Binding Pattern Analysis of Different Allosteric Inhibitors of Hepatitis C Virus (HCV) Polymerase

Hepatitis C Virus (HCV) is a positive stranded RNA virus, grouped into the family of Flaviviridae. The HCV genome encodes a single polyprotein, which is co- and posttranslationally cleaved into ten structural and non-structural (NS) proteins by cellular and viral proteases. The coding sequence is flanked by 5’ and 3’ untranslated regions (UTRs), which contain essential cis-acting elements, regulating translation and RNA synthesis, e.g. an internal ribosome entry site (IRES) for cap-independent translation. HCV RNA replication involves the synthesis of a negative strand replication intermediate, serving as a template for the generation of multiple strands of genomic RNA. This process requires a concerted action of several viral nonstructural proteins, cis-acting replication elements and host factors, and is poorly understood at the molecular level. The first part of this study aimed to characterize the viral non-structural proteins comprising the replicase complex in vitro and their mode of action during (-)-strand RNA synthesis. Since the natural 3’(+)-end is a poor template for the viral polymerase NS5B, supporting roles of the viral protease/helicase NS3 and the phosphoprotein NS5A were hypothesized. Optimal conditions for NS3 activity were established by an in vitro helicase assay. By combining the individual proteins with different RNA templates, it was observed that initiation and processivity of NS5B were stimulated by active NS3, but not by inactive mutants. Inhibition of NS3 helicase activity did not impair the stimulatory effect on NS5B, but led to an altered mode of initiation. Addition of purified NS5A further augmented the effect of NS3. In conclusion, this work demonstrates that NS3 and NS5A can improve RNA dependent RNA polymerase activity on a natural template, thereby providing an experimental model to study the molecular mechanisms governing initiation of RNA synthesis. Liver -specific microRNA (miR)-122 is an important host factor of HCV replication, and recognizes two conserved target sites within the first 45nt of the HCV 5’ UTR, close to the IRES. Previous studies suggested a role of miR-122 in RNA stability, translation, and RNA synthesis. The mechanisms, by which miR-122 exerts these functions, remain enigmatic. Insertion of a heterologous IRES element, allowing for miRindependent translation of the non-structural proteins, was sufficient to enable replication in miR122deficient Hep3B cells, suggesting a substantial role of miR-122 in IRES-dependent translation. The miR122 binding region is engaged in a strong secondary in the complementary negative strand. Additionally, we found that a similar structure was predicted inthe positive strand, which would interfere with IRES formation. We therefore hypothesized that miR-122 binding in this region might prevent such alternative structures, thereby facilitating IRES-mediated translation. Indeed, mutations in the miR-122 binding region, but not the IRES sequence, which were designed to stabilize or destabilize the IRES, enhanced or decreased initial translation, respectively, independent of miR-122. Translation enhancement was independent from RNA stability, but short-lived, suggesting additional roles of miR-122, e.g. recruitment of host proteins facilitating steady state translation. Moreover, structural analysis suggested that the HCV IRES folds into a number of conformers in solution, which can be modified by miR-122 under certain conditions. Apart from the 5’ UTR, HCV also contains several seed-matches for miR-122 in the coding sequence of NS5B, and the 3’ UTR, with unknown functional significance. Two novel sites were identified to be conserved over a number of genotypes. The functional characterization of these miR-122 binding sites was evaluated by insertion of point mutations, abrogating miR-122 binding to single and multiple sites, revealing a previously unappreciated role in virion assembly or release. However, assembly of the mutants could not be rescued by a corresponding mutant miR, suggesting a specific need for wild type miR-122. Conclusively, this study provides evidence for miR-122 involvement in almost every intracellular stage of HCV infection, and defines translation enhancement by suppression of RNA structures interfering with IRES activity as a key function of miR-122.

Application of molecular docking in sorption of hydrophobic organic contaminants on black carbon.

