Computational Evaluation and Multi-Criteria Optimization of Natural Compound Analogs Targeting SARS-CoV-2 Proteases

It is well known that vital enzymes in the replication process of the coronavirus are the SARS-CoV-2 PLpro and SARS-CoV-2 3CLpro, both of which are important targets in the search for anti-coronavirus agents. These two enzymes are responsible for cleavage at various polyprotein sites in the SARS-CoV-2 lifecycle. Herein, the dynamics of the polyprotein cleavage sequences for the boundary between non-structural proteins Nsp1 and Nsp2 (CS1) and between Nsp2 and Nsp3 (CS2) in complex with both the papain-like protein PLpro and the main protease 3CLpro were explored using computational methods. The post dynamics analysis reveals that CS1 and CS2 both have greater stability when complexed with PLpro. Of these two, greater stability is observed for the CS1-PLpro complex, while destabilization resulting in loss of CS2 from the PLpro active site is observed for CS2-PLpro, suggesting the rate of exchange by the papain-like protease is faster for CS2 compared to CS1. On the other hand, the 3CLpro main protease also reveals stability for CS1 suggesting that the main protease could also play a potential role in the cleavage at point CS1. However, destabilization occurs early in the simulation for the complex CLpro-CS2 suggesting a poor interaction and non-plausible protease cleavage of the polyprotein at CS2 by the main protease. These findings could be used as a guide in the development and design of potent COVID-19 antiviral inhibitors that mimic the CS1 cleavage site.

SARS-CoV-2 main protease (Mpro or 3CLpro) and papain-like protease (PLpro) are common viral targets for repurposed drugs to combat COVID-19 disease. Recently, several antidepressants (such as fluoxetine, venlafaxine and citalopram) belonging to the Selective Serotonin Reuptake Inhibitors (SSRIs) and the Serotonin-Norepinephrine Reuptake Inhibitors (SNRI) classes have been shown to in vitro inhibit viral replication. Investigate a possible action of fluoxetine and derivatives on SARS-CoV-2 protease sites. Molecular docking was performed using AutoDock Vina. Both protease structures and different drug conformations were used to explore the possibility of SARS-CoV-2 inhibition on a Mpro or PLpro related pathway. Drug structures were obtained by optimization with the Avogadro software and MOPAC using the PM6 method. Results were analysed on Discovery Studio Visualizer. The results indicated that Mpro interacted in a thermodynamically favorable way with fluoxetine, venlafaxine, citalopram, atomoxetine, nisoxetine and norfluoxetine in the region of the active site, whether PLpro conformers did not come close to the active site. In an in silico perspective, it is likely that the SSRIs and other anti-depressants could interact with Mpro and cause the enzyme to malfunction. Unfortunately, the same drugs did not present similar results on PLpro crystal, therefore, no inhibition is expected in an in vitro trial. Anyway, in vitro tests are necessary for a better understanding of the links between SARS-CoV-2 proteases and antidepressants.

Novel therapies to treat infection by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the cause of respiratory coronavirus disease 2019 (COVID-19), would be of great clinical value to combat the current and future pandemics. Two viral proteases, papain-like protease (PLpro) and the main protease 3-chymotrypsin-like protease (3CLpro), are vital in processing the SARS-CoV-2 polyproteins (pp1a and pp1ab) and in releasing 16 nonstructural proteins, making them attractive antiviral drug targets. In this study, we investigated the degradation of the SARS-CoV-2 main protease 3CLpro by matrix metalloproteinase-14 (MMP14). MMP14 is known to recognize over 10 distinct substrate cleavage sequences. Through sequence analysis, we identified 17 and 10 putative MMP14 cleavage motifs within the SARS-CoV-2 3CLpro and PLpro proteases, respectively. Despite the presence of potential sites in both proteins, our in vitro proteolysis assays demonstrated that MMP14 selectively binds to and degrades 3CLpro, but not PLpro. This selective proteolysis by MMP14 results in the complete loss of 3CLpro enzymatic activity. In addition, SARS-CoV-2 pseudovirus replication was inhibited in 293 T cells when either full-length MMP14 or its catalytic domain (cat-MMP14) were overexpressed, presumably due to 3CLpro degradation by MMP14. Finally, to prevent MMP14 from degrading off-target proteins, we propose a new recombinant pro-PL-MMP14 construct that can be activated only by another SARS-CoV-2 protease, PLpro. These findings could open the potential of an alternative therapeutic strategy against SARS-CoV-2 infection.

