Main Protease Research Articles

Identification of compounds from natural Peruvian sources as potential inhibitors of SARS-CoV-2 Mpro mutations by virtual screening and computational simulations

Background Although the COVID-19 pandemic has diminished in intensity, the virus continues to circulate globally. The SARS-CoV-2 main protease (Mpro) is a key enzyme in the life cycle of the virus, making it important for the development of treatments against future variants of the virus. In this work, Peruvian natural compounds were evaluated against different mutations of the SARS-CoV-2 Mpro. Methods In silico techniques such as virtual screening, all-atom molecular dynamics simulations, and energy estimation analysis were applied. Results Of the tested compounds by virtual screening, rutin was identified as the best binding agent against the different proposed Mpro mutations. In addition, computational simulations and energy estimation analysis demonstrated the high structural and energetic stability between the Mpro-rutin systems. Conclusions Overall, our study identified rutin as the most promising compound with a strong affinity for various Mpro mutations, potentially playing a key role in the development of new treatments for emerging viral variants.

Computational Assessment of Clinical Drugs against SARS-CoV-2: Foreseeing Molecular Mechanisms and Potent Mpro Inhibitors.

The emergence of new SARS-CoV-2 variants of concern (VOC) is a propulsion for accelerated potential therapeutic discovery. SARS-CoV-2's main protease (Mpro), essential for host cell viral replication, is a pre-eminent druggable protein target. Here, we perform extensive drug re-profiling of the comprehensive Excelra database, which compiles various under-trial drug candidates for COVID-19 treatment. For mechanistic understanding, the most promising screened-out molecules with targets are subjected to molecular dynamics (MD) simulations. Post-MD analyses demonstrate Darunavir, Ponatinib, and Tomivosertib forming a stable complex with Mpro, characterized by less fluctuation of Cα atoms, smooth and stable root-mean-square deviation (RMSD), and robust contact with the active site residues. Likewise, they all have lower binding free energy with Mpro, demonstrating strong affinity. In free energy landscape profiles, the distances from His41 and Cys145 exhibit a single energy minima basin, implying their preponderance in proximity to Mpro's catalytic dyad. Overall, the computational assessment earmarks promising candidates from the Excelra database, emphasizing on carrying out exhaustive biochemical experiments along with clinical trials. The work lays the foundation for potential therapeutic interventions in treating COVID-19.

Rational Design of Macrocyclic Noncovalent Inhibitors of SARS-CoV-2 Mpro from a DNA-Encoded Chemical Library Screening Hit That Demonstrate Potent Inhibition against Pan-Coronavirus Homologues and Nirmatrelvir-Resistant Variants.

The recent global COVID-19 pandemic has highlighted treatments for coronavirus infection as an unmet medical need. The main protease (Mpro) has been an important target for the development of SARS-CoV-2 direct-acting antivirals. Nirmatrelvir as a covalent Mpro inhibitor was the first such approved therapy. Although Mpro inhibitors of various chemical classes have been reported, they are generally less active against nirmatrelvir-resistant variants and have limited pan-coronavirus potential, presenting a significant human health risk upon future outbreaks. We here present a novel approach and utilized DNA-encoded chemical library screening to identify the noncovalent Mpro inhibitor 5, which demonstrated a distinct binding mode to nirmatrelvir. A macrocyclization strategy designed to lock the active conformation resulted in lactone 12 with significantly improved antiviral activity. Further optimization led to the potent lactam 26, which demonstrated exceptional potency against nirmatrelvir-resistant variants as well as against a panel of viral main proteases from other coronaviruses.

Identification of catechin as main protease inhibitor of SARS-CoV-2 Omicron variant using molecular docking, molecular dynamics, PCA, DCCM, MM/GBSA and ADMET profiling

The Omicron variant of SARS-CoV-2 spreads more rapidly than other variants and can affect even vaccinated individuals. The Omicron variant main protease (Mpro), crucial for viral replication and transcription, is an attractive target for antiviral drug discovery. This research aims to investigate non-toxic flavonoids that follow Lipinski’s rule of five (RO5) and inhibit the Omicron variant Mpro. Molecular docking was performed on 35 flavonoids screened by analysing their medicinal values and adherence to RO5. Catechin (2-(3,4-dihydroxyphenol) chroman-3,5,7-triol), a non-toxic natural compound having predicted toxicity class 6 and LD50 value 10,000 mg/kg, exhibited a docking score of −7.1 kcal/mol with Mpro. The Mpro-catechin complex remained stable during 250 ns MD simulations. The post-MD free energy (MM/GBSA) calculations showed a binding energy of −20.5 kcal/mol, indicating strong interactions with the active amino acid residues. These findings suggest that catechin is a promising drug candidate against the Omicron variant.

