Search Results (214)

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

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 SARS-CoV-2 proteases M^pro and PL^pro are critical targets for antiviral drug development for the treatment of COVID-19. The 1,2,4-thiadiazole functional group is an inhibitor of cysteine proteases, such as papain and cathepsins. This chemical moiety is also present in ceftaroline fosamil (CF), an FDA-approved fifth-generation cephalosporin antibiotic. This study investigates the interactions between CF, its primary metabolites (M1 is dephosphorylated CF and M2 is an opened β-lactam ring) and derivatives (protonated M1H and M2H), and its open 1,2,4-thiadiazole rings derivatives (open-M1H and open-M2H) with SARS-CoV-2 proteases and evaluates CF’s effects on in vitro viral replication. In silico analyses (molecular docking and molecular dynamics (MD) simulations) demonstrated that CF and its metabolites are potential inhibitors of PL^pro and M^pro. Docking analysis indicated that the majority of the ligands were more stable with M^pro than PL^pro; however, in vitro biochemical analysis indicated PL^pro as the preferred target for CF. CF inhibited viral replication in the human Calu-3 cell model at submicromolar concentrations when added to cell culture medium at 12 h. Our results suggest that CF should be evaluated as a potential repurposing agent for COVID-19, considering not only viral proteases but also other viral targets and relevant cellular pathways. Additionally, the reactivity of sulfur in the 1,2,4-thiadiazole moiety warrants further exploration for the development of viral protease inhibitors. Full article

Introduction: Currently, there are no effective medications for treating all the clinical conditions of patients with COVID-19. We aimed to evaluate the antiviral activity of compounds derived from L-tyrosine against the B.1 lineage of SARS-CoV-2 in vitro and in silico. Methodology: The cytotoxicities of 15 halogenated compounds derived from L-tyrosine were evaluated in Vero-E6 cells by the MTT assay. The antiviral activity of the compounds was evaluated using four strategies, and viral quantification was performed by a plaque assay and qRT-PCR. The toxicity of the compounds was evaluated by ADMET predictor software. The affinity of these compounds for viral or cellular proteins and the stability of their conformations were determined by docking and molecular dynamics, respectively. Results: TODC-3M, TODI-2M, and YODC-3M reduced the viral titer >40% and inhibited the replication of viral RNA without significant cytotoxicity. In silico analyses revealed that these compounds presented low toxicity and binding energies between −4.3 and −5.2 Kcal/mol for three viral proteins (spike, Mpro, and RdRp). TODC-3M and YODC-3M presented the most stable conformations with the evaluated proteins. Conclusions: The most promising compounds were TODC-3M, TODI-2M, and YODC-3M, which presented low in vitro and in silico toxicity, antiviral potential through different strategies, and favorable affinities for viral targets. Therefore, they are candidates for in vivo studies. 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

Since 2020, the attention of the scientific community has been focused on the overwhelming COVID-19 pandemic, the infectious disease caused by the coronavirus that has affected populations worldwide. The alarming number of deaths and the severity of the symptoms have driven studies aimed at combating this disease. One of the key components in the development of this disease is the protein M^Pro, responsible for the replication and transcription of the virus, making it an excellent biological target in research efforts seeking an effective treatment for the disease. Furthermore, studies have shown that vanadium complexes, such as bis(N′,N′-dimethylbiguanide)oxovanadium (IV), VO(metf)₂∙H₂O, exhibit highly promising effects for the treatment of COVID-19. This molecule contains a ligand known as metformin, which also holds a prominent place as a potential agent in the treatment of this disease due to its antiviral properties. Therefore, an investigation into the interactions between these two systems (M^Pro+Vanadium Complex and M^Pro+Metformin) is pertinent given the significance of these two molecules. Thus, computational studies such as molecular docking and classical molecular dynamics are considered advantageous, assisting in this comparative study, as well as providing a deeper understanding of the interactions that occur within each of them. 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

