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Nguyen DP, Nguyen HX, Van Nguyen H, Dang LM, Nguyen MT, Minh Nguyen HT. On the steroids extracted from soft corals against the NS3/4A protease of hepatitis C virus. J Mol Graph Model 2025; 136:108936. [PMID: 39793475 DOI: 10.1016/j.jmgm.2024.108936] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/11/2024] [Revised: 12/16/2024] [Accepted: 12/26/2024] [Indexed: 01/13/2025]
Abstract
The Hepatitis C virus (HCV) causes a variety of liver diseases, making it a global health issue that affects millions of people in the world. The NS3/4A protease has been considered a common target for anti-HCV treatments using direct-acting antiviral agents and their derivatives. Of the natural products that have been proposed for novel therapeutic product alternatives, the soft coral compounds are found to contain steroids with various bioactive properties for effective HCV treatments. They are screened to search for HCV inhibitors using computational approaches to screen for potential HCV inhibitors from the extracts of soft corals. Among 188 steroids considered, the five top compounds are selected for evaluation of binding affinities and stabilities using molecular docking and dynamics simulations, as well as with absorption, distribution, metabolism, excretion, and toxicity to assess the drug's performance.
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Affiliation(s)
- Duy Phuong Nguyen
- Faculty of Chemistry and Center for Computational Science, Hanoi National University of Education, Hanoi, Viet Nam.
| | - Ha Xuan Nguyen
- Institute of Natural Products Chemistry, Vietnam Academy of Science and Technology, Hanoi, Viet Nam
| | - Hue Van Nguyen
- Faculty of Chemistry and Center for Computational Science, Hanoi National University of Education, Hanoi, Viet Nam
| | - Linh Mai Dang
- Faculty of Chemistry and Center for Computational Science, Hanoi National University of Education, Hanoi, Viet Nam
| | - Minh Tho Nguyen
- Laboratory for Chemical Computation and Modeling, Institute for Computational Science and Artificial Intelligence, Van Lang University, Ho Chi Minh City, Viet Nam; Faculty of Applied Technology, School of Technology, Van Lang University, Ho Chi Minh City, Viet Nam
| | - Hue Thi Minh Nguyen
- Faculty of Chemistry and Center for Computational Science, Hanoi National University of Education, Hanoi, Viet Nam; Institute of Natural Sciences, Hanoi National University of Education, Hanoi, Viet Nam
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2
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Baig S, Berikkara A, Khalid R, Subhan SA, Abbas T, Abidi SH. In silico analysis of the effect of HCV genotype-specific polymorphisms in Core, NS3, NS5A, and NS5B proteins on T-cell epitope processing and presentation. Front Microbiol 2025; 15:1498069. [PMID: 39881992 PMCID: PMC11774985 DOI: 10.3389/fmicb.2024.1498069] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/18/2024] [Accepted: 11/20/2024] [Indexed: 01/31/2025] Open
Abstract
Background HCV genotypes are 30-35% polymorphic at the nucleotide level, while subtypes within the same genotype differ by nearly 20%. Although previous studies have shown the immune escape potential of several mutations within the HCV proteins, little is known about the effect of genotype/subtype-specific gene polymorphism on T-cell immunity. Therefore, this study employed several in silico methods to examine the impact of genotype/subtype-specific polymorphisms in Core, NS3, NS5A, and NS5B sequences on T cell epitope processing and HLA-epitope interactions. Methods For this study, 8,942, 17,700, 14,645, and 3,277 HCV Core, NS3, NS5A, and NS5B sequences, respectively, from eight genotypes and 21 subtypes were retrieved from the Los Alamos HCV Database. Next, the NetCTL tool was employed to predict Cytotoxic T Lymphocyte (CTL) epitopes based on combined proteasomal cleavage, TAP efficacy, and HLA class I receptor binding