Background: The recent outbreak of the COVID-19 pandemic has raised a global health concern due to the unavailability of any vaccines or drugs. The repurposing of traditional herbs with broad-spectrum anti-viral activity can be explored to control or prevent a pandemic. Objective: The 3-chymotrypsin-like main protease (3CLpro), also referred to as the “Achilles’ heel” of the coronaviruses (CoVs), is highly conserved among CoVs and is a potential drug target. 3CLpro is essential for the virus’ life cycle. The objective of the study was to screen and identify broad- -spectrum natural phytoconstituents against the conserved active site and substrate-binding site of 3CLpro of HCoVs. Methods: Herein, we applied the computational strategy based on molecular docking to identify potential phytoconstituents for the non-covalent inhibition of the main protease 3CLpro from four different CoVs, namely, SARS-CoV-2, SARS-CoV, HCoV-HKU1, and HCoV-229E. Results: Our study shows that natural phytoconstituents in Triphala (a blend of Emblica officinalis fruit, Terminalia bellerica fruit, and Terminalia chebula fruit), namely chebulagic acid, chebulinic acid, and elagic acid, exhibited the highest binding affinity and lowest dissociation constants (Ki), against the conserved 3CLpro main protease of SARSCoV-2, SARS-CoV, HCoV-HKU1, and HCoV-229E. Besides, phytoconstituents of other herbs like Withania somnifera, Glycyrrhiza glabra, Hyssopus officinalis, Camellia sinensis, Prunella vulgaris, and Ocimum sanctum also showed good binding affinity and lower Ki against the active site of 3CLpro. The top-ranking phytoconstituents’ binding interactions clearly showed strong and stable interactions with amino acid residues in the catalytic dyad (CYS-HIS) and substrate-binding pocket of the 3CLpro main proteases. Conclusion: This study provides a valuable scaffold for repurposing traditional herbs with anti-- CoV activity to combat SARS-CoV-2 and other HCoVs until the discovery of new therapies.

TAT-peptide conjugated repurposing drug against SARS-CoV-2 main protease (3CLpro): Potential therapeutic intervention to combat COVID-19

The global public health has been compromised since the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) emerged in late December 2019. There are no specific antiviral drugs available to combat SARS-CoV-2 infection. Besides the rapid dissemination of SARS-CoV-2, several variants have been identified with a potential epidemiologic and pathogenic variation. This fact has forced antiviral drug development strategies to stay innovative, including new drug discovery protocols, combining drugs, and establishing new drug classes. Thus, developing novel screening methods and direct-targeting viral enzymes could be an attractive strategy to combat SARS-CoV-2 infection. In this study, we designed, optimized, and validated a cell-based assay protocol for high-throughput screening (HTS) antiviral drug inhibitors against main viral protease (3CLpro). We applied the split-GFP complementation to develop GFP-split-3CLpro HTS system. The system consists of GFP-based reporters that become fluorescent upon cleavage by SARS-CoV-2 protease 3CLpro. We generated a stable GFP-split-3CLpro HTS system valid to screen large drug libraries for inhibitors to SARS-CoV-2 main protease in the bio-safety level 2 laboratory, providing real-time antiviral activity of the tested compounds. Using this assay, we identified a new class of viral protease inhibitors derived from quinazoline compounds that worth further in vitro and in vivo validation.

