Search Results (180)

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

The global impact of the COVID-19 crisis has underscored the need for novel therapeutic candidates capable of efficiently targeting essential viral proteins. Existing therapeutic strategies continue to encounter limitations such as reduced efficacy against emerging variants, safety concerns, and suboptimal pharmacodynamics, which emphasize the potential of natural-origin compounds as supportive agents with immunomodulatory, anti-inflammatory, and antioxidant benefits. The present study significantly advances prior molecular docking research through comprehensive virtual screening of structurally related analogs derived from antiviral phytochemicals. These compounds were evaluated specifically against the SARS-CoV-2 main protease (3CLpro) and papain-like protease (PLpro). Utilizing chemical similarity algorithms via the ChEMBL database, over 600 candidate molecules were retrieved and subjected to automated docking, interaction pattern analysis, and comprehensive ADMET profiling. Several analogs showed enhanced binding scores relative to their parent scaffolds, with CHEMBL1720210 (a shogaol-derived analog) demonstrating strong interaction with PLpro (−9.34 kcal/mol), and CHEMBL1495225 (a 6-gingerol derivative) showing high affinity for 3CLpro (−8.04 kcal/mol). Molecular interaction analysis revealed that CHEMBL1720210 forms hydrogen bonds with key PLpro residues including GLY163, LEU162, GLN269, TYR265, and TYR273, complemented by hydrophobic interactions with TYR268 and PRO248. CHEMBL1495225 establishes multiple hydrogen bonds with the 3CLpro residues ASP197, ARG131, TYR239, LEU272, and GLY195, along with hydrophobic contacts with LEU287. Gene expression predictions via DIGEP-Pred indicated that the top-ranked compounds could influence biological pathways linked to inflammation and oxidative stress, processes implicated in COVID-19’s pathology. Notably, CHEMBL4069090 emerged as a lead compound with favorable drug-likeness and predicted binding to PLpro. Overall, the applied in silico framework facilitated the rational prioritization of bioactive analogs with promising pharmacological profiles, supporting their advancement toward experimental validation and therapeutic exploration against SARS-CoV-2. 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

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

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

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

Hand, foot, and mouth disease (HFMD) represents an emerging health concern whose main causative agents are Coxsackievirus A6 (CVA6) and enterovirus A71 (EV71). The lack of a CVA6 vaccine and the rise of new HFMD-causing strains due to the containment of established HFMD-causing viruses necessitates the search for alternative vaccine technologies, including virus-like particle (VLP) vaccine candidates. While studies have demonstrated that production of enterovirus-like particles in various organisms can be achieved by expression of the viral P1 structural proteins and the 3CD protease, optimization based on the interplay between the three most commonly altered infection parameters (multiplicity of infection (MOI), viable cell density at the time of infection (VCD), and the infection period) is often not investigated. To address this challenge, we have performed Design of Experiments (DoE) to optimize the production of CVA6 and EV71 VLPs. Our results indicate that CVA6 VLP production peaks at high MOI, high VCD, and long infection periods. Our subsequent downstream purification processes yielded 38 mg and 158 mg of purified CVA6 and EV71 VLPs from 1 L crude harvest, respectively. This translates into thousands of potential vaccine doses and highlights the economic potential of enterovirus-like particles for vaccine purposes. 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

