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We have synthesized a novel and highly selective severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) main protease peptide mimetic inhibitor mimicking the replicase 1ab recognition sequence -Val-Leu-Gln- and utilizing a cysteine selective acyloxymethyl ketone as the electrophilic warhead to target the active site Cys145. Utilizing a constrained cyclic peptide that locks the conformation between the P3 (Val) and P2 (Leu) residues, we identified a highly selective inhibitor that fills the P2 pocket occupied by the leucine residue sidechain of PF-00835231 and the dimethyl-3-azabicyclo-hexane motif in nirmatrelvir (PF-07321332). This strategy resulted in potent and highly selective Mpro inhibitors without inhibiting essential host cathepsin cysteine or serine proteases. The lead prototype compound 1 (MPro IC 50 = 230 ± 18 nM) also inhibits the replication of multiple SARS-CoV-2 variants in vitro , including SARS-CoV-2 variants of concern, and can synergize at lower concentrations with the viral RNA polymerase inhibitor, remdesivir, to inhibit replication. It also reduces SARS-CoV-2 replication in SARS-CoV-2 Omicron-infected Syrian golden hamsters without obvious toxicities, demonstrating in vivo efficacy. This novel lead structure provides the basis for optimization of improved agents targeting evolving SARS-CoV-2 drug resistance that can selectively act on Mpro versus host proteases and are less likely to have off-target effects due to non-specific targeting. Developing inhibitors against the active site of the main protease (Mpro), which is highly conserved across coronaviruses, is expected to impart a higher genetic barrier to evolving SARS-CoV-2 drug resistance. Drugs that selectively inhibit the viral Mpro are less likely to have off-target effects warranting efforts to improve this therapy., Competing Interests: J.M.S. receives funding from the NIH P30-CA010815-53; has patents pending to Wistar; owns equity in Alliance Discovery, Inc., Barer Institute, and Context Therapeutics; and consults for Syndeavor Therapeutics, Inc. All other authors declare no competing interests.

Aedes aegypti is the primary vector for the transmission of the dengue virus, which causes dengue fever, dengue hemorrhagic illness and dengue shock syndrome. There is now no antiviral medication available to treat DENV, which kills thousands of people each year and infects millions of individuals. A possible target for the creation of fresh and efficient dengue treatments is the DENV-3 NS5 MTase. So, Nigella sativa quinones were examined using in silico methods to find natural anti-DENV compounds. The in silico docking was conducted utilising the Discovery Studio software on the quinones of N. sativa and the active site of the target protein DENV-3 NS5 MTase. In addition, the druggability and pharmacokinetics of the lead compound were assessed. Dithymoquinone was comparable to the reference compound in terms of its ability to bind to the active site of target protein. Dithymoquinone met the requirements for drug likeness and Lipinski's principles, as demonstrated by the ADMET analysis and drug likeness results. The current study indicated that the dithymoquinone from N. sativa had anti-DENV activity, suggesting further drug development and dengue treatment optimisation.Communicated by Ramaswamy H. Sarma.

NS3-4A, a serine protease, is a primary target for drug development against Hepatitis C Virus (HCV). However, the effectiveness of potent next-generation protease inhibitors is limited by the emergence of mutations and resulting drug resistance. To address this, in this study a structure-based drug design approach is employed to screen a large library of 7320 natural compounds against both wild-type and mutant variants of NS3-4A protease. Telaprevir, a widely used protease inhibitor, was recruited as the control drug. The top 10 compounds with favorable binding affinities underwent drug-likeness evaluation. Based on ADMET studies, complexes of NP_024762 and NP_006776 were selected for molecular dynamic simulations. Principal component analysis (PCA) was employed to explore the conformational space and protein dynamics of the protein-ligand complex using a Free Energy Landscape (FEL) approach. The cosine values obtained from FEL analysis ranged from 0 to 1, and eigenvectors with cosine values below 0.2 were chosen for further analysis. To forecast binding free energies and evaluate energy contributions per residue, the MM-PBSA method was employed. The results highlighted the crucial role of amino acids in the catalytic domain for the binding of the protease with phytochemicals. Stable associations between the top compounds and the target protease were confirmed by the formation of hydrogen bonds in the binding pocket involving residues: His1057, Gly1137, Ser1139, and Ala1157. These findings suggest the potential of these compounds for further validation through biological evaluation.Communicated by Ramaswamy H. Sarma.

Dengue virus (DENV2) is the cause of dengue disease and a worldwide health problem. DENV2 replicates in the host cell using polyproteins such as NS3 protease in conjugation with NS2B cofactor, making NS3 protease a promising antiviral drug-target. This study investigated the efficacy of 'Niloticin' against NS2B/NS3-protease. In silico and in vitro analyses were performed which included interaction of niloticin with NS2B/NS3-protease, protein stability and flexibility, mutation effect, betweenness centrality of residues and analysis of cytotoxicity, protein expression and WNV NS3-protease activity. Similar like acyclovir, niloticin forms strong H-bonds and hydrophobic interactions with residues LEU149, ASN152, LYS74, GLY148 and ALA164. The stability of the niloticin-NS2B/NS3-protease complex was found to be stable compared to the apo NS2B/NS3-protease in structural deviation, PCA, compactness and FEL analysis. The IC 50 value of niloticin was 0.14 μM in BHK cells based on in vitro cytotoxicity analysis and showed significant activity at 2.5 μM in a concentration-dependent manner. Western blotting and qRT-PCR analyses showed that niloticin reduced DENV2 protein transcription in a dose-dependent manner. Besides, niloticin confirmed the inhibition of NS3-protease by the SensoLyte 440 WNV protease detection kit. These promising results suggest that niloticin could be an effective antiviral drug against DENV2 and other flaviviruses., Competing Interests: Declaration of competing interest The authors declare that there are no conflicts of interest regarding the publication of this article., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Researchers worldwide are looking for molecules that might disrupt the COVID-19 life cycle. Endoribonuclease, which is responsible for processing viral RNA to avoid detection by the host defense system, and helicase, which is responsible for unwinding the RNA helices for replication, are two key non-structural proteins. This study performs a hierarchical structure-based virtual screening approach for NSP15 and helicase to reach compounds with high binding probabilities. In this investigation, we incorporated a variety of filtering strategies for predicting compound interactions. First, we evaluated 756,275 chemicals from four databases using a deep learning method (NCI, Drug Bank, Maybridge, and COCONUT). Following that, two docking techniques (extra precision and induced fit) were utilized to evaluate the compounds' binding affinity, followed by molecular dynamic simulation supported by the MM-GBSA free binding energy calculation. Remarkably, two compounds (90616 and CNP0111740) exhibited high binding affinity values of -66.03 and -12.34 kcal/mol for helicase and NSP15, respectively. The VERO-E6 cell line was employed to test their in vitro therapeutic impact. The CC 50 for CNP0111740 and 90616 were determined to be 102.767 μg/ml and 379.526 μg/ml, while the IC 50 values were 140.176 μg/ml and 5.147 μg/ml, respectively. As a result, the selectivity index for CNP0111740 and 90616 is 0.73 and 73.73, respectively. Finally, these compounds were found to be novel, effective inhibitors for the virus; however, further in vivo validation is needed.Communicated by Ramaswamy H. Sarma.

Human coronaviruses are a group of pathogens that primarily cause respiratory and intestinal diseases. Infection can easily cause respiratory symptoms, as well as a variety of serious complications. There are several types of human coronaviruses, such as SARS-CoV, MERS-CoV, HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, and SARS-CoV-2. The prevalence of COVID-19 has led to a growing focus on drug research against human coronaviruses. The main protease (M pro ) from human coronaviruses is a relatively conserved that controls viral replication. X77 was discovered to have extremely high inhibitory activity against SARS-CoV-2 M pro through the use of computer-simulated docking. In this paper, we have resolved the crystal structure of the HCoV-NL63 M pro complexed with X77 and analyzed their interaction in detail. This data provides essential information for solving their binding modes and their structural determinants. Then, we compared the binding modes of X77 with SARS-CoV-2 M pro and HCoV-NL63 M pro in detail. This study illustrates the structural basis of HCoV-NL63 M pro binding to the inhibitor X77. The structural insights derived from this study will inform the development of new drugs with broad-spectrum resistance to human coronaviruses., Competing Interests: Declaration of competing interest The authors declare no conflicts of interest., (Copyright © 2024 Elsevier Inc. All rights reserved.)