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

Multistep rational molecular design and combined docking for discovery of novel classes of inhibitors of SARS-CoV-2 main protease 3CLpro

The COVID-19 has now been declared a global pandemic by the World Health Organization. No approved drug is currently available; therefore, an urgent need has been developed for any antiviral therapy for COVID-19. Main protease 3CLpro of this novel Coronavirus (SARS-CoV-2) play a critical role in the disease propagation, and hence represent a crucial target for the drug discovery. Herein, we have applied a bioinformatics approach for drug repurposing to identify the possible potent inhibitors of SARS-CoV-2 main proteases 3CLpro (6LU7). In search of the anti-COVID-19 compound, we selected 145 phyto-compounds from Kabasura kudineer (KK), a poly-herbal formulation recommended by AYUSH for COVID-19 which are effective against fever, cough, sore throat, shortness of breath (similar to SARS-CoV2-like symptoms). The present study aims to identify molecules from natural products which may inhibit COVID-19 by acting on the main protease (3CLpro). Obtained results by molecular docking showed that Acetoside (−153.06), Luteolin 7 -rutinoside (−134.6) rutin (−133.06), Chebulagic acid (−124.3), Syrigaresinol (−120.03), Acanthoside (−122.21), Violanthin (−114.9), Andrographidine C (−101.8), myricetin (−99.96), Gingerenone -A (−93.9), Tinosporinone (−83.42), Geraniol (−62.87), Nootkatone (−62.4), Asarianin (−79.94), and Gamma sitosterol (−81.94) are main compounds from KK plants which may inhibit COVID-19 giving the better energy score compared to synthetic drugs. Based on the binding energy score, we suggest that these compounds can be tested against Coronavirus and used to develop effective antiviral drugs.

Objectives: The global burden of the current severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causing the corona virus disease-19 (COVID-19) is enormous. No definitive treatment and prophylactic guidelines for COVID-19 currently exist except for physical distancing and aerial barriers between individuals. This work explored the natural compound-binding efficiency of SARS-CoV-2 proteins essential for host cell interaction and infection. Methods: The binding activity of artemisinin to SARS-CoV-2 spike glycoprotein (Protein Data Bank (PDB) ID: 6VYB), SARS-CoV-2 main protease (3C-like main protease (3CLpro); PDB ID: 6Y84) and SARS-CoV-2 papain-like protease (PLpro; PDB ID: 6W9C), were tested using in silico methods. Moreover, chloroquine and hesperidin were used as the positive control of binding affinity and proven therapeutic effect, respectively. Results: The highest affinities for binding to all tested SARS-CoV-2 proteins are observed for hesperidin (?5.8,?10.0, and ?8.1 kcal/mol), then for artemisinin (?4.8,?8.3, and ?6.0 kcal/mol), and the lowest for chloroquine (?4.1,?8.2, and ?4.8 kcal/mol). Artemisinin, hesperidin, and chloroquine had similar positioning toward targeted proteins at specific sites when these interactions were visualized. Conclusion: This study shows that artemisinin has the potential to bind and inhibit the SARS-CoV-2 spike protein, the 3CLpro main protease, and PLpro proteinase similar to hesperidin and chloroquine that have been proven as antivirals in previous preclinical and clinical studies. Keywords: Artemisinin, molecular docking study, SARS-CoV-2 proteins

