the known antiviral agents and compounds synthesized de novo in order to bind and inhibit the coronavirus main protease using computer simulation tools.Results and discussion. COVID-19 coronavirus has become a worldwide challenge in recent months. Taking into account the rapid spread and severity of COVID-19 among a significant part of the population there is an urgent need to develop effective medicines and appropriate treatment protocols, which, unfortunately, are not yet available. Currently, the search for molecules with an acceptable toxicity profile that are able to inhibit and/or stop coronavirus SARS-CoV-2 replication in the human body is very relevant. In this study, the virtual screening and molecular docking of both antiviral agents known and new compounds synthesized have been performed based on the structure of the main protease Mpro of SARS-CoV-2. The regularities identified during our study can be useful for searching and developing new antiviral drugs to control COVID-19 and other coronavirus infections. The analysis of the results of calculations of physicochemical characteristics of antiviral agents, as well as the determination of their binding sites with the main viral protease Mpro gives an optimistic assessment of the possibility to develop new drugs based on the structures of the known antiviral drugs or their modified analogs.Experimental part. Based on recent studies of the crystal structure of the virus main protease Mpro in the complex with various inhibitors (Protein Data Bank http://www.rcsb.org/pdb, the structure code – 6LU7) the virtual screening and molecular docking of 100 known antiviral agents and 50 novel compounds synthesized were performed. The screening data for the in vitro antimalarial activity of the compounds synthesized were presented. The following binding and physicochemical parameters of the ligand–protein interaction for all virus main protease potential inhibitors were calculated: binding affinity score (BAS), binding energy, lipophilicity (clogP) and topological polar surface area (TPSA). The protein and ligand structures were studied using Jmol, PyMol, and Avogadro graphics software packages. The virtual screening and molecular docking, as well as the analysis of the results were performed using a LigandScout 4.4 software package. Data on the antimalarial activity of 50 compounds synthesized were obtained from the Laboratory of Microbiology, Parasitology and Hygiene of theUniversity ofAntwerp (Belgium).Conclusions. According to the results of the virtual screening and molecular docking with protein 6LU7 it has been found that a number of the known antiviral drugs have a certain potential for their use as inhibitors of SARS-CoV-2 coronavirus main protease. Remdesivir and ritonavir substances have shown higher activity than the reference compound of the 6LU7 complex. The molecular docking of a series of compounds recently synthesized with the proven in vitro antimalarial activity has revealed that L1 – L6 compounds are promising candidates for further modification and development of new antiviral drugs to control coronavirus infection.Received: 02.04.2020Revised: 23.05.2020Accepted: 29.05.2020

Insights into the structural properties of SARS-CoV-2 main protease.

Background: Molecular docking has been used recently in pharma industry for drug designing, it’s a powerful tool to find ligand-substrate interactions at molecules level. Since urgent need to develop anti-viral drug that target new coronavirus main proteins, in silico docking has been used to achieve this purpose.
 Materials and Methods: Thirteen herbs are known for their antioxidants and antiviral properties have been selected to investigate their abilities in inhibiting SARS-COV2 spike protein and main protease Mpro. pdb files for RBD (Receptor Binding Domain) region of spike protein and for Mpro and mol2 files for all herbs understudy were uploaded for swiss dock online server, the docking results were analyzed using chimera software. Full fitness energy and hydrogens bonds interactions were considered for docking evaluation. Pharma kinetic properties for compounds have good binding results were evaluated through AMES and ADMET tests.
 Results: All compounds showed negative full fitness energy that means they are able to complex with both SARS-COV2 spike protein and main protease, however some of the herbs form very powerful hydrogen bonding with the RBD site of the spike protein and the catalytic site of Mpro such as coumarylquinic acid, while stigmasterol has strong binding with the spike protein only. Both compounds appear to be safe drugs for human according to AMES test results.
 Conclusion: Coumarylquinic acid and stigmasterol have powerful binding in silico, further in vitro studies include using viral infected human lung cells and testing above compounds ability for inhibiting viral entry and replication should be proceed to confirm the study results.

