Search Results (243)

Background: There is growing evidence suggesting that SARS-CoV-2 may contribute to metabolic dysfunction. SARS-CoV-2 infection is associated with systemic inflammation, oxidative stress, and metabolic dysregulation, all of which may impair liver function and promote glucose intolerance. This study investigated the role of SARS-CoV-2, specifically its Main Protease (M^pro), in accelerating insulin resistance and metabolic dysfunction in HepG2 cells in vitro. Methods: HepG2 cells were treated with varying concentrations of M^pro (2.5, 5, 10, 20, 40, 80, and 160 nmol/mL) for 24 h to assess cytotoxicity and glucose uptake. Based on initial findings, subsequent assays focused on higher concentrations (40, 80, and 160 nmol/mL). The effects of M^pro on cell viability, protein kinase B (AKT) expression, matrix metallopeptidase-1 (MMP1), dipeptidyl peptidase 4 (DPP4), interleukin-6 (IL-6) expression, and lipid peroxidation were investigated. Results: Our findings reveal that the SARS-CoV-2 M^pro treatment led to a concentration-dependent reduction in glucose uptake in HepG2 cells. Additionally, the M^pro treatment was associated with reduced insulin-stimulated AKT activation, particularly at higher concentrations. Inflammatory markers such as IL-6 were elevated in the extracellular medium, while DPP4 expression was decreased. However, extracellular soluble DPP4 (sDPP4) levels did not show a significant change. Despite these changes, cell viability remained relatively unaffected, suggesting that the HepG2 cells were able to maintain overall metabolic functions under M^pro exposure. Conclusions: This study demonstrated the concentration-dependent impairment of hepatic glucose metabolism, insulin signaling, and inflammatory pathways in HepG2 cells acutely exposed to the SARS-CoV-2 M^pro. These findings warrant further investigation to explore the long-term metabolic effects of SARS-CoV-2 and its proteases in the liver and to develop potential therapeutic approaches for post-viral metabolic complications. Full article

Background/Objectives: Combination therapies using traditional Chinese medicine and Western drugs have gained attention for their enhanced therapeutic effects and reduced side effects. Toujie Quwen Granules (TQG), known for its antiviral properties, particularly against respiratory viruses, could offer new treatment strategies when combined with antiviral drugs like arbidol, especially for diseases such as Coronavirus disease. This study investigates the synergistic mechanisms between arbidol and components from TQG against the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) main protease (M^pro). Methods: We identified compounds from TQG via existing data. Multi-ligand molecular docking, pharmacokinetic/toxicity screening, and preliminary simulations were performed to assess potential synergistic compounds with arbidol. UPLC-Q-Exactive Orbitrap-MS verified the presence of these compounds. Extended simulations and in vitro assays, including Luciferase and surface plasmon resonance, validated the findings. Results: Five compounds interacted with arbidol in synergy based on docking and preliminary dynamics simulation results. Only Baicalein (HQA004) could be identified in the herbal remedy by untargeted metabolomics, with ideal pharmacokinetic properties, and as a non-toxic compound. Extended simulations revealed that HQA004 enhanced arbidol’s antiviral activity via a “Far” Addition Mechanism #2, with an optimal 2:1 arbidol:HQA004 ratio. The movements of arbidol (diffusion and intramolecular conformational shifts) in the system were significantly reduced by HQA004, which may be the main reason for the synergism that occurred. In vitro experiments confirmed an increased inhibition of M^pro by the combination. Conclusions: HQA004 demonstrated synergistic potential with arbidol in inhibiting M^pro. The development of combination therapies integrating Western and herbal medicine is supported by these findings for effective antiviral treatments. Full article