scores. PEP-FOLD was used to model selected epitopes, followed by peptide-HLA docking using HPEPDOCK. Finally, molecular dynamics simulations were conducted for 200 ns using Desmond software to analyze differences in HLA-epitope (from different HCV genotypes) interaction kinetics and dynamics. Results A total of 3,410, 8,054, 6,532, and 14,015 CTL epitopes were observed in the HCV Core, NS3, NS5A, and NS5B sequences, respectively. Significant genotype/subtype-specific variations in CTL values and docking scores were observed among NS3, NS5A, and NS5B proteins. In silico results reveal that epitopes from genotype 6b (NS3), 6d/r (NS5B), 6o and 6 k (NS5A) exhibit higher immunogenicity than other genotypes, forming more energetically stable complexes with host receptors. These epitopes, compared to those from the same positions but different genotypes, showed binding energies of -144.24 kcal/mol, -85.30 kcal/mol, and - 43 kcal/mol, respectively. Over a 200 ns MD simulation, GT 6b and 6d/r epitopes displayed up to a 40% stronger binding energy with the HLA receptor. These findings suggest that patients infected with GT 6 may experience enhanced T cell responsiveness and broader immunogenicity. Conclusion Our study suggests that genotype/subtype-specific polymorphism in HCV may result in altered immune responses by modulating T-cell epitope processing and interaction with HLA receptors. Further experimental studies can be performed to confirm the effect of genotype/subtype-specific polymorphisms on T cell-mediated immune response.
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Affiliation(s)
- Samina Baig
- Department of Microbiology, University of Karachi, Karachi, Pakistan
- Dow Institute of Medical Technology, Dow University of Health Sciences, Karachi, Pakistan
| | - Assel Berikkara
- Department of Biomedical Sciences, Nazarbayev University School of Medicine, Astana, Kazakhstan
| | - Ramsha Khalid
- Department of Biochemistry, University of Karachi, Karachi, Pakistan
| | - Syed A. Subhan
- Department of Microbiology, University of Karachi, Karachi, Pakistan
| | - Tanveer Abbas
- Department of Microbiology, University of Karachi, Karachi, Pakistan
| | - Syed Hani Abidi
- Department of Biomedical Sciences, Nazarbayev University School of Medicine, Astana, Kazakhstan
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3
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Hung JH, Teng CF, Hung HC, Chen YL, Chen PJ, Ho CL, Chuang CH, Huang W. Genomic instabilities in hepatocellular carcinoma: biomarkers and application in immunotherapies. Ann Hepatol 2024; 29:101546. [PMID: 39147130 DOI: 10.1016/j.aohep.2024.101546] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 01/26/2024] [Revised: 05/16/2024] [Accepted: 06/18/2024] [Indexed: 08/17/2024]
Abstract
Hepatocellular carcinoma (HCC) is one of the deadliest cancers. For patients with advanced HCC, liver function decompensation often occurs, which leads to poor tolerance to chemotherapies and other aggressive treatments. Therefore, it remains critical to develop effective therapeutic strategies for HCC. Etiological factors for HCC are complex and multifaceted, including hepatitis virus infection, alcohol, drug abuse, chronic metabolic abnormalities, and others. Thus, HCC has been categorized as a "genomically unstable" cancer due to the typical manifestation of chromosome breakage and aneuploidy, and oxidative DNA damage. In recent years, immunotherapy has provided a new option for cancer treatments, and the degree of genomic instability positively correlates with immunotherapy efficacies. This article reviews the endogenous and exogenous causes that affect the genomic stability of liver cells; it also updates the current biomarkers and their detection methods for genomic instabilities and relevant applications in cancer immunotherapies. Including genomic instability biomarkers in consideration of cancer treatment options shall increase the patients' well-being.