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genome encodes 29 proteins including four structural, 16 nonstructural (nsps), and nine accessory proteins (https://epimedlab.org/sars-cov-2-proteome/). Many of these proteins contain potential targetable sites for the development of antivirals. Despite the widespread use of vaccinations, the emergence of variants necessitates the investigation of new therapeutics and antivirals. Here, the EpiMed Coronabank Chemical Collection (https://epimedlab.org/crl/) was utilized to investigate potential antivirals against the nsp14 exoribonuclease (ExoN) domain. Molecular docking was performed to evaluate the binding characteristics of our chemical library against the nsp14 ExoN site. Based on the initial screen, trisjuglone, ararobinol, corilagin, and naphthofluorescein were identified as potential lead compounds. Molecular dynamics (MD) simulations were subsequently performed, with the results highlighting the stability of the lead compounds in the nsp14 ExoN site. Protein-RNA docking revealed the potential for the lead compounds to disrupt the interaction with RNA when bound to the ExoN site. Moreover, hypericin, cyanidin-3-O-glucoside, and rutin were previously identified as lead compounds targeting the papain-like protease (PL pro ) naphthalene binding site. Through performing MD simulations, the stability and interactions of lead compounds with PL pro were further examined. Overall, given the critical role of the exonuclease activity of nsp14 in ensuring viral fidelity and the multifunctional role of PL pro in viral pathobiology and replication, these nsps represent important targets for antiviral drug development. Our databases can be utilized for in silico studies, such as the ones performed here, and this approach can be applied to other potentially druggable SARS-CoV-2 protein targets., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Three new series of macrocyclic active site-directed inhibitors of the Zika virus (ZIKV) NS2B-NS3 protease were synthesized. First, attempts were made to replace the basic P3 lysine residue of our previously described inhibitors with uncharged and more hydrophobic residues. This provided numerous compounds with inhibition constants between 30 and 50 nM. A stronger reduction of the inhibitory potency was observed when the P2 lysine was replaced by neutral residues, all of these inhibitors possess K i values >1 µM. However, it is possible to replace the P2 lysine with the less basic 3-aminomethylphenylalanine, which provides a similarly potent inhibitor of the ZIKV protease (K i  = 2.69 nM). Crystal structure investigations showed that the P2 benzylamine structure forms comparable interactions with the protease as lysine. Twelve additional structures of these inhibitors in complex with the protease were determined, which explain many, but not all, SAR data obtained in this study. All individual modifications in the P2 or P3 position resulted in inhibitors with low antiviral efficacy in cell culture. Therefore, a third inhibitor series with combined modifications was synthesized; all of them contain a more hydrophobic d-cyclohexylalanine in the linker segment. At a concentration of 40 µM, two of these compounds possess similar antiviral potency as ribavirin at 100 µM. Due to their reliable crystallization in complex with the ZIKV protease, these cyclic compounds are very well suited for a rational structure-based development of improved inhibitors., (© 2024 The Authors. Archiv der Pharmazie published by Wiley‐VCH GmbH on behalf of Deutsche Pharmazeutische Gesellschaft.)

Chronic hepatitis caused by the hepatitis C virus (HCV) is closely linked with the advancement of liver disease. The research hypothesis suggests that the NS5B enzyme (non-structural 5B protein) of HCV plays a pivotal role in facilitating viral replication within host cells. Hence, the objective of the present investigation is to identify the binding interactions between the structurally diverse phytotherapeutics and those of the catalytic residue of the target NS5B polymerase protein. Results of our docking simulations reveal that compounds such as arjunolic acid, sesamin, arjungenin, astragalin, piperic acid, piperidine, piperine, acalyphin, adhatodine, amyrin, anisotine, apigenin, cuminaldehyde, and curcumin exhibit a maximum of three interactions with the catalytic residues (Asp 220, Asp 318, and Asp 319) present on the Hepatitis C virus NS5B polymerase of HCV. Molecular dynamic simulation, particularly focusing on the best binding lead compound, arjunolic acid (-8.78 kcal/mol), was further extensively analyzed using RMSD, RMSF, Rg, and SASA techniques. The results of the MD simulation confirm that the NS5B-arjunolic acid complex becomes increasingly stable from 20 to 100 ns. The orientation of both arjunolic acid and sofosbuvir triphosphate (standard) within the active site was investigated through DCCM, PCA, and FEL analysis, indicating highly stable interactions of the lead arjunolic acid with the catalytic region of the NS5B enzyme. The findings of our current investigation suggest that bioactive therapeutics like arjunolic acid could serve as promising candidates for limiting the NS5B polymerase activity of the hepatitis C virus, offering hope for the future of HCV treatment., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Zika virus (ZIKV) is a mosquito-borne human flavivirus responsible that causing emergency outbreaks in Brazil. ZIKV is suspected of causing Guillain-Barre syndrome in adults and microcephaly. The NS2B-NS3 protease and NS5 RNA-dependent RNA polymerase (RdRp), central to ZIKV multiplication, have been identified as attractive molecular targets for drugs. We performed a structure-based virtual screening of 2,659 FDA-approved small molecule drugs in the DrugBank database using AutoDock Vina in PyRx v0.8. Accordingly, 15 potential drugs were selected as ZIKV inhibitors because of their high values (binding affinity - binding energy) and we analyzed the molecular interactions between the active site amino acids and the compounds. Among these drugs, tamsulosin was found to interact most efficiently with NS2B/NS3 protease, as indicated by the lowest binding energy value (-8.27 kJ/mol), the highest binding affinity (-5.7 Kcal/mol), and formed H-bonds with amino acid residues TYRB130, SERB135, TYRB150. Furthermore, biotin was found to interact most efficiently with NS5 RdRp with a binding energy of -150.624 kJ/mol, a binding affinity of -5.6 Kcal/mol, and formed H-bonds with the amino acid residues ASPA665 and ASPA540. In vitro , in vivo , and clinical studies are needed to demonstrate anti-ZIKV safety and the efficacy of these FDA-approved drug candidates.Communicated by Ramaswamy H. Sarma.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for nearly 7 million deaths worldwide since its outbreak in late 2019. Even with the rapid development and production of vaccines and intensive research, there is still a huge need for specific anti-viral drugs that address the rapidly arising new variants. To address this concern, the National Institute of Allergy and Infectious Diseases (NIAID) established nine Antiviral Drug Discovery (AViDD) Centers, tasked with exploring approaches to target pathogens with pandemic potential, including SARS-CoV-2. In this study, we sought inhibitors of SARS-CoV2 non-structural protein 13 (nsP13) as potential antivirals, first developing a HTS-compatible assay to measure SARS-CoV2 nsP13 helicase activity. Here we present our effort in implementing the assay in a 1,536 well-plate format and in identifying nsP13 inhibitor hit compounds from a ∼650,000 compound library. The primary screen was robust (average Z' = 0.86 ± 0.05) and resulted in 7,009 primary hits. 1,763 of these compounds upon repeated retests were further confirmed, showing consistent inhibition. Following in-silico analysis, an additional orthogonal assay and titration assays, we identified 674 compounds with IC 50 <10 μM. We confirmed activity of independent compound batches from de novo powders while also incorporating multiple counterscreen assays. Our study highlights the potential of this assay for use on HTS platforms to discover novel compounds inhibiting SARS-CoV2 nsP13, which merit further development as an effective SARS-CoV2 antiviral., Competing Interests: Declaration of competing interest There are no conflicts of interest amongst any of the authors and the work pertained in this manuscript., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Main proteases (M pro s) are a class of conserved cysteine hydrolases among coronaviruses and play a crucial role in viral replication. Therefore, M pro s are ideal targets for the development of pan-coronavirus drugs. X77, previously developed against SARS-CoV M pro , was repurposed as a non-covalent tight binder inhibitor against SARS-CoV-2 M pro during COVID-19 pandemic. Many novel inhibitors with favorable efficacy have been discovered using X77 as a reference, suggesting that X77 could be a valuable scaffold for drug design. However, the broad-spectrum performance of X77 and underlying mechanism remain less understood. Here, we reported the crystal structures of M pro s from SARS-CoV-2, SARS-CoV, and MERS-CoV, and several M pro mutants from SARS-CoV-2 variants bound to X77. A detailed analysis of these structures revealed key structural determinants essential for interaction and elucidated the binding modes of X77 with different coronaviral M pro s. The potencies of X77 against these investigated M pro s were further evaluated through molecular dynamic simulation and binding free energy calculation. These data provide molecular insights into broad-spectrum inhibition against coronaviral M pro s by X77 and the similarities and differences of X77 when bound to various M pro s, which will promote X77-based design of novel antivirals with broad-spectrum efficacy against different coronaviruses and SARS-CoV-2 variants., Competing Interests: Declaration of competing interest The authors declare that there is no conflicts of interests., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Orthoflaviviruses, including zika (ZIKV), West Nile (WNV), and dengue (DENV) virus, induce severely debilitating infections and contribute significantly to the global disease burden, yet no clinically approved antiviral treatments exist. This review offers a comprehensive analysis of small-molecule drug development targeting orthoflaviviral infections, with a focus on NS2B-NS3 inhibition. We systematically examined clinical trials, preclinical efficacy studies, and modes of action for various viral replication inhibitors, emphasizing allosteric and orthosteric drugs inhibiting NS2B-NS3 protease with in vivo efficacy and in vitro-tested competitive NS2B-NS3 inhibitors with cellular efficacy. Our findings revealed that several compounds with in vivo preclinical efficacy failed to show clinical antiviral efficacy. NS3-NS4B inhibitors, such as JNJ-64281802 and EYU688 , show promise, recently entering clinical trials, underscoring the importance of developing novel viral replication inhibitors targeting viral machinery. To date, the only NS2B-NS3 inhibitor that has undergone clinical trials is doxycycline , however, its mechanism of action and clinical efficacy as viral growth inhibitor require additional investigation. SYC-1307 , an allosteric inhibitor, exhibits high in vivo efficacy, while temoporfin and methylene blue represent promising orthosteric non-competitive inhibitors. Compound 71 , a competitive NS2B-NS3 inhibitor, emerges as a leading preclinical candidate due to its high cellular antiviral efficacy, minimal cytotoxicity, and favorable in vitro pharmacokinetic parameters. Challenges remain in developing competitive NS2B-NS3 inhibitors, including appropriate biochemical inhibition assays as well as the selectivity and conformational flexibility of the protease, complicating effective antiviral treatment design.