The SARS-CoV-2 main protease (Mpro) is a validated therapeutic target for inhibiting viral replication. Few compounds have advanced clinically, underscoring the difficulty in optimizing both target affinity and drug-like properties. To address this challenge, we integrated machine learning (ML), molecular docking, and molecular dynamics (MD) simulations to investigate the balance between pharmacodynamic (PD) and pharmacokinetic (PK) properties in Mpro inhibitor design. We developed ML models to classify Mpro inhibitors based on experimental IC₅₀ data, combining molecular descriptors with structural insights from MD simulations. Our Support Vector Machine (SVM) model achieved strong performance (training accuracy = 0.84, ROC AUC = 0.91; test accuracy = 0.79, ROC AUC = 0.86), while our Logistic Regression model (training accuracy = 0.78, ROC AUC = 0.85; test accuracy = 0.76, ROC AUC = 0.83). Notably, PK descriptors often exhibited opposing trends to binding affinity: hydrophilic features enhanced binding affinity but compromised PK properties, whereas hydrogen bonding, hydrophobic, and π–π interactions in Mpro subsites S2 and S3/S4 are fundamental for binding affinity. Our findings highlight the need for a balanced approach in Mpro inhibitor design, strategically targeting these subsites may balance PD and PK properties. For the first time, we demonstrate antagonistic trends between pharmacokinetic (PK) and pharmacodynamic (PD) features through the integrated application of ML/MD. This study provides a computational framework for rational Mpro inhibitors, combining ML and MD to investigate the complex interplay between enzyme inhibition and drug likeness. These insights may guide the hit-to-lead optimization of the novel next-generation Mpro inhibitors of SARS-CoV-2 with preclinical and clinical potential. Full article

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus responsible for COVID-19, remains a major global health threat. The virus enters host cells by binding to the angiotensin-converting enzyme 2 (ACE2) receptor. Several small-molecule antiviral drugs, including molnupiravir, favipiravir, remdesivir, and nirmatrelvir have been shown to inhibit SARS-CoV-2 replication and are approved for treating SARS-CoV-2 infections. Nirmatrelvir inhibits the viral main protease (M^pro), a key enzyme for processing polyproteins in viral replication. In contrast, molnupiravir, favipiravir, and remdesivir are prodrugs that target RNA-dependent RNA polymerase (RdRp), which is crucial for genome replication and subgenomic RNA production. However, undergoing extensive metabolism profoundly impacts their therapeutic effects. Carboxylesterases (CES) are a family of enzymes that play an essential role in the metabolism of many drugs, especially prodrugs that require activation through hydrolysis. Molnupiravir is activated by carboxylesterase-2 (CES2), while remdesivir is hydrolytically activated by CES1 but inhibits CES2. Nirmatrelvir and remdesivir are oxidized by the same cytochrome P450 (CYP) enzyme. Additionally, various transporters are involved in the uptake or efflux of these drugs and/or their metabolites. It is well established that drug-metabolizing enzymes and transporters are differentially expressed depending on the cell type, and these genes exhibit significant polymorphisms. In this review, we examine how CES-related cellular and genetic factors influence the therapeutic activities of these widely used COVID-19 medications. This article highlights implications for improving product design, targeted inhibition, and personalized medicine by exploring genetic variations and their impact on drug metabolism and efficacy. Full article

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the coronavirus disease of 2019 (COVID-19) and has persistently caused infections since its emergence in late 2019. The main protease (Mpro) of SARS-CoV-2 plays a crucial role in its life-cycle; thus, it is an important target for drug development. One of the first virus-specific drugs that has been approved for the treatment of COVID-19 patients is Paxlovid, which contains nirmatrelvir, a covalent inhibitor of Mpro. Screening of inhibitor candidates and specificity studies also rely on efficient substrates and activity assays. Casein is one of the most commonly applied universal substrates that can be used to study a wide range of proteases, including SARS-CoV-2 Mpro. Casein is a known substrate for Mpro in vitro, but the specific casein isoform cleaved by Mpro remained unidentified, and the cleavage sites have yet to be determined. This work studied cleavage of α-, β- and κ-isoforms of bovine casein by SARS-CoV-2 Mpro, using in vitro and in silico approaches. The candidate cleavage sites were predicted in silico based on the protein sequences, and the cleavage positions were identified based on mass spectrometric analysis of cleavage fragments. Based on our results, only β-casein contains cleavage sites for Mpro and thus can be used as its substrate in vitro. The newly identified cleavage site sequences further widen the knowledge about the specificity of SARS-CoV-2 Mpro. Full article

Coronaviruses (CoVs) have recently emerged as significant causes of respiratory disease outbreaks, with the novel coronavirus pneumonia of 2019, known as COVID-19, being highly infectious and triggered by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Understanding virus–host interactions and molecular targets in host cell death signalling is crucial for inhibitor development. Among the promising targets for inhibitor development is the main protease (Mpro), which is essential for viral replication. While current research has focused mainly on covalent inhibitors, growing attention is being given to non-covalent inhibitors due to their potential for lower toxicity and improved resistance to viral mutations. This literature review provides an in-depth analysis of recent in silico approaches used to identify and optimise non-covalent inhibitors of SARS-CoV-2 Mpro. It focuses on molecular docking and robust molecular dynamics (MD) simulation technologies to discover novel scaffolds with better binding affinities. The article summarises recent studies that pre-screened several potential non-covalent inhibitors, including natural constituents like alkaloids, flavonoids, terpenoids, diarylheptanoids, and anthraquinones, using in silico methods. The in silico approach, pivotal to developing small molecules of Mpro non-covalent inhibitors, provides an efficient avenue to guide future research efforts toward developing high-performance Mpro inhibitors for SARS-CoV-2 Mpro, representing the latest advancements in drug design. Full article