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Affiliation(s)
- Jui-Hsiang Hung
- Department of Biotechnology, Chia Nan University of Pharmacy & Science, Tainan, Taiwan
| | - Chiao-Feng Teng
- Graduate Institute of Biomedical Sciences, China Medical University, Taichung, Taiwan; Organ Transplantation Center, China Medical University Hospital, Taichung, Taiwan; Program for Cancer Biology and Drug Development, China Medical University, Taichung, Taiwan; Research Center for Cancer Biology, China Medical University, Taichung, Taiwan
| | - Hsu-Chin Hung
- Institute of Basic Medical Science, College of Medicine, National Cheng Kung University, Tainan, Taiwan
| | - Yi-Lin Chen
- Department of Pathology, National Cheng Kung University Hospital, Tainan, Taiwan
| | - Pin-Jun Chen
- Institute of Molecular Medicine, College of Medicine, National Cheng Kung University, Tainan, Taiwan
| | - Chung-Liang Ho
- Department of Pathology, National Cheng Kung University Hospital, Tainan, Taiwan; Institute of Molecular Medicine, College of Medicine, National Cheng Kung University, Tainan, Taiwan; Institute of Basic Medical Science, College of Medicine, National Cheng Kung University, Tainan, Taiwan
| | - Cheng-Hsiang Chuang
- Department of Life Science, College of Life Sciences and Medicine, National Tsing Hua University, Hsinchu, Taiwan
| | - Wenya Huang
- Department of Pathology, National Cheng Kung University Hospital, Tainan, Taiwan; Institute of Basic Medical Science, College of Medicine, National Cheng Kung University, Tainan, Taiwan; Department of Medical Laboratory Science and Biotechnology, College of Medicine, National Cheng Kung University, Tainan, Taiwan; Center of Infectious Diseases and Signal Transduction, National Cheng Kung University, Tainan, Taiwan..
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Iman K, Mirza MU, Sadia F, Froeyen M, Trant JF, Chaudhary SU. Pharmacophore-Assisted Covalent Docking Identifies a Potential Covalent Inhibitor for Drug-Resistant Genotype 3 Variants of Hepatitis C Viral NS3/4A Serine Protease. Viruses 2024; 16:1250. [PMID: 39205224 PMCID: PMC11359326 DOI: 10.3390/v16081250] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/13/2024] [Revised: 07/28/2024] [Accepted: 07/30/2024] [Indexed: 09/04/2024] Open
Abstract
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.
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Affiliation(s)
- Kanzal Iman
- Biomedical Informatics & Engineering Research Laboratory, Department of Life Sciences, Lahore University of Management Sciences, Lahore 36000, Pakistan; (K.I.); (F.S.)
| | - Muhammad Usman Mirza
- Department of Chemistry & Biochemistry, University of Windsor, Windsor, ON N9B 3P4, Canada;
| | - Fazila Sadia
- Biomedical Informatics & Engineering Research Laboratory, Department of Life Sciences, Lahore University of Management Sciences, Lahore 36000, Pakistan; (K.I.); (F.S.)
| | - Matheus Froeyen
- Department of Pharmaceutical and Pharmacological Sciences, Rega Institute for Medical Research, KU Leuven—University of Leuven, B-3000 Leuven, Belgium;
| | - John F. Trant
- Department of Chemistry & Biochemistry, University of Windsor, Windsor, ON N9B 3P4, Canada;
| | - Safee Ullah Chaudhary
- Biomedical Informatics & Engineering Research Laboratory, Department of Life Sciences, Lahore University of Management Sciences, Lahore 36000, Pakistan; (K.I.); (F.S.)