Traditional medicine offers a wide range of application for in silico study techniques. This drug research and development strategy is embryonic in the West African context, particularly in Burkina Faso, which is increasingly faced with emerging diseases such as dengue fever. Circulation of the 4 serotypes of this virus has been documented in the country. This study aims to evaluate the therapeutic potential of phytocompounds contained in the West African pharmacopoeia against dengue virus NS2B/NS3 protein, using computational methods integrating several software packages and databases. Based on a literature review, we identified 191 molecules from 30 plants known for their antiviral effects. Five met the inclusion criteria for molecular docking: patulin from calotropis procera, resiniferonol from Euphorbia poissonii, Securinol A from Flueggea virosa, Shikimic acid and Methyl gallate from Terminalia macroptera. The best binding scores were observed between resiniferonol and the serotypes 1, 2 and 4 NS2B/NS3 protease, with binding energies of -7.4 Kcal/mol, -6.8 Kcal/mol and -7.3 Kcal/mol respectively; while the NS2B/NS3 protease of serotype 3 had the best affinity for securinol A (-7 Kcal/mol). This study points the way to further research in computer aided drug design field and calls for multidisciplinary collaboration to promote West African medicinal plants against health challenges.

The Alphavirus genus includes viruses that cause encephalitis due to neuroinvasion and viruses that cause arthritis due to acute and chronic inflammation. There is no approved therapeutic for alphavirus infections, but significant efforts are ongoing, more so in recent years, to develop vaccines and therapeutics for alphavirus infections. This review article highlights some of the major advances made so far to identify small molecules that can selectively target the structural and the nonstructural proteins in alphaviruses with the expectation that persistent investigation of an increasingly expanding chemical space through a variety of structure-based design and high-throughput screening strategies will yield candidate drugs for clinical studies. While most of the works discussed are still in the early discovery to lead optimization stages, promising avenues remain for drug development against this family of viruses.

Microcephaly, Guillain-Barré syndrome, and potential sexual transmission stand as prominent complications associated with Zika virus (ZIKV) infection. The absence of FDA-approved drugs or vaccines presents a substantial obstacle in combatting the virus. Furthermore, the inclusion of pregnancy in the pharmacological screening process complicates and extends the endeavor to ensure molecular safety and minimal toxicity. Given its pivotal role in viral assembly and maturation, the NS2B-NS3 viral protease emerges as a promising therapeutic target against ZIKV. In this context, a dipeptide inhibitor was specifically chosen as a control against 200 compounds for docking analysis. Subsequent molecular dynamics simulations extending over 200 ns were conducted to ascertain the stability of the docked complex and confirm the binding of the inhibitor at the protein's active site. The simulation outcomes exhibited conformity to acceptable thresholds, encompassing parameters such as root mean square deviation (RMSD), root mean square fluctuation (RMSF), ligand-protein interaction analysis, ligand characterization, and surface area analysis. Notably, analysis of ligand angles bolstered the identification of prospective ligands capable of inhibiting viral protein activity and impeding virus dissemination. In this study, the integration of molecular docking and dynamics simulations has pinpointed the dipeptide inhibitor as a potential candidate ligand against ZIKV protease, thereby offering promise for therapeutic intervention against the virus., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Ullah et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The emergence of drug-resistance-inducing mutations in Hepatitis C virus (HCV) coupled with genotypic heterogeneity has made targeting NS3/4A serine protease difficult. In this work, we investigated the mutagenic variations in the binding pocket of Genotype 3 (G3) HCV NS3/4A and evaluated ligands for efficacious inhibition. We report mutations at 14 positions within the ligand-binding residues of HCV NS3/4A, including H57R and S139P within the catalytic triad. We then modelled each mutational variant for pharmacophore-based virtual screening (PBVS) followed by covalent docking towards identifying a potential covalent inhibitor, i.e., cpd-217. The binding stability of cpd-217 was then supported by molecular dynamic simulation followed by MM/GBSA binding free energy calculation. The free energy decomposition analysis indicated that the resistant mutants alter the HCV NS3/4A-ligand interaction, resulting in unbalanced energy distribution within the binding site, leading to drug resistance. Cpd-217 was identified as interacting with all NS3/4A G3 variants with significant covalent docking scores. In conclusion, cpd-217 emerges as a potential inhibitor of HCV NS3/4A G3 variants that warrants further in vitro and in vivo studies. This study provides a theoretical foundation for drug design and development targeting HCV G3 NS3/4A.

Dengue fever, prevalent in Southeast Asian countries, currently lacks effective pharmaceutical interventions for virus replication control. This study employs a strategy that combines machine learning (ML)-based quantitative-structure-activity relationship (QSAR), molecular docking, and molecular dynamics simulations to discover potential inhibitors of the NS3 protease of the dengue virus. We used nine molecular fingerprints from PaDEL to extract features from the NS3 protease dataset of dengue virus type 2 in the ChEMBL database. Feature selection was achieved through the low variance threshold, F-Score, and recursive feature elimination (RFE) methods. Our investigation employed three ML models - support vector machine (SVM), random forest (RF), and extreme gradient boosting (XGBoost) - for classifier development. Our SVM model, combined with SVM-RFE, had the best accuracy (0.866) and ROC&#95;AUC (0.964) in the testing set. We identified potent inhibitors on the basis of the optimal classifier probabilities and docking binding affinities. SHAP and LIME analyses highlighted the significant molecular fingerprints (e.g. ExtFP69, ExtFP362, ExtFP576) involved in NS3 protease inhibitory activity. Molecular dynamics simulations indicated that amphotericin B exhibited the highest binding energy of -212 kJ/mol and formed a hydrogen bond with the critical residue Ser196. This approach enhances NS3 protease inhibitor identification and expedites the discovery of dengue therapeutics.