Chapter 16 - Docking strategies

Background: The application of molecular docking and Machine Learning (ML) calculations in evaluating peptide-based inhibitors allows for the systematic investigation of sequenceactivity relationships, guiding the design of potent peptides with optimal binding characteristics. Objective: This study aimed to screen short peptides using computational simulation to identify promising inhibitors against SARS-CoV-2 Mpro. Methods: Short peptides were screened using molecular docking to identify promising candidates. The ML model was applied to confirm the docking outcome. The PreADME server was then used to analyze the HIA and toxicity of the peptides. Results: 168,420 short peptides were docked to identify 5 tetrapeptides with promising docking scores against SARS-CoV-2 Mpro including, PYPW, WWPF, WWPY, HYPW, and WYPF. The obtained results were also confirmed via ML calculations. The analyses highlighted the importance of residues Thr190 and Asn142 that are crucial in the binding process. All of top-lead peptides adopt low toxicity and can be absorbed via the human intestine. They can also cross the blood brain barier. Conclusion: This work enhances our understanding of Mpro interactions and informs future ligand design, contributing to the development of therapeutic strategies against COVID-19.

Hepatitis C virus (HCV) has a positive-strand RNA genome and is grouped into the family of Flaviviridae. Similar to other positive-stranded RNA viruses, HCV RNA replication takes place in the cytoplasm. The sites of viral replication are designated “membranous web” and represented by an accumulation of vesicular structures, which are induced by the viral non-structural proteins and probably originate from membranes of the Endoplasmic Reticulum. The aim of this work was to purify and characterize these viral replication complexes (RCs) in vitro and to identify potential host factors of viral replication. First a purification strategy for enzymatically active viral replication complexes was developed to determine associated cellular proteins by proteomics. Thereby, several potential host factors of viral replication were identified and the most reproducible, Annexin II (ANXA2) was further characterized. In immunofluorescence analyses, ANXA2 strongly colocalized to the sites of viral replication in all applicable cell lines supporting HCV replication, in HCV-transfected as well as in infected cells. In contrast, we found no obvious colocalization of HCV proteins with Annexin I, IV or V or with p11 (also denoted S100A10), a common cellular ligand of Annexin II. Specificity of the ANXA2-HCV interaction was further indicated by the lack of colocalization with replication sites of other positive-strand RNA viruses, namely Dengue virus and Semliki-Forest-Virus. By individual expression of the viral non-structural (NS) proteins we found that NS5A colocalized with Annexin II, indicating that NS5A might be involved in the recruitment of ANXA2. SiRNA-mediated silencing clearly reduced Annexin II levels but failed to block HCV replication. However, FACS analyses revealed a strong correlation of intracellular HCV and ANXA2 levels even in presence of ANXA2 siRNA, suggesting that Annexin II expression was induced by HCV, thereby counteracting siRNA-mediated knockdown. Still, ANXA2 silencing moderately reduced the number of HCV positive cells. Interestingly, the presence of replicating HCV sequences in HepG2 cells, harboring very little endogenous ANXA2, clearly induced Annexin II expression to detectable levels perfectly colocalizing with the viral NS proteins. However, the role and function of ANXA2 in the HCV life cycle has yet to be defined. In a second line of investigations, a detailed stoichiometric analysis of HCV RCs was performed. Thus, the ratio of non-structural proteins to RNA that is required for HCV RNA replication could be determined. Almost the entire negative- and positive-strand RNA but <5% of the non-structural proteins present in HCV-harboring cells were protected against nuclease and protease treatments. Nevertheless, this protease-resistant portion of NS proteins accounted for the full in vitro replicase activity. Therefore, only a minor fraction of the HCV non-structural proteins was actively involved in RNA synthesis. However, due to the high amounts present in replicon cells, this still represented a huge excess compared to the viral RNA. Based on the comparison of nuclease-resistant viral RNA to protease-resistant viral proteins, an active HCV replication complex probably consists of one negative-strand RNA, two to ten positive-strand RNAs, and several hundred non-structural protein copies. These might be required as structural components of the vesicular compartments that are the site of HCV replication.