The main proteinase (Mpro), or 3CLpro, is a critical enzyme in the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) lifecycle and is responsible for breaking down and releasing vital functional viral proteins crucial for virus development and transmission. As a catalytically active dimer, its dimerization interface has become an attractive target for antiviral drug development. Recent research has extensively investigated the enzymatic activity of Mpro, focusing on its role in regulating the coronavirus replication complex and its significance in virus maturation and infectivity. Computational investigations have identified four druggable pockets, suggesting potential allosteric sites beyond the substrate-binding region. Empirical validation through site-directed alanine mutagenesis has targeted residues in both the active and allosteric regions and corroborated these predictions. Structural studies of drug target proteins can inform therapeutic approaches, with metadynamics simulations shedding light on the role of H163 in regulating Mpro function and providing insights into its dynamic equilibrium to the wild-type enzyme. Despite the efficacy of vaccines and drugs in mitigating SARS-CoV-2 spread, its ongoing viral evolution, selective pressures, and continued transmission pose challenges, potentially leading to resistant mutations. Phylogenetic analyses have indicated the existence of several resistant variations predating drug introduction to the human population, emphasizing the likelihood of drug spread. Hydrogen/deuterium-exchange mass spectrometry reveals the structural influence of the mutation. At the same time, clinical trials on 3CLPro inhibitors underscore the clinical significance of reduced enzymatic activity and offer avenues for future therapeutic exploration. Understanding the implications of 3CLPro mutations holds promise for shaping forthcoming therapeutic strategies against COVID-19. This review delves into factors influencing mutation rates and identifies areas warranting further investigation, providing a comprehensive overview of Mpro mutations, categorization, and terminology. Moreover, we examine their associations with clinical outcomes, illness severity, unresolved issues, and future research prospects, including their impact on vaccine efficacy and potential therapeutic targeting. 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

Endemic and pandemic viruses represent significant public health challenges, leading to substantial morbidity and mortality over time. The COVID-19 pandemic has underscored the urgent need for the development and discovery of new, potent antiviral agents. In this study, we present the synthesis and anti-SARS-CoV-2 activity of a series of benzocarbazoledinones, assessed using cell-based screening assays. Our results indicate that four compounds (4a, 4b, 4d, and 4i) exhibit EC50 values below 4 μM without cytotoxic effects in Calu-3 cells. Mechanistic investigations focused on the inhibition of the SARS-CoV-2 main protease (Mpro) and papain-like protease (PLpro) have used enzymatic assays. Notably, compounds 4a and 4b showed Mpro inhibition activity with IC50 values of 0.11 ± 0.05 and 0.37 ± 0.05 µM, respectively. Furthermore, in silico molecular docking, physicochemical, and pharmacokinetic studies were conducted to validate the mechanism and assess bioavailability. Compound 4a was selected for preliminary drug-likeness analysis and in vivo pharmacokinetics investigations, which yielded promising results and corroborated the in vitro and in silico findings, reinforcing its potential as an anti-SARS-CoV-2 lead compound. Full article

The pathogen of COVID-19, SARS-CoV-2, has caused a severe global health crisis. So far, while COVID-19 has been suppressed, the continuous evolution of SARS-CoV-2 variants has reduced the effectiveness of vaccines such as mRNA-1273 and drugs such as Remdesivir. To uphold the effectiveness of vaccines and drugs prior to potential coronavirus outbreaks, it is necessary to explore the underlying mechanisms between biomolecules and nanodrugs. The experimental study reported that acrylamide fragments covalently attached to Cys145, the main protease enzyme (Mpro) of SARS-CoV-2, and occupied the substrate binding pocket, thereby disrupting protease dimerization. However, the potential mechanism linking them is unclear. The purpose of this work is to complement and validate experimental results, as well as to facilitate the study of novel antiviral drugs. Based on our experimental studies, we identified two acrylamide fragments and constructed corresponding protein-ligand complex models. Subsequently, we performed molecular dynamics (MD) simulations to unveil the crucial interaction mechanisms between these nanodrugs and SARS-CoV-2 Mpro. This approach allowed the capture of various binding conformations of the fragments on both monomeric and dimeric Mpro, revealing significant conformational dissociation between the catalytic and helix domains, which indicates the presence of allosteric targets. Notably, Compound 5 destabilizes Mpro dimerization and acts as an effective inhibitor by specifically targeting the active site, resulting in enhanced inhibitory effects. Consequently, these fragments can modulate Mpro’s conformational equilibrium among extended monomeric, compact, and dimeric forms, shedding light on the potential of these small molecules as novel inhibitors against coronaviruses. Overall, this research contributes to a broader understanding of drug development and fragment-based approaches in antiviral covalent therapeutics. Full article