Infections with Flaviviridae viruses, such as hepatitis C (HCV), dengue (DENV), and yellow fever (YFV) viruses, are major public health problems worldwide. In the case of HCV, treatment is associated with drug resistance and high costs, while there is no clinically approved therapy for DENV and YFV. Consequently, there is still a need for new chemotherapies with alternative modes of action. We have previously identified novel 2-hydroxypyrazino[1,2- a ]indole-1,3(2 H ,4 H )-diones as metal-chelating inhibitors targeting HCV RNA replication. Here, by utilizing a structure-based approach, we rationally designed a second series of compounds by introducing various substituents at the indole core structure and at the imidic nitrogen, to improve specificity against the RNA-dependent RNA polymerase (RdRp). The resulting derivatives were evaluated for their potency against HCV genotype 1b, DENV2, and YFV-17D using stable replicon cell lines. The most favorable substitution was nitro at position 6 of the indole ring (compound 36 ), conferring EC 50 1.6 μM against HCV 1b and 2.57 μΜ against HCV 1a, with a high selectivity index. Compound 52 , carrying the acetohydroxamic acid functionality (-CH 2 CONHOH) on the imidic nitrogen, and compound 78 , the methyl-substituted molecule at the position 4 indolediketopiperazine counterpart, were the most effective against DENV and YFV, respectively. Interestingly, compound 36 had a high genetic barrier to resistance and only one resistance mutation was detected, T181I in NS5B, suggesting that the compound target HCV RdRp is in accordance with our predicted model.

The quest to identify antiviral drug candidates for dengue and rabies viral diseases is a great challenge for the researchers. While different research is being conducted on the repurposed drugs against these two viruses, no drug compound has gained success in treating them. Therefore, in this study, 3, 4-dihydroxy complexes have been virtually designed to investigate their antiviral properties and analyze their efficiency in interaction with the concerned viral diseases. DFT calculations are carried out to study the electronic and thermodynamic properties to understand the stability and reactivity of the reported compounds. These compounds were subjected to molecular docking studies to understand the binding interactions with NS5 Dengue virus mRNA 2'-O-methyltransferase and phosphoprotein C-terminal domain of Rabies virus. MD simulation, hydrogen bond analysis, and MM/PBSA were performed at 100 ns to support the obtained docking results.Communicated by Ramaswamy H. Sarma.

During the first years of COVID-19 pandemic, X-ray structures of the coronavirus drug targets were acquired at an unprecedented rate, giving hundreds of PDB depositions in less than a year. The main protease (Mpro) of severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) is the primary validated target of direct-acting antivirals. The selection of the optimal ensemble of structures of Mpro for the docking-driven virtual screening campaign was thus non-trivial and required a systematic and automated approach. Here we report a semi-automated active site RMSD based procedure of ensemble selection from the SARS-CoV-2 Mpro crystallographic data and virtual screening of its inhibitors. The procedure was compared with other approaches to ensemble selection and validated with the help of hand-picked and peer-reviewed activity-annotated libraries. Prospective virtual screening of non-covalent Mpro inhibitors resulted in a new chemotype of thienopyrimidinone derivatives with experimentally confirmed enzyme inhibition., (© 2024 Wiley-VCH GmbH.)

The SARS-CoV-2 pandemic has bolstered unprecedented research efforts to better understand the pathogenesis of coronavirus (CoV) infections and develop effective therapeutics. We here focus on non-structural protein nsp15, a hexameric component of the viral replication-transcription complex (RTC). Nsp15 possesses uridine-specific endoribonuclease (EndoU) activity for which some specific cleavage sites were recently identified in viral RNA. By preventing accumulation of viral dsRNA, EndoU helps the virus to evade RNA sensors of the innate immune response. The immune-evading property of nsp15 was firmly established in several CoV animal models and makes it a pertinent target for antiviral therapy. The search for nsp15 inhibitors typically proceeds via compound screenings and is aided by the rapidly evolving insight in the protein structure of nsp15. In this overview, we broadly cover this fascinating protein, starting with its structure, biochemical properties and functions in CoV immune evasion. Next, we summarize the reported studies in which compound screening or a more rational method was used to identify suitable leads for nsp15 inhibitor development. In this way, we hope to raise awareness on the relevance and druggability of this unique CoV protein., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

SARS-COV-2 is responsible for the COVID-19 pandemic, which has infected more than 767 million people worldwide including about 7 million deaths till 5 June 2023. Despite the emergency use of certain vaccines, deaths due to COVID-19 have not yet stopped completed. Therefore, it is imperative to design and develop drugs that can be used to treat patients suffering from COVID-19. Here, two peptide inhibitors derived from nsp7 and nsp8 cofactors of nsp12 have been shown to block different substrate binding sites of nsp12 that are mainly responsible for the replication of the viral genome of SARS-CoV-2. By using the docking, molecular dynamics (MD), and MM/GBSA techniques, it is shown that these inhibitors can bind to multiple binding sites of nsp12, such as the interface of nsp7 and nsp12, interface of nsp8 and nsp12, RNA primer entry site, and nucleoside triphosphate (NTP) entry site. The relative binding free energies of the most stable protein-peptide complexes are found to lie between ∼-34.20 ± 10.07 to -59.54 ± 9.96 kcal/mol. Hence, it is likely that these inhibitors may bind to different sites of nsp12 to block the access of its cofactors and the viral genome, thereby affecting the replication. It is thus proposed that these peptide inhibitors may be further developed as potential drug candidates to suppress the viral loads in COVID-19 patients.Communicated by Ramaswamy H. Sarma.

DENV-2 strain is the most fatal and infectious of the five dengue virus serotypes. The non-structural protein NS1 encoded by its genome is the most significant protein required for viral pathogenesis and replication inside the host body. Thus, targeting the NS1 protein and designing an inhibitor to limit its stability and secretion is a propitious attempt in our fight against dengue. Four novel inhibitors are designed to target the conserved cysteine residues (C55, C313, C316, and C329) and glycosylation sites (N130 and N207) of the NS1 protein in an attempt to halt the spread of the dengue infection in the host body altogether. Numerous computer-aided drug designing techniques including molecular docking, molecular dynamics simulation, virtual screening, principal component analysis, and dynamic cross-correlation matrix were employed to determine the structural and functional activity of the NS1-inhibitor complexes. From our analysis, it was evident that the extent of structural and atomic level fluctuations of the ligand-bound protein exhibited a declining trend in contrast to unbound protein which was prominently noticeable through the RMSD, RMSF, R g , and SASA graphs. The ADMET analysis of the four ligands revealed a promising pharmacokinetics and pharmacodynamic profile, along with good bioavailability and toxicity properties. The proposed drugs when bound to the targeted cavities resulted in stable conformations in comparison to their unbound state, implying they have good affinity promising effective drug action. Thus, they can be tested in vitro and used as potential anti-dengue drugs.Communicated by Ramaswamy H. Sarma.

The global health crisis caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) urges the development of new antiviral agents with broad coronavirus coverage. Due to its key role in viral evasion from the host innate immune response, the coronavirus Nsp15 uridine-specific endoribonuclease (EndoU) is of high interest as a drug target. Considering that the isatin scaffold is well-known for its versatile pharmacological properties, we synthesized and evaluated a series of compounds carrying an isatin core. The initial compounds were selected on the basis of in silico predictions. After biochemical assays showed moderate inhibition of SARS-CoV-2 EndoU-mediated RNA cleavage, structural analogues were rationally designed to enhance the interaction with the target. This included the incorporation of a nitrile group since this dipole can improve ADME and facilitate polar interactions with proteins and can operate as hydroxy or carboxy surrogate. A straightforward solvent free and green, microwave-assisted synthetic process was established to achieve the development of the different target compounds. The best compound exhibited inhibitory activity in enzymatic EndoU assays, and reduced the SARS-CoV-2 viral RNA load by almost 68,000-fold in the low micromolar range similarly to the established antiviral agent GS-441524., Competing Interests: Declaration of competing interest All authors have approved the final submission to the journal. The authors declare no conflict of interest., (Copyright © 2024. Published by Elsevier Masson SAS.)