Inhibitory Effects of Mangifera indica Secondary Metabolites and Their Synthetic Derivatives against SARS-CoV-2 Mpro and NS2B/NS3 (ZIKV and DENV-2).

Chemical studies of Mangifera indica twigs yielded two compounds, identified as taraxerol (1) and methyl gallate (2). The galloyl moiety was suggested as a potential scaffold that can interfere with proteases by previous biological investigations on SARS-CoV-2 main protease (Mpro) inhibitors in combination with docking studies. Therefore, a series of 13 gallate esters were prepared by treating gallic acid with natural and non-natural alcohols. Their inhibitory effects were evaluated against Mpro and NS2B/NS3 of Zika and Dengue viruses. Among the obtained compounds, 2e and 2i were the most potent against Mpro with IC50 values of 2.60 and 4.0 μM, respectively. Compounds 2f and 2g were more potent than others against ZIKV protease with IC50 values of 2.7 and 1.9 μM, respectively. The bioactivity profile against DENV NS2B/NS3 was different with 2e and 2f showing moderate inhibition with IC50 values of 66.0 and 59.29 μM, respectively. It was found that 2f and 2g inhibited ZIKV NS2B/NS3 via a noncompetitive mechanism. The study also showed that 2e and 2f could exert noncompetitive inhibition at the previously described allosteric pocket of flaviviral NS2B/NS3 proteases. Molecular docking revealed different types of interactions among the most prominent were hydrogen bonding with the galloyl moiety.

Artemisia campestris L. as a promising source of potential antiviral drugs for SARS-CoV-2: Docking and dynamic simulation studies

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been considered as global public health security threats. Due to its rapid spread, high mortality rate and unavailability of treatment, we need to find a potent drug against SARS-CoV-2. Natural products are useful agents for the discovery of new potential drugs to combat coronavirus. Artemisia campestris, an aromatic plant widely used in traditional medicine, particularly in southern region of Algeria, is recognized for its essential oils and phenolics compounds possessing a wide range of biological activities. This study selected sixty-nine compounds from this plant to determine their binding interactions with the SARS-CoV-2 main protease (Mpro) and receptor-binding domain of the spike (S-RBD) protein, by using computational methods. Rutin, isoquercetin, and quercetin-3-O-glucuronide were shown to be the most potent inhibitors for Mpro and S-RBD with docking scores and Ki values ranging from -16.06 to -10.67 Kcal/mol and 0.005 to 30.01 nM, respectively. Evaluation of ADMET pharmacokinetic properties and the drug likeness in silico revealed that only 3,5-dicaffeoylquinic acid, 3–4–5-tricaffeoylquinic acid, isorhamnetin-3-O-glucoside, and rubescensin A could be more effective drugs against COVID-19. Molecular dynamics (MD) simulations (100 ns) and MM-GBSA calculations confirmed the stability of ligand-protein complexes via hydrogen bonding interactions with crucial residues. The analysis of structural parameters (RMSD, RMSF, H-bonds, Rg, and SASA) indicates that 3,4,5-tricaffeoylquinic acid and rubescensin A compounds have good stability and significant binding affinity with the Mpro and S-RBD protein. Taken together, our findings confirm that Artemisia campestris as a plausible source of anti-SARS-COV-2 phytochemicals and suggest that may play important role in this activity.

Design, Synthesis, Biological Evaluation and Molecular Docking Studies of New Thiazolidinone Derivatives as NNRTIs and SARS-CoV-2 Main Protease Inhibitors.