The ongoing threat of viral pandemics such as COVID-19 highlights the urgent need for novel antiviral therapeutics targeting conserved viral proteins. In this study, peptides of 10–30 kDa derived from the marine diatom Phaeodactylum tricornutum were identified as potential inhibitors of SARS-CoV-2 main protease (Mpro), a key enzyme in viral replication. Peptides less than 60 amino acids in length were retrieved from the UniProt database and aligned with reference antiviral sequences using the Biopython pairwise2 algorithm. Six candidates were selected for structural modeling using AlphaFold2 and Swiss-Model, followed by molecular docking using ClusPro2. LigPlot+ was used to assess molecular interactions, while NetMHCpan 4.1 and AVPpred evaluated immunogenicity and antiviral potential, respectively. Molecular dynamics simulations over 100 ns were conducted using OpenMM. These peptides demonstrated stable binding interactions with key catalytic residues of Mpro. Specifically, peptide A0A8J9SA87 interacted with Cys145 and Glu166, while peptide A0A8J9SDW0 exhibited interactions with His41 and Phe140, both of which are known to be essential for Mpro inhibition. Although peptide A0A8J9X3P8 also interacted with catalytic residues, it exhibited greater structural fluctuations during molecular dynamics simulations and achieved lower AVPpred scores, suggesting lower overall antiviral potential. Therefore, A0A8J9SA87 and A0A8J9SDW0 were identified as the most promising candidates. Molecular dynamics simulations further supported the high structural stability of these peptide-Mpro complexes over a 100 ns timescale, reinforcing their potential as effective inhibitors. These findings support P. tricornutum as a valuable source of antiviral peptides and demonstrate the feasibility of in silico pipelines for identifying therapeutic candidates against SARS-CoV-2. Full article

Flavone derivatives have emerged as promising antiviral agents, with baicalein demonstrating the potent inhibition of the SARS-CoV-2 main protease (Mpro). In this study, the unique electronic and structural properties of 3-hydroxyflavone (3-HF) were investigated using the density functional theory (B3PW91/cc-pVTZ), providing insights into its potential as a bioactive scaffold. Among mono-hydroxyflavone (n-HF) isomers, 3-HF exhibits an extensive intramolecular hydrogen-bonding network linking the phenyl B-ring to the A- and γ-pyrone C-rings, enabled by the distinctive C3-OH substitution. Despite a slight non-planarity (dihedral angle: 15.4°), this hydrogen-bonding network enhances rigidity and influences the electronic environment. A ¹³C-NMR chemical shift analysis revealed pronounced quantum mechanical effects of the C3-OH group, diverging from the trends observed in other flavones. A natural bond orbital (NBO) analysis highlighted an unusual charge distribution, with predominantly positive charges on the γ-pyrone C-ring carbons, in contrast to the typical negative charges in flavones. These effects impact C1s orbital energies, suggesting that the electronic structure plays a more significant role in ¹³C-NMR shifts than simple ring assignments. Given the established antiviral activity of hydroxylated flavones, the distinct electronic properties of 3-HF may enhance its interaction with SARS-CoV-2 Mpro, making it a potential candidate for further investigation. This study underscores the importance of quantum mechanical methods in elucidating the structure–activity relationships of flavones and highlights 3-HF as a promising scaffold for future antiviral drug development. Full article