Hepatitis C virus (HCV) is a global health concern and the NS5B RNA-dependent RNA polymerase (RdRp) of HCV is an attractive target for drug discovery due to its role in viral replication. This study focuses on NS5B thumb site II inhibitors, specifically phenylalanine derivatives, and explores bioisosteric replacement and prodrug strategies to overcome limitations associated with carboxylic acid functionality. The synthesized compounds demonstrated antiviral activity, with compound 6d showing the most potent activity with an EC 50 value of 3.717 μM. The hydroxamidine derivatives 7a-d showed EC 50 values ranging from 3.9 μM to 11.3 μM. However, the acidic heterocyclic derivatives containing the oxadiazolone (8a-d) and oxadiazolethione (9a-d) rings did not exhibit measurable activity. A methylated heterocycle 10b showed a hint of activity at 8.09 μM. The pivaloyloxymethyl derivatives 11a and 11b did not show antiviral activity. Further studies are warranted to fully understand the effects of these modifications and to explore additional strategies for developing novel therapeutic options for HCV., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Masson SAS. All rights reserved.)

The main proteases M pro are a group of highly conserved cysteine hydrolases in β-coronaviruses. They have been demonstrated to play an unavoidable role in viral replication, and consequently they have been suggested as key targets for treating coronavirus-caused infectious diseases, mainly from the COVID-19 epidemic. Since the most functional form for M pro enzymatic activity is associated to its homodimer, compounds inhibiting dimerization should also inhibit catalytic activity. We show how PIR-SEIRA (Plasmonic Internal Reflection-Surface Enhanced InfraRed Absorption) spectroscopy can be a noteworthy technique to study proteins subtle structural variations associated to inhibitor binding. Nanoantennas arrays can selectively confine and enhance electromagnetic field via localized plasmonic resonances, thus promoting ultrasensitive detection of biomolecules in close proximity of nanoantenna arrays and enabling the effective investigation of protein monolayers. By adopting this approach, reflection measurements conducted under back illumination of nanoantennas allow to probe anchored protein monolayers, with minimum contribution of environmental buffer molecules. PIR-SEIRA spectroscopy on M pro was carried out by ad hoc designed devices, resonating in the spectral region of Amide I and Amide II bands. We evaluated here the structure of anchored monomers and dimers in different buffered environment and in presence of a newly designed M pro inhibitor. Experimental results show that dimerization is not associated to relevant backbone rearrangements of the protein at secondary structure level, and even if the compound inhibits the dimerization, it is not effective at breaking preformed dimers., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

Advancing climate change increases the risk of future infectious disease outbreaks, particularly of zoonotic diseases, by affecting the abundance and spread of viral vectors. Concerningly, there are currently no approved drugs for some relevant diseases, such as the arboviral diseases chikungunya, dengue or zika. The development of novel inhibitors takes 10-15 years to reach the market and faces critical challenges in preclinical and clinical trials, with approximately 30% of trials failing due to side effects. As an early response to emerging infectious diseases, CavitOmiX allows for a rapid computational screening of databases containing 3D point-clouds representing binding sites of approved drugs to identify candidates for off-label use. This process, known as drug repurposing, reduces the time and cost of regulatory approval. Here, we present potential approved drug candidates for off-label use, targeting the ADP-ribose binding site of Alphavirus chikungunya non-structural protein 3. Additionally, we demonstrate a novel in silico drug design approach, considering potential side effects at the earliest stages of drug development. We use a genetic algorithm to iteratively refine potential inhibitors for (i) reduced off-target activity and (ii) improved binding to different viral variants or across related viral species, to provide broad-spectrum and safe antivirals for the future.

The benzofuran core inhibitors HCV-796 , BMS-929075 , MK-8876 , compound 2 , and compound 9B exhibit good pan-genotypic activity against various genotypes of NS5B polymerase. To elucidate their mechanism of action, multiple molecular simulation methods were used to investigate the complex systems of these inhibitors binding to GT 1a , 1b , 2a , and 2b NS5B polymerases. The calculation results indicated that these five inhibitors can not only interact with the residues in the palm II subdomain of NS5B polymerase, but also with the residues in the palm I subdomain or the palm I/III overlap region. Interestingly, the binding of inhibitors with longer substituents at the C5 position ( BMS-929075 , MK-8876 , compound 2 , and compound 9B ) to the GT 1a and 2b NS5B polymerases exhibits different binding patterns compared to the binding to the GT 1b and 2a NS5B polymerases. The interactions between the para-fluorophenyl groups at the C2 positions of the inhibitors and the residues at the binding pockets, together with the interactions between the substituents at the C5 positions and the residues at the reverse β-fold (residues 441-456), play a key role in recognition and the induction of the binding. The relevant studies could provide valuable information for further research and development of novel anti-HCV benzofuran core pan-genotypic inhibitors.

Dengue fever represents a significant medical and socio-economic burden in (sub)tropical regions, yet antivirals for treatment or prophylaxis are lacking. JNJ-A07 was described as highly active against the different genotypes within each serotype of the disease-causing dengue virus (DENV). Based on clustering of resistance mutations it has been assumed to target DENV non-structural protein 4B (NS4B). Using a photoaffinity labeling compound with high structural similarity to JNJ-A07, here we demonstrate binding to NS4B and its precursor NS4A-2K-NS4B. Consistently, we report recruitment of the compound to intracellular sites enriched for these proteins. We further specify the mechanism-of-action of JNJ-A07, which has virtually no effect on viral polyprotein cleavage, but targets the interaction between the NS2B/NS3 protease/helicase complex and the NS4A-2K-NS4B cleavage intermediate. This interaction is functionally linked to de novo formation of vesicle packets (VPs), the sites of DENV RNA replication. JNJ-A07 blocks VPs biogenesis with little effect on established ones. A similar mechanism-of-action was found for another NS4B inhibitor, NITD-688. In summary, we unravel the antiviral mechanism of these NS4B-targeting molecules and show how DENV employs a short-lived cleavage intermediate to carry out an early step of the viral life cycle., (© 2024. The Author(s).)

The recent pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) highlighted a critical need to discover more effective antivirals. While therapeutics for SARS-CoV-2 exist, its nonstructural protein 13 (Nsp13) remains a clinically untapped target. Nsp13 is a helicase responsible for unwinding double-stranded RNA during viral replication and is essential for propagation. Like other helicases, Nsp13 has two active sites: a nucleotide binding site that hydrolyzes nucleoside triphosphates (NTPs) and a nucleic acid binding channel that unwinds double-stranded RNA or DNA. Targeting viral helicases with small molecules, as well as the identification of ligand binding pockets, have been ongoing challenges, partly due to the flexible nature of these proteins. Here, we use a virtual screen to identify ligands of Nsp13 from a collection of clinically used drugs. We find that a known ion channel inhibitor, IOWH-032, inhibits the dual ATPase and helicase activities of SARS-CoV-2 Nsp13 at low micromolar concentrations. Kinetic and binding assays, along with computational and mutational analyses, indicate that IOWH-032 interacts with the RNA binding interface, leading to displacement of nucleic acid substrate, but not bound ATP. Evaluation of IOWH-032 with microbial helicases from other superfamilies reveals that it is selective for coronavirus Nsp13. Furthermore, it remains active against mutants representative of observed SARS-CoV-2 variants. Overall, this work provides a new inhibitor for Nsp13 and provides a rationale for a recent observation that IOWH-032 lowers SARS-CoV-2 viral loads in human cells, setting the stage for the discovery of other potent viral helicase modulators.