HIV-1 remains a major health problem worldwide since the virus has developed drug-resistant strains, so, the need for novel agents is urgent. The protein reverse transcriptase plays fundamental role in the viruses' replication cycle. FDA approved Delavirdine bearing a sulfonamide moiety, while thiazolidinone has demonstrated significant anti-HIV activity as a core heterocycle or derivative of substituted heterocycles. In this study, thirty new thiazolidinone derivatives (series A, B and C) bearing sulfonamide group were designed, synthesized and evaluated for their HIV-1 RT inhibition activity predicted by computer program PASS taking into account the best features of available NNRTIs as well as against SARS-COV-2 main protease. Seven compounds showed good anti-HIV inhibitory activity, with two of them, C1 and C2 being better (IC50 0.18 μΜ & 0.12 μΜ respectively) than the reference drug nevirapine (IC50 0.31 μΜ). The evaluation of molecules to inhibit the main protease revealed that 6 of the synthesized compounds exhibited excellent to moderate activity with two of them (B4 and B10) having better IC50 values (0.15 & 0.19 μΜ respectively) than the reference inhibitor GC376 (IC50 0.439 μΜ). The docking studies is coincides with experimental results, showing good binding mode to both enzymes.

Revealing SARS-CoV-2 Mpro mutation cold and hot spots: Dynamic residue network analysis meets machine learning

Deciphering the effect of evolutionary mutations of viruses and predicting future mutations is crucial for designing long-lasting and effective drugs. While understanding the impact of current mutations on protein drug targets is feasible, predicting future mutations due to natural evolution of viruses and environmental pressures remains challenging. Here, we leveraged existing mutation data during the evolution of the SARS-CoV-2 protein drug target main protease (Mpro) to test the predictive power of dynamic residue network (DRN) analysis in identifying mutation cold and hot spots. We conducted molecular dynamics simulations on the Mpro of SARS-CoV-2 (Wuhan strain) and calculated eight DRN metrics (averaged BC, CC, DC, EC, ECC, KC, L, PR), each of which identifies a unique network feature within the protein. The sets of residues with the highest and lowest values for each metric, comprising potential cold and hot spots, were compared to published biochemical analyses and per residue mutation frequencies observed across five SARS-CoV-2 lineages, encompassing a total of 191,878 sequences. Individual DRN metrics displayed only modest power to predict the mutation frequency of individual residues. However, integrating the eight DRN metrics with additional structural and sequence-derived metrics allowed us to develop machine learning models which significantly improved the prediction of residue mutation frequency. While further refinements should enhance accuracy, we demonstrated a robust method to understand pathogen evolution. This approach can also guide the development of long-lasting drugs by targeting functional residues located in and near active site, and allosteric sites, that are less prone to mutations.

DFT, Molecular Docking, ADME, and Cardiotoxicity Studies of Persuasive Thiazoles as Potential Inhibitors of the Main Protease of SARS-CoV-2.

This study explores the capability of thiazoles as potent inhibitors of SARS-CoV-2 Mpro. Seventeen thiazoles (1-17) were screened for their linking affinity with the active site of SARS-CoV-2 Mpro and compared with the FDA-recommended antiviral drugs, Remdesivir and Baricitinib. Density Functional Theory (DFT) calculations provided electronic and energetic properties of these ligands, shedding light on their stability and reactivity. Molecular docking analysis revealed that thiazole derivatives exhibited favorable linking affinities with various functional sites of SARS-CoV-2 proteins, including spike receptor-linking zone, nucleocapsid protein N-terminal RNA linking zone, and Mpro. Notably, compounds 3, 10, and 12 displayed the best interaction with 6LZG as compared to FDA-approved antiviral drugs Remdesivir and Baricitinib, while compounds 1, 10, and 8 exhibited strong linking with 6 M3 M and also better than Remdesivir and Baricitinib. Additionally, compounds 3, 1, and 6 showed promising interactions with 6LU7 but only compound 3 performed better than Baricitinib. An ADME (Absorption, Distribution, Metabolism, and Excretion) study provided insights into the pharmacokinetics and drug-likeness of these compounds, with all ligands demonstrating good physicochemical characteristics, lipophilicity, water solubility, pharmacokinetics, drug-likeness, and medicinal chemistry attributes. The results suggest that these selected thiazole derivatives hold promise as potential candidates for further drug development.