Concern has been expressed worldwide about the rising prevalence of HCV induced acute hepatitis and chronic liver diseases with associated cirrhosis and liver cancers. However, the available synthetic drugs are ineffective for all the HCV genotypes especially in genotype-1 patients with about 40% viral response rates and numerous side effects. Besides, the availability of veritable bioinformatics tools which includes molecular docking and virtual screening studies have shown that computational generated models nowadays assists in modern drug design and development of novel and more potent inhibitors through the understanding of protein (receptors) -ligand (drugs) interaction mechanisms. Non-structural proteins especially the 5B (NS5B) is an RNA-dependent RNA polymerase implicated in the synthesis and replication of the HCV RNA; and has been a potential target for its inhibitory activities. Due to the paucity of knowledge, we aimed to determine the differential inhibitory activity of essential oils present in the crude Sesame leaves extracts on HCV-NS5B RNA dependent RNA polymerase. Using in-silico studies- a Microsoft pharmacophore-based virtual screening and molecular docking tools on the iGEMDOCK vs 2.0 software was used to dock the essential oil ligands on the generated HCV NS5B (PDB ID: 4EO6) RNA-dependent RNA polymerase protein. GC-MS of the leaves confirmed carboxylic acids and phenolic groups in the essential oils especially some potent antioxidants like alpha-linolenic acid, linoleic acid, oleic acid, etc. Moreover, Alpha-Linolenic acid/ALA (-102.2/-103.4 kcal/mol) and Linoleic acid/LA (-94.8/-109.8 kcal/mol) showed higher inhibitory impacts among the six top different docked ligands, selected based on their high differential binding affinity and pharmacological interaction energy profiles against HCV NS5B RNA polymerase activities, by forming more H-bond interactions than the NS5B co-crystallized ligand. ADMET showed that ALA is well tolerated without any apparent toxicity in the body. Hence, ALA having the highest inhibitory impacts against the HCV NS5B goes to confirm the beneficial impacts of carboxylic acids from Sesame plant in maintaining liver cellular integrity and as a natural inhibitor of HCV NS5B RNA dependent RNA Polymerase enzyme activities.

ChemistrySelectVolume 4, Issue 23 p. 6887-6887 CorrigendumFree Access Corrigendum: Regioselective Synthesis, Antibacterial, Molecular Docking and Fingerprint Applications of 1-Benzhydrylpiperazine Derivatized 1,4-Disubstituted 1,2,3-Triazoles This article corrects the following: Regioselective Synthesis, Antibacterial, Molecular Docking and Fingerprint Applications of 1-Benzhydrylpiperazine Derivatized 1,4-Disubstituted 1,2,3-Triazoles Shivaraja Govindaiah Swamy Sreenivasa Ramesha Andagar Ramakrishna Tadimety Madhu Chakrapani Rao Hanumanthappa Nagabhushana Volume 3Issue 28ChemistrySelect pages: 8111-8117 First Published online: July 25, 2018 Shivaraja Govindaiah, Search for more papers by this authorDr. Swamy Sreenivasa, Search for more papers by this authorDr. Ramesha Andagar Ramakrishna, Search for more papers by this authorTadimety Madhu Chakrapani Rao, Search for more papers by this authorProf. Hanumanthappa Nagabhushana, Search for more papers by this author Shivaraja Govindaiah, Search for more papers by this authorDr. Swamy Sreenivasa, Search for more papers by this authorDr. Ramesha Andagar Ramakrishna, Search for more papers by this authorTadimety Madhu Chakrapani Rao, Search for more papers by this authorProf. Hanumanthappa Nagabhushana, Search for more papers by this author First published: 18 June 2019 https://doi.org/10.1002/slct.201902075AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onEmailFacebookTwitterLinked InRedditWechat No abstract is available for this article. Volume4, Issue23June 21, 2019Pages 6887-6887 RelatedInformation