Resistance-associated substitutions (RASs) of hepatitis C virus (HCV) affect the efficacy of direct-acting antivirals (DAAs). In this study, we aimed to clarify the susceptibility of the coexistence of nonstructural (NS) 5A Q24K/L28M/R30Q (or R30E)/A92K RASs, which were observed in patients with DAAs re-treatment failure and to consider new therapeutic agents. We used a subgenomic replicon system in which HCV genotype 1B strain 1B-4 was electroporated into OR6c cells derived from HuH-7 cells (Wild-type [WT]). We converted WT genes to NS5A Q24K/L28M/R30Q/A92K or Q24/L28K/R30E/A92K. Compared with the WT, the Q24K/L28M/R30Q/A92K RASs was 36,000-fold resistant to daclatasvir, 440,000-fold resistant to ledipasvir, 6300-fold resistant to velpatasvir, 3100-fold resistant to elbasvir, and 1.8-fold resistant to pibrentasvir. Compared with the WT, the Q24K/L28M/R30E/A92K RASs was 640,000-fold resistant to daclatasvir and ledipasvir, 150,000-fold resistant to velpatasvir, 44,000-fold resistant to elbasvir, and 1500-fold resistant to pibrentasvir. The Q24K/L28M/R30E/A92K RASs was 816.3 times more resistant to pibrentasvir than the Q24K/L28M/R30Q/A92K RASs. Furthermore, a combination of pibrentasvir and sofosbuvir showed therapeutic efficacy against these RASs. Combination regimens may eradicate HCV with NS5A Q24K/L28M/R30E/A92K RASs., (© 2024. The Author(s).)

The RNA viruses SARS-CoV, SARS-CoV-2 and MERS-CoV encode the non-structural Nsp16 (2'-O-methyltransferase) that catalyzes the transfer of a methyl group from S-adenosylmethionine (SAM) to the first ribonucleotide in mRNA. Recently, it has been found that breaking the bond between Nsp16 and SAM substrate results in the cessation of mRNA virus replication. To date, only a limited number of such inhibitors have been identified, which can be attributed to a lack of an effective "recipe". The aim of our study was to propose and verify a rapid and effective screening protocol dedicated to such purposes. We proposed four new indices describing structure-binding strength (structure-binding affinity, structure-hydrogen bonding, structure-steric and structure-protein-ligand indices) were then applied and shown to be extremely helpful in determining the degree of increase or decrease in binding affinity in response to a relatively small change in the ligand structure. After initial pre-selection, based on similarity to SAM, we limited the study to 967 compounds, so-called molecular chameleons. They were then docked in the Nsp16 protein pocket, and 10 candidate ligands were selected using the novel structure-binding affinity index. Subsequently the selected 10 candidate ligands and 8 known inhibitors and were docked to Nsp16 pockets from SARS-CoV-2, MERS-CoV and SARS-CoV. Based on the four new indices, the best ligands were selected and a new one was designed by tuning them. Finally, ADMET profiling and molecular dynamics simulations were performed for the best ligands. The new structure-binding strength indices can be successfully applied not only to screen and tune ligands, but also to determine the effectiveness of the ligand in response to changes in the target viral entity, which is particularly useful for assessing drug effectiveness in the case of alterations in viral proteins. The developed approach, the so-called chameleon strategy, has the capacity to introduce a novel universal paradigm to the field of drugs design, including RNA antivirals.

Dengue virus is a mosquito-borne pathogen that causes a variety of illnesses ranging from mild fever to severe and fatal dengue haemorrhagic fever or dengue shock syndrome. One of the major clinical manifestations of severe dengue infection is thrombocytopenia. The dengue non-structural protein 1 (NS1) is the primary protein that stimulates immune cells via toll-like receptor 4 (TLR4), induces platelets, and promotes aggregation, which could result in thrombocytopenia. The leaf extracts of Carica papaya seem to have therapeutic benefits in managing thrombocytopenia associated with dengue. The present study focuses on understanding the underlying mechanism of the use of papaya leaf extracts in treating thrombocytopenia. We have identified 124 phytocompounds that are present in the papaya leaf extract. The pharmacokinetics, molecular docking, binding free energy calculations, and molecular dynamic simulations were performed to investigate the drug-like properties, binding affinities, and interaction of phytocompounds with NS1 protein as well as the interactions of NS1 with TLR4. Three phytocompounds were found to bind with the ASN130, a crucial amino acid residue in the active site of the NS1 protein. Thus, we conclude that Rutin, Myricetin 3-rhamnoside, or Kaempferol 3-(2''-rhamnosylrutinoside) may serve as promising molecules by ameliorating thrombocytopenia in dengue-infected patients by interfering the interaction of NS1 with TLR4. These molecules can serve as drugs in the management of dengue-associated thrombocytopenia after verifying their effectiveness and assessing the drug potency, through additional in-vitro assays.Communicated by Ramaswamy H. Sarma.

Zika virus (ZIKV) continues to pose a significant global public health threat, with recurring regional outbreaks and potential for pandemic spread. Despite often being asymptomatic, ZIKV infections can have severe consequences, including neurological disorders and congenital abnormalities. Unfortunately, there are currently no approved vaccines or antiviral drugs for the prevention or treatment of ZIKV. One promising target for drug development is the ZIKV NS2B-NS3 protease due to its crucial role in the virus life cycle. In this study, we established a cell-based ZIKV protease inhibition assay designed for high-throughput screening (HTS). Our assay relies on the ZIKV protease's ability to cleave a cyclised firefly luciferase fused to a natural cleavage sequence between NS2B and NS3 protease within living cells. We evaluated the performance of our assay in HTS setting using the pharmacologic controls (JNJ-40418677 and MK-591) and by screening a Library of Pharmacologically Active Compounds (LOPAC). The results confirmed the feasibility of our assay for compound library screening to identify potential ZIKV protease inhibitors., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Inc.)

Background: The Zika virus (ZIKV) infection has emerged as a global health threat. The causal reasoning is that Zika infection is linked to the development of microcephaly in newborns and Guillain-Barré syndrome in adults. With no clinically approved antiviral treatment for ZIKV, the need for the development of potential inhibitors against the virus is essential. In this study, we aimed to screen phytochemicals from papaya ( Carica papaya L. ) against NS5 protein domains of ZIKV., Methods: Approximately 193 phytochemicals from an online database (IMPACT) were subjected to molecular docking using AutoDock Vina against the NS5-MTase protein domain (5WXB) and -RdRp domain (5U04)., Results: Our results showed that β-sitosterol, carpaine, violaxanthin, pseudocarpaine, Δ7-avenasterols, Rutin, and cis-β-carotene had the highest binding affinity to both protein domains, with β-sitosterol having the most favorable binding energy. Furthermore, ADMET analysis revealed that selected compounds had good pharmacokinetic properties and were nontoxic., Conclusions: Our findings suggest that papaya-derived phytochemicals could be potential candidates for developing antiviral drugs against ZIKV. However, further experimental studies using cell lines and in vivo models are needed to validate their efficacy and safety., Competing Interests: No competing interests were disclosed., (Copyright: © 2024 Kumaree KK et al.)