New insights in the mechanism of the SARS-CoV-2 Mpro inhibition by benzisoselenazolones and diselenides.

Although global vaccination campaigns alleviated the SARS-CoV-2 pandemic in terms of morbidity and mortality, the ability of the virus to originate mutants may reduce the efficacy of vaccines, posing a serious risk of a renewed pandemic. There is therefore a need to develop small molecules capable of targeting conserved viral targets, such as the main protease (Mpro). Here, a series of benzisoselenazolones and diselenides were tested for their ability to inhibit Mpro; then the most potent compounds were measured for antiviral activity in vitro, and the mechanism of action was investigated. Density functional theory calculations, molecular docking and molecular dynamics simulations were also used to elucidate the protein/drug interaction. Finally, a bio-organic model was established to study the reaction between selenorganic compounds and biologically relevant thiols to unveil possible metabolic pathways of such compounds. The overall results contribute to the identification of a series of novel Se-containing molecules active against SARS-CoV-2 and to the clarification of some important aspects in the mechanisms of action of such inhibitors targeting SARS-CoV-2 Mpro.

Naphthalen-1-ylethanamine–containing small molecule inhibitors of the papain-like protease of SARS-CoV-2

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which causes coronavirus disease 2019 (COVID-19), has not yet been eradicated. SARS-CoV-2 has two types of proteases, a main protease (Mpro) and a papain-like protease (PLpro), which together process two translated non-structural polyproteins, pp1a and pp1ab, to produce functional viral proteins. In this study, effective inhibitors against PLpro of SARS-CoV-2 were designed and synthesized using GRL-0048 as a lead. A docking simulation of GRL-0048 and SARS-CoV-2 PLpro showed that GRL-0048 noncovalently interacts with PLpro, and there is a newly identified binding pocket in PLpro. Structure-activity relationship studies were next performed on GRL-0048, resulting in the development of several inhibitors, specifically compounds 1, 2b, and 3h, that have more potent inhibitory activity than GRL-0048.

Recognition and Cleavage of Human tRNA Methyltransferase TRMT1 by the SARS-CoV-2 Main Protease.

The SARS-CoV-2 main protease (Mpro, or Nsp5) is critical for the production of functional viral proteins during infection and, like many viral proteases, can also target host proteins to subvert their cellular functions. Here, we show that the human tRNA methyltransferase TRMT1 can be recognized and cleaved by SARS-CoV-2 Mpro. TRMT1 installs the N2,N2-dimethylguanosine (m2,2G) modification on mammalian tRNAs, which promotes global protein synthesis and cellular redox homeostasis. We find that Mpro can cleave endogenous TRMT1 in human cell lysate, resulting in removal of the TRMT1 zinc finger domain. TRMT1 proteolysis results in elimination of TRMT1 tRNA methyltransferase activity and reduced tRNA binding affinity. Evolutionary analysis shows that the TRMT1 cleavage site is highly conserved in mammals, except in Muroidea, where TRMT1 is likely resistant to cleavage. In primates, regions outside the cleavage site with rapid evolution could indicate adaptation to ancient viral pathogens. Furthermore, we determined the structure of a TRMT1 peptide in complex with Mpro, revealing a substrate binding conformation distinct from the majority of available Mpro-peptide complexes. Kinetic parameters for peptide cleavage show that the TRMT1(526-536) sequence is cleaved with comparable efficiency to the Mpro-targeted nsp8/9 viral cleavage site. Mutagenesis studies and molecular dynamics simulations together indicate that kinetic discrimination occurs during a later step of Mpro-mediated proteolysis that follows substrate binding. Our results provide new information about the structural basis for Mpro substrate recognition and cleavage, the functional roles of the TRMT1 zinc finger domain in tRNA binding and modification, and the regulation of TRMT1 activity by SARS-CoV-2 Mpro. These studies could inform future therapeutic design targeting Mpro and raise the possibility that proteolysis of human TRMT1 during SARS-CoV-2 infection suppresses protein translation and oxidative stress response to impact viral pathogenesis.