Protein structural heterogeneity: A hypothesis for the basis of proteolytic recognition by the main protease of SARS-CoV and SARS-CoV-2

The main proteases (Mpro), also termed 3‐chymotrypsin‐like proteases (3CLpro), are a class of highly conserved cysteine hydrolases in β‐coronaviruses. Increasing evidence has demonstrated that 3CLpros play an indispensable role in viral replication and have been recognized as key targets for preventing and treating coronavirus‐caused infectious diseases, including COVID‐19. This review is focused on the structural features and biological function of the severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) main protease Mpro (also known as 3CLpro), as well as recent advances in discovering and developing SARS‐CoV‐2 3CLpro inhibitors. To better understand the characteristics of SARS‐CoV‐2 3CLpro inhibitors, the inhibition activities, inhibitory mechanisms, and key structural features of various 3CLpro inhibitors (including marketed drugs, peptidomimetic, and non‐peptidomimetic synthetic compounds, as well as natural compounds and their derivatives) are summarized comprehensively. Meanwhile, the challenges in this field are highlighted, while future directions for designing and developing efficacious 3CLpro inhibitors as novel anti‐coronavirus therapies are also proposed. Collectively, all information and knowledge presented here are very helpful for understanding the structural features and inhibitory mechanisms of SARS‐CoV‐2 3CLpro inhibitors, which offers new insights or inspiration to medicinal chemists for designing and developing more efficacious 3CLpro inhibitors as novel anti‐coronavirus agents.

Binding kinetics of ten small-molecule drug candidates on SARS-CoV-2 3CLpro revealed by biomolecular simulations

Black queen cell virus is one of the members belonging to Picornavirus, which possesses a 3C-like protease. Viral replication and capsid assembly in the order Picornavirales require polyprotein proteolysis processing, which is performed by 3C or 3C-like (3CL) proteases. Therefore, viral proteases are attractive targets for anti-viral therapy. The complete 3C-like protease gene from Black queen cell virus was cloned into pGEM-3Zf(+) vector, and the amino acid sequence revealed a high homology (99.1%) to 3C-like protease deposited in a reference Genbank EF517515.1. For the expression, 3C-like protease gene was subcloned into pMAL-C2 vector system. The expression level of MBP-BQCV-3CL protein was evaluated under different standards of cell optical density, IPTG concentration, induction time, and induction temperature. As the result, the optimal condition was confirmed as the induction with 0.1mM IPTG at 25°C for 6 hours, and a purification step using amylose resin reached a high protein yield of approximately 2.22mg per 1ml of cultured cells. Purified MBP-BQCV-3CL proteins can provide an important antigen source to generate monoclonal antibody and will become a potential candidate for anti-viral drugs, which can inactivate 3C-like protease function as well as BQCV replication.

CORDITE: The Curated CORona Drug InTERactions Database for SARS-CoV-2.

New cadmium(II) and zinc(II) complexes (1–6) with two NS-donor thiosemicarbazones, HL1 (benzaldehyde-3-hexamethyleneiminyl), HL2 (3-methoxybenzaldehyde-3-hexamethyleneiminyl) and H2L3 (salicylaldehyde-3-tetramethyleneiminyl, an ONS donor) were synthesized using pyridine, γ-picoline, and 2,2′-bipyridine as coligands. Coordination modes were confirmed through elemental analysis, FT-IR, 1H NMR, and UV–vis spectroscopy. Using density functional theoretical (DFT) study, frontier molecular orbital analysis of all the compounds were carried out and the quantum chemical parameters were also calculated. Molecular docking study indicates that compared to the three ligands all the new complexes are having greater binding affinity against the SARS-CoV-2 main protease Mpro. Moreover, the complex 6 is found to have better propensity by the DFT and molecular docking in silico studies.