The SARS-CoV-2 main protease (Mpro), essential for viral replication, remains a prime target for antiviral drug design against COVID-19 and related coronaviruses. In this study, we present a systematic investigation into the molecular determinants of Mpro inhibition using an integrated approach combining large-scale data mining, cheminformatics, and quantum chemical calculations. A curated dataset comprising 963 high-resolution structures of Mpro–ligand complexes—348 covalent and 615 non-covalent inhibitors—was mined from the Protein Data Bank. Cheminformatics analysis revealed distinct physicochemical profiles for each inhibitor class: covalent inhibitors tend to exhibit higher hydrogen bonding capacity and sp³ character, while non-covalent inhibitors are enriched in aromatic rings and exhibit greater aromaticity and lipophilicity. A novel descriptor, Weighted Hydrogen Bond Count (WHBC), normalized for molecular size, revealed a notable inverse correlation with aromatic ring count, suggesting a compensatory relationship between hydrogen bonding and π-mediated interactions. To elucidate the energetic underpinnings of molecular recognition, 40 representative inhibitors (20 covalent, 20 non-covalent) were selected based on principal component analysis and aromatic ring content. Quantum mechanical calculations at the double-hybrid B2PLYP/def2-QZVP level quantified non-bonded interaction energies, revealing that covalent inhibitors derive binding strength primarily through hydrogen bonding (~63.8%), whereas non-covalent inhibitors depend predominantly on π–π stacking and CH–π interactions (~62.8%). Representative binding pocket analyses further substantiate these findings: the covalent inhibitor F2F-2020198-00X exhibited strong hydrogen bonds with residues such as Glu166 and His163, while the non-covalent inhibitor EDG-MED-10fcb19e-1 engaged in extensive π-mediated interactions with residues like His41, Met49, and Met165. The distinct interaction patterns led to the establishment of pharmacophore models, highlighting key recognition motifs for both covalent and non-covalent inhibitors. Our findings underscore the critical role of aromaticity and non-bonded π interactions in driving binding affinity, complementing or, in some cases, substituting for hydrogen bonding, and offer a robust framework for the rational design of next-generation Mpro inhibitors with improved selectivity and resistance profiles. Full article

The molecular approach to understanding the mechanisms of emerging diseases, like COVID-19, has largely accelerated the search for successful therapeutical strategies. In this work, we present an extensive molecular dynamics (MD) analysis of two forms of the SARS-CoV-2 main protease M^Pro. We analyzed the free form (apo) and compared the results with those coming from the (holo) form bound to the inhibitor Boceprevir, an FDA-approved drug repurposed for COVID-19 therapy. We applied Dynamic Cross Correlation (DCC) analysis to the MD simulations to trace the concerted motion patterns within the protein structure. Although symmetric, the homodimer in the bound form showed clearly asymmetric dynamical behavior. In particular, the presence of concerted motions was detected in the protomer where the expulsion of the substrate from the active site happened. Such behavior was not observed in the same time lapses in the apo form. These results highlight a sort of ‘symmetry breaking’, making a symmetric structure to display functional induced asymmetric behavior in response to a perturbation. This highly coordinated dynamics in response to an external cue confirms the character of ‘complex molecular machines’ of biopolymers. Full article

We have designed, synthesized, and characterized a small library of shikonin derivatives and demonstrated their inhibitory activity against the main protease, M^pro, of SARS-CoV-2. One analog, 5,8-dimethyl shikonin oxime (15), exhibited the highest activity against SARS-CoV-2 M^pro with an IC₅₀ value of 12.53 ± 3.59 μM. It exhibited much less toxicity as compared with the parent compound, shikonin, in both in vitro and in vivo models. Structure–activity relationship analysis indicated that the oxime moieties on the naphthalene ring and the functional groups attached to the oxygen atom on the side chain play a pivotal role in enzymatic inhibitory activity. Molecular docking results implied that the inhibitor 15 is perfectly settled in the core of the substrate-binding pocket of M^pro by possibly interacting with three catalytic residues, His41, Cys145, and Met165. Overall, the shikonin oxime derivative 15 deserves further investigation as an antiviral agent against SARS-CoV-2. Full article

The main protease (M^pro or 3CL^pro or nsp5) of SARS-CoV-2 is crucial to the life cycle and pathogenesis of SARS-CoV-2, making it an attractive drug target to develop antivirals. This study employed the virtual screening of a few phytochemicals, and the resultant best compound, Scopoletin, was further investigated by a FRET-based enzymatic assay, revealing an experimental IC₅₀ of 15.75 µM. The impact of Scopoletin on M^pro was further investigated by biophysical and MD simulation studies. Fluorescence spectroscopy identified a strong binding constant of 3.17 × 10⁴ M⁻¹ for Scopoletin binding to M^pro, as demonstrated by its effective fluorescence quenching of M^pro. Additionally, CD spectroscopy showed a significant reduction in the helical content of M^pro upon interaction with Scopoletin. The findings of thermodynamic measurements using isothermal titration calorimetry (ITC) supported the spectroscopic data, indicating a tight binding of Scopoletin to M^pro with a K_A of 2.36 × 10³ M⁻¹. Similarly, interaction studies have also revealed that Scopoletin forms hydrogen bonds with the amino acids nearest to the active site, and this has been further supported by molecular dynamics simulation studies. These findings indicate that Scopoletin may be developed as a potential antiviral treatment for SARS-CoV-2 by targeting M^pro. Full article