Carnivorous pitcher plants from the genus Nepenthes are renowned for their ethnobotanical uses. This research explores the therapeutic potential of Nepenthes miranda leaf extract against nonstructural protein 9 (Nsp9) of SARS-CoV-2 and in treating human non-small cell lung carcinoma (NSCLC) cell lines. Nsp9, essential for SARS-CoV-2 RNA replication, was expressed and purified, and its interaction with ssDNA was assessed. Initial tests with myricetin and oridonin, known for targeting ssDNA-binding proteins and Nsp9, respectively, did not inhibit the ssDNA-binding activity of Nsp9. Subsequent screenings of various N. miranda extracts identified those using acetone, methanol, and ethanol as particularly effective in disrupting Nsp9's ssDNA-binding activity, as evidenced by electrophoretic mobility shift assays. Molecular docking studies highlighted stigmast-5-en-3-ol and lupenone, major components in the leaf extract of N. miranda , as potential inhibitors. The cytotoxic properties of N. miranda leaf extract were examined across NSCLC lines H1975, A549, and H838, focusing on cell survival, apoptosis, and migration. Results showed a dose-dependent cytotoxic effect in the following order: H1975 > A549 > H838 cells, indicating specificity. Enhanced anticancer effects were observed when the extract was combined with afatinib, suggesting synergistic interactions. Flow cytometry indicated that N. miranda leaf extract could induce G2 cell cycle arrest in H1975 cells, potentially inhibiting cancer cell proliferation. Gas chromatography-mass spectrometry (GC-MS) enabled the tentative identification of the 19 most abundant compounds in the leaf extract of N. miranda . These outcomes underscore the dual utility of N. miranda leaf extract in potentially managing SARS-CoV-2 infection through Nsp9 inhibition and offering anticancer benefits against lung carcinoma. These results significantly broaden the potential medical applications of N. miranda leaf extract, suggesting its use not only in traditional remedies but also as a prospective treatment for pulmonary diseases. Overall, our findings position the leaf extract of N. miranda as a promising source of natural compounds for anticancer therapeutics and antiviral therapies, warranting further investigation into its molecular mechanisms and potential clinical applications.

Nsp1 in SARS-CoV-2 is a key protein that increases the virus's pathogenicity and virulence by binding to the host ribosome and blocks the 40S ribosomal subunit channel, which effectively impedes the mRNA translation as well as crippling the host immune system. Previous studies revealed that the N-terminal in Nsp1 is part and parcel of Nsp1 efficiency, and mutations in its core residues have weakened the protein's. This knowledge persuades us to carry out the in silico screening on plant compounds of Piper sarmentosum Roxb. against the five target residues which are Glu36, Glu37, Arg99, Arg124 and Lys125. Potential compounds were tested for their druggability. As a result, we identified five out of 112 compounds including stigmasterol, N-feruloyltyramine, beta-Sitosterol, 13-(1,3-benzodioxol-5-yl)- N-(2methylpropyl) trideca-2,4,12-trienamide and N-(2-methylpropyl) octadeca-2-4dienamide in Piper sarmentosum Roxb. as potential inhibitors for Nsp1. These compounds formed at least a hydrophobic, hydrogen bonding or π-cation interactions with the protein. Furthermore, SwissADME analysis and the number of bindings to the target residues suggest that N-feruloyltyramine is the ideal inhibitor candidate against SARS-CoV-2 at its N-terminal of Nsp1. Lastly, the interaction with N-feruloyltyramine increased flexibility in the loop regions of N-terminal Nsp1, especially residues 54 to 70, with residue 59 showing the highest fluctuation, potentially affecting the protein's stability and function due to the correlation between RMSF and protein function.

Global warming and climate change have made dengue disease a global health issue. More than 50 % of the world's population is at danger of dengue virus (DENV) infection, according to the World Health Organization (WHO). Therefore, a clinically approved dengue fever vaccination and effective treatment are needed. Peptide medication development is new pharmaceutical research. Here we intend to recognize the structural features inhibiting the DENV NS2B/NS3 serine protease for a series of peptide-hybrid inhibitors (R 1 -R 2 -Lys-R 3 -NH 2 ) by the 3D-QSAR technique. Comparative molecular field analysis (q 2  = 0.613, r 2  = 0.938, r 2 pred  = 0.820) and comparative molecular similarity indices analysis (q 2  = 0.640, r 2  = 0.928, r 2 pred  = 0.693) were established, revealing minor, electropositive, H-bond acceptor groups at the R 1 position, minor, electropositive, H-bond donor groups at the R 2 position, and bulky, hydrophobic groups at the R 3 position for higher inhibitory activity. Docking studies revealed extensive H-bond and hydrophobic interactions in the binding of tripeptide analogues to the NS2B/NS3 protease. This study provides an insight into the key structural features for the design of peptide-based inhibitors of DENV NS2B/NS3 protease., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Approximately 3.9 billion individuals are vulnerable to dengue infection, a prevalent cause of tropical diseases worldwide. Currently, no drugs are available for preventing or treating Flavivirus diseases, including Dengue, West Nile, and the more recent Zika virus. The highly conserved Flavivirus NS2B-NS3 protease, crucial for viral replication, is a promising therapeutic target. This study employed in-silico methodologies to identify novel and potentially effective anti-dengue small molecules. A pharmacophore model was constructed using an experimentally validated NS2B-NS3 inhibitor, with the Gunner Henry score confirming the model's validity. The Natural Product Activity and Species Source (NPASS) database was screened using the validated pharmacophore model, yielding a total of 60 hits against the NS2B-NS3 protease. Furthermore, the docking finding reveals that our newly identified compounds from the NPASS database have enhanced binding affinities and established significant interactions with allosteric residues of the target protein. MD simulation and post-MD analysis further validated this finding. The free binding energy was computed in terms of MM-GBSA analysis, with the total binding energy for compound 1 (-57.3 ± 2.8 and - 52.9 ± 1.9 replica 1 and 2) indicating a stronger binding affinity for the target protein. Overall, this computational study identified these compounds as potential hit molecules, and these findings can open up a new avenue to explore and develop inhibitors against Dengue virus infection., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

Discovering an alternative therapy with a long-lasting effect on symptoms caused by chikungunya virus (CHIKV) infection is prompted by the lack of a vaccine and the absence of safe, effective and non-toxic medications. One potential strategy is synthesizing or identifying small compounds that can specifically target the active site of an essential enzyme and prevent virus replication. Previous site-directed mutagenesis studies have demonstrated the crucial role of the macrodomain, which is a part of non-structural protein 3 (nsP3), in virus replication. Exploiting this fact, the macrodomain can be targeted to discover a natural substance that can inhibit its function and thereby impede virus replication. With this aim, the present study focused on potential CHIKV nsP3 macrodomain (nsP3 MD ) inhibitors through in silico, in vitro and cell-based methods. Through virtual screening of the natural compound library, nine nsP3 MD inhibitors were initially identified. Molecular dynamics (MD) simulations were employed to evaluate these nine compounds based on the stability of their ligand-receptor complexes and energy parameters. Target analysis and ADMET (i.e. absorption, distribution, metabolism, excretion and toxicity) prediction of the selected compounds revealed their drug-like characteristics. Subsequent in vitro investigation allowed us to narrow the selection down to one compound, N-[2-(5-methoxy-1H-indol-3-yl) ethyl]-2-oxo-1,2-dihydroquinoline-4-carboxamide, which exhibited potent inhibition of CHIKV growth. This molecule effectively inhibited CHIKV replication in the stable embryonal rhabdomyosarcoma cell line capable of producing CHIKV. Our findings demonstrate that the selected compound possesses substantial anti-CHIKV nsP3 MD activity both in vitro and in vivo. This work provides a promising molecule for further preclinical studies to develop a potential drug against the CHIKV., (© 2024 Federation of European Biochemical Societies.)

We have assembled a computational pipeline based on virtual screening, docking techniques, and nonequilibrium molecular dynamics simulations, with the goal of identifying possible inhibitors of the SARS-CoV-2 NSP13 helicase, catalyzing by ATP hydrolysis the unwinding of double or single-stranded RNA in the viral replication process inside the host cell. The druggable sites for broad-spectrum inhibitors are represented by the RNA binding sites at the 5' entrance and 3' exit of the central channel, a structural motif that is highly conserved across coronaviruses. Potential binders were first generated using structure-based ligand techniques. Their potency was estimated by using four popular docking scoring functions. Common docking hits for NSP13 were finally tested using advanced nonequilibrium alchemical techniques for binding free energy calculations on a high-performing parallel cluster. Four potential NSP13 inhibitors with potency from submicrimolar to nanomolar were finally identified., (© 2024 Wiley-VCH GmbH.)