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5
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Brewitz L, Schofield CJ. Fixing the Achilles Heel of Pfizer's Paxlovid for COVID-19 Treatment. J Med Chem 2024; 67:11656-11661. [PMID: 38967233 DOI: 10.1021/acs.jmedchem.4c01342] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 07/06/2024]
Abstract
Nirmatrelvir (PF-07321332), a first-in-class inhibitor of the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) main protease (Mpro), was developed by Pfizer under intense pressure during the pandemic to treat COVID-19. A weakness of nirmatrelvir is its limited metabolic stability, which led to the development of a combination therapy (paxlovid), involving coadministration of nirmatrelvir with the cytochrome P450 inhibitor ritonavir. However, limitations in tolerability of the ritonavir component reduce the scope of paxlovid. In response to these limitations, researchers at Pfizer have now developed the second-generation Mpro inhibitor PF-07817883 (ibuzatrelvir). Structurally related to nirmatrelvir, including with the presence of a trifluoromethyl group, albeit located differently, ibuzatrelvir manifests enhanced oral bioavailability, so it does not require coadministration with ritonavir. The development of ibuzatrelvir is an important milestone, because it is expected to enhance the treatment of COVID-19 without the drawbacks associated with ritonavir. Given the success of paxlovid in treating COVID-19, it is likely that ibuzatrelvir will be granted approval as an improved drug for treatment of COVID-19 infections, so complementing vaccination efforts and improving pandemic preparedness. The development of nirmatrelvir and ibuzatrelvir dramatically highlights the power of appropriately resourced modern medicinal chemistry to very rapidly enable the development of breakthrough medicines. Consideration of how analogous approaches can be used to develop similarly breakthrough medicines for infectious diseases such as tuberculosis and malaria is worthwhile.
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Affiliation(s)
- Lennart Brewitz
- Chemistry Research Laboratory, Department of Chemistry and the Ineos Oxford Institute for Antimicrobial Research, University of Oxford, 12 Mansfield Road, OX1 3TA Oxford, United Kingdom
| | - Christopher J Schofield
- Chemistry Research Laboratory, Department of Chemistry and the Ineos Oxford Institute for Antimicrobial Research, University of Oxford, 12 Mansfield Road, OX1 3TA Oxford, United Kingdom
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6
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Martino SD, Petri GL, De Rosa M. Hepatitis C: The Story of a Long Journey through First, Second, and Third Generation NS3/4A Peptidomimetic Inhibitors. What Did We Learn? J Med Chem 2024; 67:885-921. [PMID: 38179950 DOI: 10.1021/acs.jmedchem.3c01971] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/06/2024]
Abstract
Hepatitis C viral (HCV) infection is the leading cause of liver failure and still represents a global health burden. Over the past decade, great advancements made HCV curable, and sustained viral remission significantly improved to more than 98%. Historical treatment with pegylated interferon alpha and ribavirin has been displaced by combinations of direct-acting antivirals. These regimens include drugs targeting different stages of the HCV life cycle. However, the emergence of viral resistance remains a big concern. The design of peptidomimetic inhibitors (PIs) able to fit and fill the conserved substrate envelope region within the active site helped avoid contact with the vulnerable sites of the most common resistance-associated substitutions Arg155, Ala156, and Asp168. Herein, we give an overview of HCV NS3 PIs discovered during the past decade, and we deeply discuss the rationale behind the structural optimization efforts essential to achieve pangenotypic activity.