In order to detect and respond to invading pathogens, mammals have evolved a battery of pattern recognition receptors. Among these, RIG-I-like receptors (RLR) are cytosolic RNA sensors that play an essential role in the innate immune response against RNA viruses, including coronaviruses. In return, coronaviruses have acquired diverse strategies to impair RLR-mediated immune responses to enable productive infection. Viral innate immune evasion mechanisms have been well studied for highly pathogenic human coronaviruses (HCoVs), and often, these activities are thought to be linked to the severe symptoms these viruses can cause. Whether other coronaviruses, including human common cold coronaviruses, display similar activities has remained understudied. Here, we present evidence that the main protease (Mpro) of common cold HCoV-229E acts as an interferon (IFN) and NF-κB antagonist by disrupting RLR-mediated antiviral signalling. Furthermore, we show that HCoV-229E, HCoV-OC43 and MERS-CoV Mpros are able to directly cleave NEMO. We also show that HCoV-229E Mpro induces the cleavage and/or degradation of multiple other RLR pathway components, including MDA5, TBK1 and IKKε. Finally, we show that HCoV-229E infection leads to a delayed innate immune response that is accompanied by a decrease in NEMO protein levels. Our results suggest that NEMO degradation during HCoV-229E infection could be mediated, in part, by cellular degradation pathways, in addition to viral Mpro-mediated cleavage. Altogether, our research unveils innate immune evasion activities of the Mpros of low-pathogenic coronaviruses, which, despite their low pathogenicity, appear to share functionalities previously described for highly pathogenic HCoVs. Full article

The three-year COVID-19 pandemic ‘has’ caused a wide range of medical, social, political, and financial implications. Since the end of 2020, various mutations and variations in SARS-CoV-2 strains, along with the immune escape phenomenon, have emerged. There is an urgent need to identify a relatively stable target for the development of universal vaccines and drugs that can effectively combat both SARS-CoV-2 strains and their mutants. Currently, the main focus in treating SARS-CoV-2 lies in disrupting the virus’s life cycle. The main protease (M^pro) is closely associated with virus replication and maturation and plays a crucial role in the early stages of infection. Consequently, it has become an important target for the development of SARS-CoV-2-specific drugs. This review summarizes the recent research progress on the novel coronavirus’s main proteases, including the pivotal role of M^pro in the virus’s life cycle, the structure and catalytic mechanism of M^pro, the self-maturation mechanism of M^pro, the role of M^pro in virus immune escape, the current methods of developing antiviral drugs targeting M^pro, and the key drugs that have successfully entered clinical trials. The aim is to provide researchers involved in the development of antiviral drugs targeting M^pro with systematic and comprehensive information. Full article

The global burden of COVID-19 continues to rise, and despite significant progress in vaccine development, there remains a critical need for effective treatments for the severe inflammation and acute lung injury associated with SARS-CoV-2 infection. In this study, we explored the antiviral properties of a plant-derived complex consisting of flavonol and hydroxyorganic acid compounds. Our research focused on the ability of the flavonol and hydroxyorganic acid complex to suppress the activity of several key proteins involved in the replication and maturation of SARS-CoV-2. These proteins include ACE2 protein, HRV 3C Protease, and Mpro (Main Protease). It was shown that the plant-based complex effectively inhibited the activity of these viral proteins. In addition to its effects on viral proteins, the flavonol and hydroxyorganic acid complex were shown to suppress viral replication in Vero E6 cells. At a dose of 22 μg/mL, the drug demonstrated maximum antiviral activity, significantly reducing the replication of SARS-CoV-2 in vitro. In preliminary studies, the complex showed both prophylactic and therapeutic potential, suggesting that it may be useful for preventing infection, as well as reducing the severity of disease once an individual has been infected with SARS-CoV-2. Based on the compelling results of this study, we propose the flavonol and hydroxyorganic acid complex as a potential therapeutic compound for SARS-CoV-2. Its ability to inhibit key viral proteins, suppress viral replication and exhibit protective and therapeutic effects positions it as a valuable candidate for further research and clinical evaluation. As the global fight against SARS-CoV-2 continues, plant-based therapies like this complex could complement existing treatments and provide new options for managing and treating the disease. Full article