The ongoing COVID-19 pandemic still threatens human health around the world. The methyltransferases (MTases) of SARS-CoV-2, specifically nsp14 and nsp16, play crucial roles in the methylation of the N7 and 2'-O positions of viral RNA, making them promising targets for the development of antiviral drugs. In this work, we performed structure-based virtual screening for nsp14 and nsp16 using the screening workflow (HTVS, SP, XP) of Schrödinger 2019 software, and we carried out biochemical assays and molecular dynamics simulation for the identification of potential MTase inhibitors. For nsp14, we screened 239,000 molecules, leading to the identification of three hits A1-A3 showing N7-MTase inhibition rates greater than 60% under a concentration of 50 µM. For the SAM binding and nsp10-16 interface sites of nsp16, the screening of 210,000 and 237,000 molecules, respectively, from ZINC15 led to the discovery of three hit compounds B1-B3 exhibiting more than 45% of 2'-O-MTase inhibition under 50 µM. These six compounds with moderate MTase inhibitory activities could be used as novel candidates for the further development of anti-SARS-CoV-2 drugs.

Nsp13, a non-structural protein belonging to the coronavirus family 1B (SF1B) helicase, exhibits 5'-3' polarity-dependent DNA or RNA unwinding using NTPs. Crucially, it serves as a key component of the viral replication-transcription complex (RTC), playing an indispensable role in the coronavirus life cycle and thereby making it a promising target for broad-spectrum antiviral therapies. The imidazole scaffold, known for its antiviral potential, has been proposed as a potential scaffold. In this study, a fluorescence-based assay was designed by labeling dsDNA substrates with a commercial fluorophore and monitoring signal changes upon Nsp13 helicase activity. Optimization and high-throughput screening validated the feasibility of this approach. In accordance with the structural characteristics of ADP, we employed a structural-based design strategy to synthesize three classes of imidazole-based compounds through substitution reaction. Through in vitro activity research, pharmacokinetic parameter analysis, and molecular docking simulation, we identified compounds A16 (IC 50 = 1.25 μM) and B3 (IC 50 = 0.98 μM) as potential lead antiviral compounds for further targeted drug research.

The recent COVID-19 pandemic underscored the limitations of currently available direct-acting antiviral treatments against acute respiratory RNA-viral infections and stimulated major research initiatives targeting anticoronavirus agents. Two novel nsp5 protease (MPro) inhibitors have been approved, nirmatrelvir and ensitrelvir, along with two existing nucleos(t)ide analogues repurposed as nsp12 polymerase inhibitors, remdesivir and molnupiravir, but a need still exists for therapies with improved potency and systemic exposure with oral dosing, better metabolic stability, and reduced resistance and toxicity risks. Herein, we summarize our research toward identifying nsp12 inhibitors that led to nucleoside analogues 10e and 10n , which showed favorable pan-coronavirus activity in cell-infection screens, were metabolized to active triphosphate nucleotides in cell-incubation studies, and demonstrated target (nsp12) engagement in biochemical assays.

Paritaprevir is an orally bioavailable, macrocyclic drug used for treating chronic Hepatitis C virus (HCV) infection. Its structures have been elusive to the public until recently when one of the crystal forms is solved by microcrystal electron diffraction (MicroED). In this work, the MicroED structures of two distinct polymorphic crystal forms of paritaprevir are reported from the same experiment. The different polymorphs show conformational changes in the macrocyclic core, as well as the cyclopropyl sulfonamide and methyl pyrazinamide substituents. Molecular docking shows that one of the conformations fits well into the active site pocket of the HCV non-structural 3/4A (NS3/4A) serine protease target, and can interact with the pocket and catalytic triad via hydrophobic interactions and hydrogen bonds. These results can provide further insight for optimization of the binding of acyl sulfonamide inhibitors to the HCV NS3/4A serine protease. In addition, this also demonstrates the opportunity to derive different polymorphs and distinct macrocycle conformations from the same experiments using MicroED., (© 2024 The Authors. Advanced Biology published by Wiley‐VCH GmbH.)

The influenza A virus nonstructural protein 1 (NS1), which is crucial for viral replication and immune evasion, has been identified as a significant drug target with substantial potential to contribute to the fight against influenza. The emergence of drug-resistant influenza A virus strains highlights the urgent need for novel therapeutics. This study proposes a combined theoretical criterion for the virtual screening of molecular libraries to identify candidate NS1 inhibitors. By applying the criterion to the ZINC Natural Product database, followed by ligand-based virtual screening and molecular docking, we proposed the most promising candidate as a potential NS1 inhibitor. Subsequently, the selected natural compound was experimentally evaluated, revealing measurable virus replication inhibition activity in cell culture. This approach offers a promising avenue for developing novel anti-influenza agents targeting the NS1 protein.

The Covid-19 pandemic outbreak has accelerated tremendous efforts to discover a therapeutic strategy that targets severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) to control viral infection. Various viral proteins have been identified as potential drug targets, however, to date, no specific therapeutic cure is available against the SARS-CoV-2. To address this issue, the present work reports a systematic cheminformatic approach to identify the potent andrographolide derivatives that can target methyltransferases of SARS-CoV-2, i.e. nsp14 and nsp16 which are crucial for the replication of the virus and host immune evasion. A consensus of cheminformatics methodologies including virtual screening, molecular docking, ADMET profiling, molecular dynamics simulations, free-energy landscape analysis, molecular mechanics generalized born surface area (MM-GBSA), and density functional theory (DFT) was utilized. Our study reveals two new andrographolide derivatives (PubChem CID: 2734589 and 138968421) as natural bioactive molecules that can form stable complexes with both proteins via hydrophobic interactions, hydrogen bonds and electrostatic interactions. The toxicity analysis predicts class four toxicity for both compounds with LD 50 value in the range of 500-700 mg/kg. MD simulation reveals the stable formation of the complex for both the compounds and their average trajectory values were found to be lower than the control inhibitor and protein alone. MMGBSA analysis corroborates the MD simulation result and showed the lowest energy for the compounds 2734589 and 138968421. The DFT and MEP analysis also predicts the better reactivity and stability of both the hit compounds. Overall, both andrographolide derivatives exhibit good potential as potent inhibitors for both nsp14 and nsp16 proteins, however, in-vitro and in vivo assessment would be required to prove their efficacy and safety in clinical settings. Moreover, the drug discovery strategy aiming at the dual target approach might serve as a useful model for inventing novel drug molecules for various other diseases., (© 2024. The Author(s).)

Hepatitis B and C viruses (HBV and HCV) are significant causes of chronic liver diseases, with approximately 350 million infections globally. To accelerate the finding of effective treatment options, we introduce HBCVTr, a novel ligand-based drug design (LBDD) method for predicting the inhibitory activity of small molecules against HBV and HCV. HBCVTr employs a hybrid model consisting of double encoders of transformers and a deep neural network to learn the relationship between small molecules' simplified molecular-input line-entry system (SMILES) and their antiviral activity against HBV or HCV. The prediction accuracy of HBCVTr has surpassed baseline machine learning models and existing methods, with R-squared values of 0.641 and 0.721 for the HBV and HCV test sets, respectively. The trained models were successfully applied to virtual screening against 10 million compounds within 240 h, leading to the discovery of the top novel inhibitor candidates, including IJN04 for HBV and IJN12 and IJN19 for HCV. Molecular docking and dynamics simulations identified IJN04, IJN12, and IJN19 target proteins as the HBV core antigen, HCV NS5B RNA-dependent RNA polymerase, and HCV NS3/4A serine protease, respectively. Overall, HBCVTr offers a new and rapid drug discovery and development screening method targeting HBV and HCV., (© 2024. The Author(s).)

In the global pandemic scenario, dengue and zika viruses (DENV and ZIKV, respectively), both mosquito-borne members of the flaviviridae family, represent a serious health problem, and considering the absence of specific antiviral drugs and available vaccines, there is a dire need to identify new targets to treat these types of viral infections. Within this drug discovery process, the protease NS2B/NS3 is considered the primary target for the development of novel anti-flavivirus drugs. The NS2B/NS3 is a serine protease that has a dual function both in the viral replication process and in the elusion of the innate immunity. To date, two main classes of NS2B/NS3 of DENV and ZIKV protease inhibitors have been discovered: those that bind to the orthosteric site and those that act at the allosteric site. Therefore, this perspective article aims to discuss the main features of the use of the most potent NS2B/NS3 inhibitors and their impact at the social level.