The SARS-CoV-2 main protease (Mpro, also known as 3CLpro) is a key target for antiviral therapy due to its critical role in viral replication and maturation. This study investigated the inhibitory effects of Bofutrelvir, Nirmatrelvir, and Selinexor on 3CLpro through molecular docking, molecular dynamics (MD) simulations, and free energy calculations. Nirmatrelvir exhibited the strongest binding affinity across docking tools (AutoDock Vina: −8.3 kcal/mol; DiffDock: −7.75 kcal/mol; DynamicBound: 7.59 to 7.89 kcal/mol), outperforming Selinexor and Bofutrelvir. Triplicate 300 ns MD simulations revealed that the Nirmatrelvir-3CLpro complex displayed high conformational stability, reduced root mean square deviation (RMSD), and a modest decrease in solvent-accessible surface area (SASA), indicating enhanced structural rigidity. Gibbs free energy analysis highlighted greater flexibility in unbound 3CLpro, stabilized by Nirmatrelvir binding, supported by stable hydrogen bonds. MolProphet prediction tools, targeting the Cys145 residue, confirmed that Nirmatrelvir exhibited the strongest binding, forming multiple hydrophobic, hydrogen, and π-stacking interactions with key residues, and had the lowest predicted IC₅₀/EC₅₀ (9.18 × 10⁻⁸ mol/L), indicating its superior potency. Bofutrelvir and Selinexor showed weaker interactions and higher IC₅₀/EC₅₀ values. MM/PBSA analysis calculated a binding free energy of −100.664 ± 0.691 kJ/mol for the Nirmatrelvir-3CLpro complex, further supporting its stability and binding potency. These results underscore Nirmatrelvir’s potential as a promising therapeutic agent for SARS-CoV-2 and provide novel insights into dynamic stabilizing interactions through AI-based docking and long-term MD simulations. Full article

The extremely rapid development of understanding and technology that led to the containment of the COVID-19 pandemic resulted from collaborative efforts in the fields of Betacoronavirus pandemicum (SARS-CoV-2) biology, pharmacology, vaccinology, and medicine. Perhaps surprisingly, much of the research was conducted using simple and efficient yeast models. In this manuscript, we describe how yeast, eukaryotic microorganisms, have been used to research this global challenge, focusing on the therapeutic potential of the studies discussed herein. Thus, we outline the role of yeast in studying viral protein interactions with the host cell proteome, including the binding of the SARS-CoV-2 virus spike protein to the human ACE2 receptor and its modulation. The production and exploration of viral antigens in yeast systems, which led to the development of two approved COVID-19 vaccines, are also detailed. Moreover, yeast platforms facilitating the discovery and production of single-domain antibodies (nanobodies) against SARS-CoV-2 are described. Methods guiding modern and efficient drug discovery are explained at length. In particular, we focus on studies designed to search for inhibitors of the main protease (Mpro), a unique target for anti-coronaviral therapies. We highlight the adaptability of the techniques used, providing opportunities for rapid modification and implementation alongside the evolution of the SARS-CoV-2 virus. Approaches introduced in yeast systems that may have universal potential application in studies of emerging viral diseases are also described. Full article

The conservation of the main protease in viral genomes, combined with the absence of a homologous protease in humans, makes this enzyme family an ideal target for developing broad-spectrum antiviral drugs with minimized host toxicity. GC-376, a peptidomimetic 3CL protease inhibitor, has shown significant efficacy against coronaviruses. Recently, a GC-376-based PROTAC was developed to target and induce the proteasome-mediated degradation of the dimeric SARS-CoV-2 3CL^Pro protein. Extending this approach, the current study investigates the application of the GC-376 PROTAC to the 3C^Pro protease of enteroviruses, specifically characterizing its interaction with CVB3 3C^Pro through X-ray crystallography, NMR (Nuclear Magnetic Resonance) and biochemical techniques. The crystal structure of CVB3 3C^Pro bound to the GC-376 PROTAC precursor was obtained at 1.9 Å resolution. The crystallographic data show that there are some changes between the binding of CVB3 3C^Pro and SARS-CoV-2 3CL^Pro, but the overall similarity is strong (RMSD on C-alpha 0.3 Å). The most notable variation is the orientation of the benzyloxycarbonyl group of GC-376 with the S4 subsite of the proteases. NMR backbone assignment of CVB3 3C^Pro bound and unbound to the GC-376 PROTAC precursor (80% and 97%, respectively) was obtained. This information complemented the investigation, by NMR, of the interaction of CVB3 3C^Pro with the GC-376 PROTAC, and its precursor allows us to define that the GC-376 PROTAC binds to CVB3 3C^Pro in a mode very similar to that of the precursor. The NMR relaxation data indicate that a quench of dynamics of a large part of the protein backbone involving the substrate-binding site and surrounding regions occurs upon GC-376 PROTAC precursor binding. This suggests that the substrate cavity, by sampling different backbone conformations in the absence of the substrate, is able to select the suitable one necessary to covalently bind the substrate, this being the latter reaction, which is the fundamental step required to functionally activate the enzymatic reaction. The inhibition activity assay showed inhibition potency in the micromolar range for GC-376 PROTAC and its precursor. Overall, we can conclude that the GC-376 PROTAC fits well within the binding sites of both proteases, demonstrating its potential as a broad-spectrum antiviral agent. Full article