In silico investigation of therapeutic potential of phytosteroids against SARS-CoV2 infection via inhibition of viral multiplication and attenuation of host cytokine storm

SARS-CoV2 infection overcomes host cell membrane barrier, followed by utilization of host cellular processes for viral multiplication. Simultaneously, it triggers a cytokine storm within and around infected cells and tissues. Anti-inflammatory agents that can potentially inhibit viral penetration and multiplication within the host cells may be ideal drug candidates against COVID-19. Dietary phytosteroids have significant anti-inflammatory potential. Hence, the present study intends to investigate anti-viral potential of three dietary phytosteroids, namely, brassinolide, 28-homocastasterone and 24-epibrassinolide against SARS-CoV2 proteins, S1 spike protein, nucleocapsid protein and main protease enzyme, in addition to host pro-inflammatory proteins, interleukin-1, tumour necrosis factor-α, cyclooxygenase-2 and prostaglandin synthase, as drug targets. Molecular docking studies by AutoDock version 4.0 was performed. Brassinolide, 28-homocastasterone and 24-epibrassinolide exhibits high docking score against all the seven proteins, as compared to hydroxychloroquine. Brassinolide and 28- homocastasterone has maximum binding affinity for pro-inflammatory proteins and SARS-CoV2 proteins.Dietary phytosteroids may potentially attenuate cytokine storm with simultaneous inhibition of host entry and multiplication of SARS-CoV2. In-vitro and in-vivo anti-viral studies of plant steroids may provide a clear path for the identification and development of novel drug candidates against COVID-19, that also provides evidence for the concept of reverse pharmacognosy.

MERS-CoV-nsp5 expression in human epithelial BEAS 2b cells attenuates type I interferon production by inhibiting IRF3 nuclear translocation

Middle East Respiratory Syndrome Coronavirus (MERS-CoV) is an enveloped, positive-sense RNA virus that emerged in 2012, causing sporadic cases and localized outbreaks of severe respiratory illness with high fatality rates. A characteristic feature of the immune response to MERS-CoV infection is low type I IFN induction, despite its importance in viral clearance. The non-structural proteins (nsps) of other coronaviruses have been shown to block IFN production. However, the role of nsp5 from MERS-CoV in IFN induction of human respiratory cells is unclear. In this study, we elucidated the role of MERS-CoV-nsp5, the viral main protease, in modulating the host’s antiviral responses in human bronchial epithelial BEAS 2b cells. We found that overexpression of MERS-CoV-nsp5 had a dose-dependent inhibitory effect on IFN-β promoter activation and cytokine production induced by HMW-poly(I:C). It also suppressed IFN-β promoter activation triggered by overexpression of key components in the RIG-I-like receptor (RLR) pathway, including RIG-I, MAVS, IKK-ε and IRF3. Moreover, the overexpression of MERS-CoV-nsp5 did not impair expression or phosphorylation of IRF3, but suppressed the nuclear translocation of IRF3. Further investigation revealed that MERS-CoV-nsp5 specifically interacted with IRF3. Using docking and molecular dynamic (MD) simulations, we also found that amino acids on MERS-CoV-nsp5, IRF3, and KPNA4 may participate in protein-protein interactions. Additionally, we uncovered protein conformations that mask the nuclear localization signal (NLS) regions of IRF3 and KPNA4 when interacting with MERS-CoV-nsp5, suggesting a mechanism by which this viral protein blocks IRF3 nuclear translocation. Of note, the IFN-β expression was restored after administration of protease inhibitors targeting nsp5, indicating this suppression of IFN-β production was dependent on the enzyme activity of nsp5. Collectively, our findings elucidate a mechanism by which MERS-CoV-nsp5 disrupts the host’s innate antiviral immunity and thus provides insights into viral pathogenesis.

In Silico Identification of Potential Inhibitors of SARS-CoV-2 Main Protease (Mpro).