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Affiliation(s)
- Simona Di Martino
- Drug Discovery Unit, Medicinal Chemistry Group, Ri.MED Foundation, Palermo 90133, Italy
| | - Giovanna Li Petri
- Drug Discovery Unit, Medicinal Chemistry Group, Ri.MED Foundation, Palermo 90133, Italy
| | - Maria De Rosa
- Drug Discovery Unit, Medicinal Chemistry Group, Ri.MED Foundation, Palermo 90133, Italy
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7
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Ren P, Li S, Wang S, Zhang X, Bai F. Computer-Aided Prediction of the Interactions of Viral Proteases with Antiviral Drugs: Antiviral Potential of Broad-Spectrum Drugs. Molecules 2023; 29:225. [PMID: 38202808 PMCID: PMC10780089 DOI: 10.3390/molecules29010225] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/29/2023] [Revised: 12/27/2023] [Accepted: 12/29/2023] [Indexed: 01/12/2024] Open
Abstract
Human society is facing the threat of various viruses. Proteases are promising targets for the treatment of viral infections. In this study, we collected and profiled 170 protease sequences from 125 viruses that infect humans. Approximately 73 of them are viral 3-chymotrypsin-like proteases (3CLpro), and 11 are pepsin-like aspartic proteases (PAPs). Their sequences, structures, and substrate characteristics were carefully analyzed to identify their conserved nature for proposing a pan-3CLpro or pan-PAPs inhibitor design strategy. To achieve this, we used computational prediction and modeling methods to predict the binding complex structures for those 73 3CLpro with 4 protease inhibitors of SARS-CoV-2 and 11 protease inhibitors of HCV. Similarly, the complex structures for the 11 viral PAPs with 9 protease inhibitors of HIV were also obtained. The binding affinities between these compounds and proteins were also evaluated to assess their pan-protease inhibition via MM-GBSA. Based on the drugs targeting viral 3CLpro and PAPs, repositioning of the active compounds identified several potential uses for these drug molecules. As a result, Compounds 1-2, modified based on the structures of Ray1216 and Asunaprevir, indicate potential inhibition of DENV protease according to our computational simulation results. These studies offer ideas and insights for future research in the design of broad-spectrum antiviral drugs.
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Affiliation(s)
- Pengxuan Ren
- School of Life Science and Technology, Shanghai Institute for Advanced Immunochemical Studies, ShanghaiTech University, Shanghai 201210, China; (P.R.); (S.L.); (S.W.)
| | - Shiwei Li
- School of Life Science and Technology, Shanghai Institute for Advanced Immunochemical Studies, ShanghaiTech University, Shanghai 201210, China; (P.R.); (S.L.); (S.W.)
| | - Shihang Wang
- School of Life Science and Technology, Shanghai Institute for Advanced Immunochemical Studies, ShanghaiTech University, Shanghai 201210, China; (P.R.); (S.L.); (S.W.)
| | - Xianglei Zhang
- School of Life Science and Technology, Shanghai Institute for Advanced Immunochemical Studies, ShanghaiTech University, Shanghai 201210, China; (P.R.); (S.L.); (S.W.)
| | - Fang Bai
- School of Life Science and Technology, Shanghai Institute for Advanced Immunochemical Studies, ShanghaiTech University, Shanghai 201210, China; (P.R.); (S.L.); (S.W.)
- School of Information Science and Technology, ShanghaiTech University, Shanghai 201210, China
- Shanghai Clinical Research and Trial Center, Shanghai 201210, China
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8
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Wang L, Wang S, Chiou S, Tsai J, Chai C, Tseng L, Lee J, Lin M, Huang S, Hsu S. HCV Core Protein-ISX Axis Promotes Chronic Liver Disease Progression via Metabolic Remodeling and Immune Suppression. ADVANCED SCIENCE (WEINHEIM, BADEN-WURTTEMBERG, GERMANY) 2023; 10:e2300644. [PMID: 37316966 PMCID: PMC10427408 DOI: 10.1002/advs.202300644] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 01/30/2023] [Revised: 04/13/2023] [Indexed: 06/16/2023]
Abstract
Chronic hepatitis C virus (HCV) infection is an important public health issue. However, knowledge on how the virus remodels the metabolic and immune response toward hepatic pathologic environment is limited. The transcriptomic and multiple evidences reveal that the HCV core protein-intestine-specific homeobox (ISX) axis promotes a spectrum of metabolic, fibrogenic, and immune modulators (e.g., kynurenine, PD-L1, and B7-2), regulating HCV-infection relevant pathogenic phenotype in vitro and in vivo. In a transgenic mice model, the HCV core protein-ISX axis enhance metabolic disturbance (particularly lipid and glucose metabolism) and immune suppression, and finally, chronic liver fibrosis in a high-fat diet (HFD)-induced disease model. Mechanistically, cells with HCV JFH-1 replicons upregulate ISX and, consequently, the expressions of metabolic, fibrosis progenitor, and immune modulators via core protein-induced nuclear factor-κB signaling. Conversely, cells with specific ISX shRNAi inhibit HCV core protein-induced metabolic disturbance and immune suppression. Clinically, the HCV core level is significantly correlated with ISX, IDOs, PD-L1, and B7-2 levels in HCC patients with HCV infection. Therefore, it highlights the significance of HCV core protein-ISX axis as an important mechanism in the development of HCV-induced chronic liver disease and can be a specific therapeutic target clinically.