Chapter 11 - Application of molecular docking and dynamics tools in SARS-CoV-2 drug design: ligand–protein interaction studies

The pandemic of novel coronavirus disease 2019 (COVID-19) has rampaged the world, with more than 58.4 million confirmed cases and over 1.38 million deaths across the world by 23 November 2020. There is an urgent need to identify effective drugs and vaccines to fight against the virus. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) belongs to the family of coronaviruses consisting of four structural and 16 non-structural proteins (NSP). Three non-structural proteins, main protease (Mpro), papain-like protease (PLpro), and RNA-dependent RNA polymerase (RdRp), are believed to have a crucial role in replication of the virus. We applied computational ligand-receptor binding modeling and performed comprehensive virtual screening on FDA-approved drugs against these three SARS-CoV-2 proteins using AutoDock Vina, Glide, and rDock. Our computational studies identified six novel ligands as potential inhibitors against SARS-CoV-2, including antiemetics rolapitant and ondansetron for Mpro; labetalol and levomefolic acid for PLpro; and leucal and antifungal natamycin for RdRp. Molecular dynamics simulation confirmed the stability of the ligand-protein complexes. The results of our analysis with some other suggested drugs indicated that chloroquine and hydroxychloroquine had high binding energy (low inhibitory effect) with all three proteins—Mpro, PLpro, and RdRp. In summary, our computational molecular docking approach and virtual screening identified some promising candidate SARS-CoV-2 inhibitors that may be considered for further clinical studies.

Molecular docking is a method that mimics the interactions between small ligand and its biomacromolecule receptor. The interactions between ligand and receptor are the process of molecules recognition, which include several intermolecular interactions, like hydrogen bond actions, electrostatic reactions and so on. Molecular docking can predict the binding affinity and mode of action through computational calculation so that could be used for virtual screening of drug. Its application, however, is limited on the virtual screening that is based on that the interaction between small ligand and target protein receptor is covalent bonding action. The present research took the study of interactions between Keap1 protein and Michael reaction acceptor molecules as an example to explore possibility of application of molecular docking on investigating the space matching and energy matching between small ligand and active package of protein receptor. Our results demonstrated that molecular docking could also be used for the rapid investigation of matching status between ligand and active package of protein receptor based on the calculation and analysis of action degree and mode of intermolecules, which will provide a basis for virtual screening dependent on that the action mode is covalent binding action between ligand and active package of target protein receptor. Our finding expands the field of application of the molecular docking for virtual screening.

Dengue virus type 2 (DEN2), a member of the Flaviviridae family, is a re-emerging human pathogen of global significance. DEN2 nonstructural protein 3 (NS3) has a serine protease domain (NS3-pro) and requires the hydrophilic domain of NS2B (NS2BH) for activation. NS3 is also an RNA-stimulated nucleoside triphosphatase (NTPase)/RNA helicase and a 5'-RNA triphosphatase (RTPase). In this study the first biochemical and kinetic properties of full-length NS3 (NS3FL)-associated NTPase, RTPase, and RNA helicase are presented. The NS3FL showed an enhanced RNA helicase activity compared with the NS3-pro-minus NS3, which was further enhanced by the presence of the NS2BH (NS2BH-NS3FL). An active protease catalytic triad is not required for the stimulatory effect, suggesting that the overall folding of the N-terminal protease domain contributes to this enhancement. In DEN2-infected mammalian cells, NS3 and NS5, the viral 5'-RNA methyltransferase/polymerase, exist as a complex. Therefore, the effect of NS5 on the NS3 NTPase activity was examined. The results show that NS5 stimulated the NS3 NTPase and RTPase activities. The NS5 stimulation of NS3 NTPase was dose-dependent until an equimolar ratio was reached. Moreover, the conserved motif, 184RKRK, of NS3 played a crucial role in binding to RNA substrate and modulating the NTPase/RNA helicase and RTPase activities of NS3.