The ongoing Coronavirus Disease 19 (COVID-19) pandemic has had a profound impact on the global healthcare system. As the SARS-CoV-2 virus, responsible for this pandemic, continues to spread and develop mutations in its genetic material, new variants of interest (VOIs) and variants of concern (VOCs) are emerging. These outbreaks lead to a decrease in the efficacy of existing treatments such as vaccines or drugs, highlighting the urgency of new therapies for COVID-19. Therefore, in this study, we aimed to identify potential SARS-CoV-2 antivirals using a virtual screening protocol and molecular dynamics simulations. These techniques allowed us to predict the binding affinity of a database of compounds with the virus Mpro protein. This in silico approach enabled us to identify twenty-two chemical structures from a public database (QSAR Toolbox Ver 4.5 ) and ten promising molecules from our in-house database. The latter molecules possess advantageous qualities, such as two-step synthesis, cost-effectiveness, and long-lasting physical and chemical stability. Consequently, these molecules can be considered as promising alternatives to combat emerging SARS-CoV-2 variants.

Integrative in silico evaluation of the antiviral potential of terpenoids and its metal complexes derived from Homalomena aromatica based on main protease of SARS-CoV-2

Abstract Substantial research is currently conducted focusing on the development of promising antiviral drugs employing in silico screening and drug repurposing strategies against SARS-CoV-2. The current study aims at identifying lead molecules targeting SARS-CoV-2 by the application of in silico and molecular dynamics (MD) approaches from phytoconstituents present in Homalomena aromatica. The main protease (Mpro) enzyme of SARS-CoV-2 is taken as the target protein to perform the docking analysis of 71 molecules reported from H. aromatica by the application of different modules of Discovery Studio 2018. Five molecules were taken as prospective leads namely dihydrocuminaldehyde, p-cymen-8-ol, cuminaldehyde, p-cymene, and cuminol. In the absence of known inhibitors, a comparative study was performed with the compounds reported in the literature and potent terpenoid–metal complexes were taken into account based on known efficacy as anti-viral molecules. After performing the docking studies with Mpro enzyme of SARS-CoV-2, it was observed that the –CDocker Energy of cuminaldehyde thiosemicarbazone was 29.152, indicating a significant affinity toward Mpro. The same was also supported by the MD study. Taken together, our results provided in silico evidence that secondary metabolites derived from H. aromatica could be employed as potent antiviral agents targeting SARS-CoV-2. Our findings warrant further validation of their in vitro and in vivo efficacies prior to their development into bona fide therapeutic agents.

Identification of novel and potent inhibitors of SARS-CoV-2 main protease from DNA-encoded chemical libraries.

In vitro screening of large compound libraries with automated high-throughput screening is expensive and time-consuming and requires dedicated infrastructures. Conversely, the selection of DNA-encoded chemical libraries (DECLs) can be rapidly performed with routine equipment available in most laboratories. In this study, we identified novel inhibitors of SARS-CoV-2 main protease (Mpro) through the affinity-based selection of the DELopen library (open access for academics), containing 4.2 billion compounds. The identified inhibitors were peptide-like compounds containing an N-terminal electrophilic group able to form a covalent bond with the nucleophilic Cys145 of Mpro, as confirmed by x-ray crystallography. This DECL selection campaign enabled the discovery of the unoptimized compound SLL11 (IC50 = 30 nM), proving that the rapid exploration of large chemical spaces enabled by DECL technology allows for the direct identification of potent inhibitors avoiding several rounds of iterative medicinal chemistry. As demonstrated further by x-ray crystallography, SLL11 was found to adopt a highly unique U-shaped binding conformation, which allows the N-terminal electrophilic group to loop back to the S1' subsite while the C-terminal amino acid sits in the S1 subsite. MP1, a close analog of SLL11, showed antiviral activity against SARS-CoV-2 in the low micromolar range when tested in Caco-2 and Calu-3 (EC50 = 2.3 µM) cell lines. As peptide-like compounds can suffer from low cell permeability and metabolic stability, the cyclization of the compounds will be explored in the future to improve their antiviral activity.

A computational biology approach for the identification of potential SARS-CoV-2 main protease inhibitors from natural essential oil compounds.