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Affiliation(s)
- Li‐Ting Wang
- Department of Life ScienceNational Taiwan Normal UniversityTaipei116059Taiwan
- Center of Applied GenomicsKaohsiung Medical UniversityKaohsiung80708Taiwan
| | - Shen‐Nien Wang
- Graduate Institute of MedicineCollege of MedicineKaohsiung Medical UniversityKaohsiung80708Taiwan
- Division of General and Digestive SurgeryDepartment of SurgeryKaohsiung Medical University HospitalKaohsiung80708Taiwan
- Department of SurgeryCollege of MedicineKaohsiung Medical University HospitalKaohsiung80708Taiwan
| | - Shyh‐Shin Chiou
- Center of Applied GenomicsKaohsiung Medical UniversityKaohsiung80708Taiwan
- Division of Pediatric Hematology and Oncology, Department of PediatricsKaohsiung Medical University HospitalKaohsiung80708Taiwan
- Graduate Institute of Clinical Medicine, College of MedicineKaohsiung Medical UniversityKaohsiung80708Taiwan
| | - Jhih‐Peng Tsai
- Center of Applied GenomicsKaohsiung Medical UniversityKaohsiung80708Taiwan
| | - Chee‐Yin Chai
- Department of PathologyKaohsiung Medical University HospitalKaohsiung80708Taiwan
| | - Li‐Wen Tseng
- Graduate Institute of MedicineCollege of MedicineKaohsiung Medical UniversityKaohsiung80708Taiwan
| | - Jin‐Ching Lee
- Department of BiotechnologyCollege of Life ScienceNational Sun Yat‐sen UniversityKaohsiung804201Taiwan
| | - Ming‐Hong Lin
- Department of Microbiology and ImmunologySchool of MedicineCollege of MedicineKaohsiung Medical UniversityKaohsiung City80708Taiwan
- Department of Medical ResearchKaohsiung Medical University HospitalKaohsiung Medical UniversityKaohsiung80708Taiwan
| | - Shau‐Ku Huang
- National Institute of Environmental Health SciencesNational Health Research InstitutesMiaoli County35053Taiwan
- Department of Respirology & AllergyThird Affiliated Hospital of Shenzhen UniversityShenzhen518020China
- Department of MedicineDivision of Allergy and Clinical ImmunologyJohns Hopkins University School of MedicineBaltimoreMD21287USA
| | - Shih‐Hsien Hsu
- Graduate Institute of MedicineCollege of MedicineKaohsiung Medical UniversityKaohsiung80708Taiwan
- Department of Medical ResearchKaohsiung Medical University HospitalKaohsiung Medical UniversityKaohsiung80708Taiwan
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Yildirim A, Tekpinar M. Building Quantitative Bridges between Dynamics and Sequences of SARS-CoV-2 Main Protease and a Diverse Set of Thirty-Two Proteins. J Chem Inf Model 2023; 63:9-19. [PMID: 36513349 DOI: 10.1021/acs.jcim.2c01206] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/15/2022]
Abstract
Proteases are major drug targets for many viral diseases. However, mutations can render several antiprotease drugs inefficient rapidly even though these mutations may not alter protein structures significantly. Understanding relations between quickly mutating residues, protease structures, and the dynamics of the proteases is crucial for designing potent drugs. Due to this reason, we studied relations between the evolutionary information on residues in the amino acid sequences and protein dynamics for SARS-CoV-2 main protease. More precisely, we analyzed three dynamical quantities (Schlitter entropy, root-mean-square fluctuations, and dynamical flexibility index) and their relation to the amino acid conservation extracted from multiple sequence alignments of the main protease. We showed that a quantifiable similarity can be built between a sequence-based quantity called Jensen-Shannon conservation and those three dynamical quantities. We validated this similarity for a diverse set of 32 different proteins, other than the SARS-CoV-2 main protease. We believe that establishing these kinds of quantitative bridges will have larger implications for all viral proteases as well as all proteins.