SARS-CoV-2 is a spherical, positive-sense, single-stranded RNA virus with a large genome, responsible for encoding both structural proteins, vital for the viral particle’s architecture, and non-structural proteins, critical for the virus’s replication cycle. Among the non-structural proteins, two cysteine proteases emerge as promising molecular targets for the design of new antiviral compounds. The main protease (M^pro) is a homodimeric enzyme that plays a pivotal role in the formation of the viral replication–transcription complex, associated with the papain-like protease (PL^pro), a cysteine protease that modulates host immune signaling by reversing post-translational modifications of ubiquitin and interferon-stimulated gene 15 (ISG15) in host cells. Due to the importance of these molecular targets for the design and development of novel anti-SARS-CoV-2 drugs, the purpose of this review is to address aspects related to the structure, mechanism of action and strategies for the design of inhibitors capable of targeting the M^pro and PL^pro. Examples of covalent and non-covalent inhibitors that are currently being evaluated in preclinical and clinical studies or already approved for therapy will be also discussed to show the advances in medicinal chemistry in the search for new molecules to treat COVID-19. Full article

Viral diseases of potatoes are among the main problems causing deterioration in the quality of tubers and loss of yield. The growth and development of potato plants largely depend on soil moisture. Prevention strategies require comprehensive protection against pathogens and abiotic stresses, including modeling the beneficial microbiome of agroecosystems combining microorganisms and immunostimulants. Chitosan and its derivatives have great potential for use in agricultural engineering due to their ability to induce plant immune responses. The effect of chitosan conjugate with caffeic acid (ChCA) in combination with Bacillus subtilis 47 on the transcriptional activity of PR protein genes and changes in the proteome of potato plants during potato virus Y (PVY) infection and drought was studied. The mechanisms of increasing the resistance of potato plants to PVY and lack of moisture are associated with the activation of transcription of genes encoding PR proteins: the main protective protein (PR-1), chitinase (PR-3), thaumatin-like protein (PR-5), protease inhibitor (PR-6), peroxidase (PR-9), and ribonuclease (PR-10), as well as qualitative and quantitative changes in the plant proteome. The revealed activation of the expression of marker genes of systemic acquired resistance and induced systemic resistance under the influence of combined treatment with B. subtilis and chitosan conjugate indicate that, in potato plants, the formation of resistance to viral infection in drought conditions proceeds synergistically. By two-dimensional electrophoresis of S. tuberosum leaf proteins followed by MALDI-TOF analysis, 10 proteins were identified, the content and composition of which differed depending on the experiment variant. In infected plants treated with ChCA, the synthesis of proteinaceous RNase P 1 and oxygen-evolving enhancer protein 2 was enhanced in conditions of normal humidity, and 20 kDa chaperonin and TMV resistance protein N-like was enhanced in conditions of lack of moisture. The virus coat proteins were detected, which intensively accumulated in the leaves of plants infected with potato Y-virus. ChCA treatment reduced the content of these proteins in the leaves, and in plants treated with ChCA in combination with Bacillus subtilis, viral proteins were not detected at all, both in conditions of normal humidity and lack of moisture, which suggests the promising use of chitosan derivatives in combination with B. subtilis bacteria in the regulation of plant resistance. Full article