Background: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has fomented a climate of fear worldwide due to its rapidly spreading nature, and high mortality rate. The World Health Organization (WHO) declared it as a global pandemic on 11th March, 2020. Many endeavors have been made to find appropriate medications to restrain the SARS CoV-2 infection from spreading but there is no specific antiviral therapy to date. However, a computer-aided drug design approach can be an alternative to identify probable drug candidates within a short time. SARS-CoV-2 main protease is a proven drug target, and it plays a pivotal role in viral replication and transcription. Methods: In this study, we identified a total of 114 essential oil compounds as a feasible anti-SARS-CoV-2 agent from several online reservoirs. These compounds were screened by incorporating ADMET profiling, molecular docking, and 50 ns of molecular dynamics simulation to identify potential drug candidates against the SARS-CoV-2 main protease. The crystallized SARS-CoV-2 main protease structure was collected from the RCSB PDB database (PDB ID 6LU7). Results: According to the results of the ADMET study, none of the compounds have any side effects that could reduce their druglikeness or pharmacokinetic properties. Out of 114 compounds, we selected bisabololoxide B, eremanthin, and leptospermone as our top drug candidates based on their higher binding affinity scores, and strong interaction with the Cys 145-His 41 catalytic dyad. Finally, the molecular dynamics simulation was implemented to evaluate the structural stability of the ligand-receptor complex. MD simulations disclosed that all the hits showed conformational stability compared to the positive control α-ketoamide. Conclusions: Our study showed that the top three hits might work as potential anti-SARS-CoV-2 agents, which can pave the way for discovering new drugs, but for experimental validation, they will require more in vivo trials.

Effects of SARS-CoV-2 Main Protease Mutations at Positions L50, E166, and L167 Rendering Resistance to Covalent and Noncovalent Inhibitors.

SARS-CoV-2 propagation under nirmatrelvir and ensitrelvir pressure selects for main protease (MPro) drug-resistant mutations E166V (DRM2), L50F/E166V (DRM3), E166A/L167F (DRM4), and L50F/E166A/L167F (DRM5). DRM2-DRM5 undergoes N-terminal autoprocessing to produce mature MPro with dimer dissociation constants (Kdimer) 2-3 times larger than that of the wildtype. Co-selection of L50F restores catalytic activity of DRM2 and DRM4 from ∼10 to 30%, relative to that of the wild-type enzyme, without altering Kdimer. Binding affinities and thermodynamic profiles that parallel the drug selection pressure, exhibiting significant decreases in affinity through entropy/enthalpy compensation, were compared with GC373. Reorganization of the active sites due to mutations observed in the inhibitor-free DRM3 and DRM4 structures as compared to MProWT may account for the reduced binding affinities, although DRM2 and DRM3 complexes with ensitrelvir are almost identical to MProWT-ensitrelvir. Chemical reactivity changes of the mutant active sites due to differences in electrostatic and protein dynamics effects likely contribute to losses in binding affinities.

Exploring Niclosamide as a Multi-target Drug Against SARS-CoV-2: Molecular Dynamics Simulation Studies on Host and Viral Proteins.

Niclosamide has emerged as a promising repurposed drug against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In vitro studies suggested that niclosamide inhibits the host transmembrane protein 16F (hTMEM16F), crucial for lipid scramblase activity, which consequently reduces syncytia formation that aids viral spread. Based on other in vitro reports, niclosamide may also target viral proteases such as papain-like protease (PLpro) and main protease (Mpro), essential for viral replication and maturation. However, the precise interactions by which niclosamide interacts with these multiple targets remain largely unclear. Docking and molecular dynamics (MD) simulation studies were undertaken based on a homology model of the hTMEM16F and available crystal structures of SARS-CoV-2 PLpro and Mpro. Niclosamide was observed to bind stably throughout a 400ns MD simulation at the extracellular exit gate of the hTMEM16F tunnel, forming crucial interactions with residues spanning the TM1-TM2 loop (Gln350), TM3 (Phe481), and TM5-TM6 loop (Lys573, Glu594, and Asp596). Among the SARS-CoV-2 proteases, niclosamide was found to interact effectively with conserved active site residues of PLpro (Tyr268), exhibiting better stability in comparison to the control inhibitor, GRL0617. In conclusion, our in silico analyses support niclosamide as a multi-targeted drug inhibiting viral and host proteins involved in SARS-CoV-2 infections.