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Affiliation(s)
- Ahmet Yildirim
- Department of Biology, Siirt University, 56100Siirt, Turkey
| | - Mustafa Tekpinar
- CNRS, IBPS, Laboratoire de Biologie Computationnelle et Quantitative - UMR 7238, Sorbonne University, 75005Paris, France
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10
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Mechanisms and Consequences of Genetic Variation in Hepatitis C Virus (HCV). Curr Top Microbiol Immunol 2023; 439:237-264. [PMID: 36592248 DOI: 10.1007/978-3-031-15640-3_7] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/03/2023]
Abstract
Chronic infection with hepatitis C virus (HCV) is an important contributor to the global incidence of liver diseases, including liver cirrhosis and hepatocellular carcinoma. Although common for single-stranded RNA viruses, HCV displays a remarkable high level of genetic diversity, produced primarily by the error-prone viral polymerase and host immune pressure. The high genetic heterogeneity of HCV has led to the evolution of several distinct genotypes and subtypes, with important consequences for pathogenesis, and clinical outcomes. Genetic variability constitutes an evasion mechanism against immune suppression, allowing the virus to evolve epitope escape mutants that avoid immune recognition. Thus, heterogeneity and variability of the HCV genome represent a great hindrance for the development of vaccines against HCV. In addition, the high genetic plasticity of HCV allows the virus to rapidly develop antiviral resistance mutations, leading to treatment failure and potentially representing a major hindrance for the cure of chronic HCV patients. In this chapter, we will present the central role that genetic diversity has in the viral life cycle and epidemiology of HCV. Incorporation errors and recombination, both the result of HCV polymerase activity, represent the main mechanisms of HCV evolution. The molecular details of both mechanisms have been only partially clarified and will be presented in the following sections. Finally, we will discuss the major consequences of HCV genetic diversity, namely its capacity to rapidly evolve antiviral and immunological escape variants that represent an important limitation for clearance of acute HCV, for treatment of chronic hepatitis C and for broadly protective vaccines.
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11
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Belachew B, Gao J, Byrd AK, Raney KD. Hepatitis C virus nonstructural protein NS3 unfolds viral G-quadruplex RNA structures. J Biol Chem 2022; 298:102486. [PMID: 36108740 PMCID: PMC9582721 DOI: 10.1016/j.jbc.2022.102486] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/11/2022] [Revised: 08/17/2022] [Accepted: 09/03/2022] [Indexed: 11/29/2022] Open
Abstract
Hepatitis C virus (HCV) is a major cause of liver-related diseases and hepatocellular carcinoma. The helicase domain of one of the nonstructural proteins of HCV, NS3 (nonstructural protein 3), is essential for viral replication; however, its specific biological role is still under investigation. Here, we set out to determine the interaction between a purified recombinant full length NS3 and synthetic guanine-rich substrates that represent the conserved G-quadruplex (G4)-forming sequences in the HCV-positive and HCV-negative strands. We performed fluorescence anisotropy binding, G4 reporter duplex unwinding, and G4RNA trapping assays to determine the binding and G4 unfolding activity of NS3. Our data suggest that NS3 can unfold the conserved G4 structures present within the genome and the negative strand of HCV. Additionally, we found the activity of NS3 on a G4RNA was reduced significantly in the presence of a G4 ligand. The ability of NS3 to unfold HCV G4RNA could imply a novel biological role of the viral helicase in replication.