The two proteases of SARS-CoV-2 coronavirus – the main protease (Mpro or 3CLpro) and the papain-like protease (PLpro) – are essential enzymes required for the successful replication of the virus within cells. Both proteases have become major targets in the development of antiviral drugs against SARS-CoV-2. The potential to achieve a dual inhibitory effect has sparked significant interest in creating dual inhibitors as complex therapeutic agents for this virus. In this article, we discuss the development and in silico evaluation of a series of new peptidomimetic molecules designed as dual-action inhibitors of both SARS-CoV-2 Mpro and PLpro, along with their synthesis. We implemented a combined approach that began with developing a basic molecular model, considering the substrate specificity of the active centers of each protease. Through rational in silico design, we created a series of peptidomimetics. Further analysis of how these compounds bound to the active sites of both proteases enabled us to identify several new structural hits, including hydantoin derivatives, as potential dual inhibitors of Mpro and PLpro. The aim of the study. This study aims to establish a common molecular framework for designing dual-action inhibitors targeting the SARS-CoV-2 Mpro and PLpro proteases. The research includes receptor-oriented molecular docking, in silico optimization, and the selection and synthesis of the most active candidate structures for further in vitro experimental studies. Materials and methods. LigandScout 4.5 software is used for 3D pharmacophore analysis, visualization, and molecular docking. AutoDock Vina 1.1 provides tools for molecular docking. The PLIP (Protein-Ligand Interaction Profiler) web servers are utilized to study molecular binding mechanisms. DataWarrior 6.0 software helps create a library of molecular structures, calculate physicochemical properties, and analyze molecular frameworks. SwissADME web server is used to predict ADME parameters and assess the pharmacokinetic properties of small molecules as potential drugs. Results. We analyzed the substrate specificity of the binding sites of the Mpro and PLpro proteases, which enabled us to identify a common amino acid sequence containing shared recognition elements for both proteases. By rationally modifying the functional groups in this initial base structure, utilizing the principle of isosteric replacement and incorporating non-classical bioisosteres, we developed a series of peptidomimetic frameworks. Molecular docking conducted at the active sites of both Mpro and PLpro, along with the assessment of their binding energy values (in kcal/mol), identified several structures with potential for dual inhibition. Notably, hydantoin derivatives demonstrated the strongest binding affinity to the active sites of both proteases. Conclusions. We have identified promising peptidomimetic molecular structures that demonstrate dual inhibitory activity against the SARS-CoV-2 proteases through in silico analysis. Specifically, we discovered a novel class of hydantoin derivatives that act as inhibitors for both SARS-CoV-2 Mpro and PLpro. The synthesis methods we developed allow for the preparation of these compounds for further in vitro studies

Interaction of small molecules with the SARS-CoV-2 papain-like protease: In silico studies and in vitro validation of protease activity inhibition using an enzymatic inhibition assay

Proteases of SARS Coronaviruses

SARS-CoV-2 pandemic has claimed millions of lives across the world. As of June 2020, there is no FDA approved antiviral therapy to eradicate this dreadful virus. In this study, drug re-purposing and computational approaches were employed to identify high affinity inhibitors of SARS-CoV-2 Main protease (3CLpro), Papain-like protease (PLpro) and the receptor domain of Spike protein. Molecular docking on 40 derivatives of standard drugs (Remdesivir, Lopinavir and Theophylline) led to the identification of R10, R2 and L9 as potential inhibitors of 3CLpro, PLpro and Spike protein, respectively. The binding affinity of R10, R2 and L9 towards 3CLpro, PLpro and Spike protein were 4.03 × 106, 3.72 × 104 and 1.31 × 104 M −1, respectively. These inhibitors interact with the active site or catalytic amino acid residues of 3CLpro, PLpro and Spike protein. We also examined the stability and dynamic behavior of protein-inhibitor complex by employing molecular dynamics simulation. RMSDs, RMSFs and variation in secondary structure of target proteins alone or in complex with their respective inhibitors were used to ascertain the integrity of proteins’ structure during simulation. Moreover, physicochemical and ADMET properties of R10, R2 and L9 along with Remdesivir, Lopinavir and Theophylline were determined. In vitro and In vivo studies are needed to further validate the potential of these derivatives before they can be developed into potential drug molecules. Communicated by Ramaswamy H. Sarma

Dual targeting of 3CLpro and PLpro of SARS-CoV-2: A novel structure-based design approach to treat COVID-19