For over two years, COVID-19 pandemic has been a major global health concern and threat to human life. In the SARS-CoV2 macromolecules, the 3-chymotrypsin like protease (3CLpro or main protease) has been identified to be crucial and essential for viral survival, processing of the viral polyproteins and has been explored as a target in COVID-19 drug discovery. Although vaccines and other various inhibitors have been designed and launched, the emergence of the variant of this virus has put an unrelenting effort of researchers to this end. Also, the high cost of manufacturing these molecules coupled with the occurrence of drug resistance is a concern. Herein, Pefloxacin and its derivative for the first time were screened for their inhibitory activity against the SARS-CoV2 main protease through in silico analysis and their pharmacokinetic properties were evaluated. Interestingly, from the docking results, they both bind with high affinity at the active site of the protein. Moreover, they showed excellent pharmacokinetic and drug - likeness properties. Derivatization of Pefloxacin at the C7 position prevents its blood-brain barrier permeability. Overall, the dual antibacterial and potential antiviral activities of these two molecules make them promising drug candidates for COVID-19 management.

Introduction: Severe acute respiratory syndrome coronavirus identified as the root cause of the Coronavirus disease 2019 (COVID-19) has spread all over the world. The pandemic situation caused by SARS-CoV-2 currently challenging the world. Given that the COVID-19 disease has no vaccine or therapeutic drugs to prevent the infection. In contrast, the key protease (Mpro) of SARS CoV-2 is involved in replication and proliferation of the virus and hence represents a crucial drug for the inhibition of COVID-19. Recent development shows the antiviral and anti-cancerous potential of natural products in the drug development process against various diseases which resulted in the screening of such agents to combat emergent mutants of SARS-CoV-2. Herein, we have applied a bioinformatics approach including molecular docking and a combination of molecular dynamics simulations and Poisson-Boltzmann surface area (MM/G/P/BSA) free energy calculations to identify the inhibitory potency of candidates against SARS-CoV2 main protease. Methods: In-Silico molecular docking analysis was performed for all selected anti-cancerous natural compounds with the potential drug target, PDB Id: 6W63 COVID-19 main protease in complex with a noncovalent inhibitor X77 using molecular docking and a combination of molecular dynamics simulations and Poisson-Boltzmann surface area (MM/G/P/BSA) free energy calculations. Absorption, Distribution, Metabolism, and Excretion (ADME) property as well as Lipinski's rule of five was also predicted for all the selected compounds. Among the 20 natural compounds, four natural metabolites namely, amentoflavone, guggulsterone, puerarin, and piperine were found to have strong interaction with Mpro of COVID-19. During MD simulations, all four natural compounds bound to Mpro at 50ns and MM/G/P/BSA free energy calculations showed that all four shortlisted ligands have stable and favorable energies causing strong binding with the binding site of Mpro protein. Conclusion: Although the anti-cancerous natural compounds show high binding affinity with the active site of SARS-CoV-2 main protease. Among these Amentoflavone and Guggulsterone were the top two leads showing the lowest binding energy and satisfying our studied parameters. Guggulsterone of Indian traditional ayurvedic medical plant Commiphoramukul was found to be the most suitable based on comprehensive pharmacokinetic parameters, drug-likeness, and docking analysis. Therefore, we propose anti-cancerous natural compound Guggulsterone may further be validated as potential inhibitors of COVID-19 main protease Mpro. Citation Format: Amaresh Mishra, Yamini Pathak, Gourav Choudhir, Anuj Kumar, Surabhi Kirti Mishra, Vishwas Tripathi. Anticancer natural compounds as potential inhibitors of novel coronavirus (COVID19) main protease: An in-silico study [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 712.

Coronavirus disease 2019 (COVID-19) is caused by novel severe acute respiratory syndrome coronavirus (SARS-CoV-2). Its main protease, 3C-like protease (3CLpro), is an attractive target for drug design, due to its importance in virus replication. The analysis of the radial distribution function of 159 3CLpro structures reveals a high similarity index. A study of the catalytic pocket of 3CLpro with bound inhibitors reveals that the influence of the inhibitors is local, perturbing dominantly only residues in the active pocket. A machine learning based model with high predictive ability against SARS-CoV-2 3CLpro is designed and validated. The model is used to perform a drug-repurposing study, with the main aim to identify existing drugs with the highest 3CLpro inhibition power. Among antiviral agents, lopinavir, idoxuridine, paritaprevir, and favipiravir showed the highest inhibition potential.Graphical abstractEnzyme – ligand interactions as a key ingredient for successful drug designSupplementary InformationThe online version contains supplementary material available at 10.1007/s11030-021-10355-8.