NS5B is the RNA-dependent RNA polymerase responsible for replicating hepatitis C virus (HCV) genomic RNA. Despite more than a decade of work, the formation of a highly active NS5B polymerase·RNA complex suitable for mechanistic and structural studies has remained elusive. Here, we report that through a novel way of optimizing initiation conditions, we were able to generate a productive NS5B·primer·template elongation complex stalled after formation of a 9-nucleotide primer. In contrast to previous reports of very low proportions of active NS5B, we observed that under optimized conditions up to 65% of NS5B could be converted into active elongation complexes. The elongation complex was extremely stable, allowing purification away from excess nucleotide and abortive initiation products so that the purified complex was suitable for pre-steady-state kinetic analyses of polymerase activity. Single turnover kinetic studies showed that CTP is incorporated with apparent K(d) and k(pol) values of 39 ± 3 μM and 16 ± 1 s(-1), respectively, giving a specificity constant of k(pol)/K(d) of 0.41 μM(-1) s(-1). The kinetics of multiple nucleotide incorporation during processive elongation also were determined. This work establishes a novel way to generate a highly active elongation complex of the medically important NS5B polymerase for structural and functional studies.

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

Hepatitis C virus (HCV) nonstructural protein 3 (NS3) possesses multiple enzyme activities. The N-terminal one-third of NS3 primarily functions as a serine protease, while the remaining two-thirds of NS3 serve as a helicase and nucleoside triphosphatase. Whether the multiple enzyme activities of NS3 are functionally interdependent and/or modulated by other viral NS proteins remains unclear. We performed biochemical studies to examine the functional interdependence of the NS3 protease and helicase domains and the modulation of NS3 helicase by NS5B, an RNA-dependent RNA polymerase (RdRp). We found that the NS3 protease domain of the full-length NS3 (NS3FL) enhances the NS3 helicase activity. Additionally, HCV RdRp stimulates the NS3FL helicase activity by more than sevenfold. However, the helicase activity of the NS3 helicase domain was unaffected by HCV RdRp. Glutathione S-transferase pull-down as well as fluorescence anisotropy results revealed that the NS3 protease domain is required for specific NS3 and NS5B interaction. These findings suggest that HCV RdRp regulates the functions of NS3 during HCV replication. In contrast, NS3FL does not increase NS5B RdRp activity in vitro, which is contrary to a previously published report that the HCV NS3 enhances NS5B RdRp activity.

Zika virus (ZIKV) is a re-emergent flavivirus that triggered a global health emergency from 2015 to 2017. ZIKV and other impactful flaviviruses, including dengue virus, lack the antiviral treatments and vaccines needed to diminish their threat to communities world-wide. This is in part due to gaps in our understanding of the flavivirus lifecycle; despite numerous investigations into flavivirus non-structural (NS) protein function. I hope to forge a deeper understanding of crucial NS protein functions and interactions throughout the ZIKV replication cycle. I will use alpaca-derived variable heavy-chain-only (VHH) antibody fragments, isolated from an alpaca immunized with ZIKV NS proteins, to perturb the replication cycle and therefore identify key NS protein epitopes used during replication. I individually purified NS1, NS2b, NS3, NS4b, NS5 for use in alpaca immunization, phage display panning, and enzyme-linked immunosorbent assays (ELISAs). From the alpaca peripheral blood I isolated RNA and amplified VHH genes, enriched for NS-protein binders with iterative phage display, and screened potential high binders via ELISA. I have isolated VHH sequences that interact with the ZIKV helicase, NS3, and the RNA-dependent RNA polymerase, NS5. I plan to purify each VHH and express them in ZIKA-permissive A549 cells as stable lines. Using purified VHH, I will analyze their binding ability in-vitro by ELISA and bio layer interferometry, and quantify in-vitro inhibition of NS3 protease and helicase activity for NS3 binders, and NS5 polymerase and methyltransferase activity. With the stable cells lines I will investigate each VHHs ability to impede ZIKV virus production and formation of replication complexes in infected cell lines. Support from training grant “Interactions at the Microbe-Host Interface” T32AI007472