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Affiliation(s)
- Binyam Belachew
- Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, Arkansas, USA
| | - Jun Gao
- Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, Arkansas, USA
| | - Alicia K Byrd
- Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, Arkansas, USA
| | - Kevin D Raney
- Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, Arkansas, USA.
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12
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Zhou Z, Zhang J, Zhou E, Ren C, Wang J, Wang Y. Small molecule NS5B RdRp non-nucleoside inhibitors for the treatment of HCV infection: A medicinal chemistry perspective. Eur J Med Chem 2022; 240:114595. [PMID: 35868125 DOI: 10.1016/j.ejmech.2022.114595] [Citation(s) in RCA: 6] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/14/2022] [Revised: 07/01/2022] [Accepted: 07/05/2022] [Indexed: 02/06/2023]
Abstract
Hepatitis C virus (HCV) infection has become a global health problem with enormous risks. Nonstructural protein 5B (NS5B) RNA-dependent RNA polymerase (RdRp) is a component of HCV, which can promote the formation of the viral RNA replication complex and is also an essential part of the replication complex itself. It plays a vital role in the synthesis of the positive and negative strands of HCV RNA. Therefore, the development of small-molecule inhibitors targeting NS5B RdRp is of great value for treating HCV infection-related diseases. Compared with NS5B RdRp nucleoside inhibitors, non-nucleoside inhibitors have more flexible structures, simpler mechanisms of action, and more predictable efficacy and safety of drugs in humans. Technological advances over the past decade have led to remarkable achievements in developing NS5B RdRp inhibitors. This review will summarize the non-nucleoside inhibitors targeting NS5B RdRp developed in the past decade and describe their structure optimization process and structure-activity relationship.
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Affiliation(s)
- Zhilan Zhou
- Targeted Tracer Research and Development Laboratory, Institute of Respiratory Health, Frontiers Science Center for Disease-related Molecular Network, National Clinical Research Center for Geriatrics, West China Hospital, Sichuan University, Chengdu, 610041, Sichuan, China; State Key Laboratory of Biotherapy and Cancer Center, Department of Respiratory and Critical Care Medicine, National Clinical Research Center for Geriatrics, West China Hospital, Sichuan University, Chengdu, 610041, Sichuan, China; Precision Medicine Key Laboratory of Sichuan Province & Precision Medicine Research Center, West China Hospital, Sichuan University, Chengdu, 610041, Sichuan, China
| | - Jifa Zhang
- Targeted Tracer Research and Development Laboratory, Institute of Respiratory Health, Frontiers Science Center for Disease-related Molecular Network, National Clinical Research Center for Geriatrics, West China Hospital, Sichuan University, Chengdu, 610041, Sichuan, China; State Key Laboratory of Biotherapy and Cancer Center, Department of Respiratory and Critical Care Medicine, National Clinical Research Center for Geriatrics, West China Hospital, Sichuan University, Chengdu, 610041, Sichuan, China; Precision Medicine Key Laboratory of Sichuan Province & Precision Medicine Research Center, West China Hospital, Sichuan University, Chengdu, 610041, Sichuan, China; Tianfu Jincheng Laboratory, Chengdu, 610041, Sichuan, China
| | - Enda Zhou
- Targeted Tracer Research and Development Laboratory, Institute of Respiratory Health, Frontiers Science Center for Disease-related Molecular Network, National Clinical Research Center for Geriatrics, West China Hospital, Sichuan University, Chengdu, 610041, Sichuan, China; State Key Laboratory of Biotherapy and Cancer Center, Department of Respiratory and Critical Care Medicine, National Clinical Research Center for Geriatrics, West China Hospital, Sichuan University, Chengdu, 610041, Sichuan, China
| | - Changyu Ren
- Department of Pharmacy, Chengdu Fifth People's Hospital, Chengdu, Sichuan, 611130, China
| | - Jiaxing Wang
- Department of Pharmaceutical Sciences, College of Pharmacy, University of Tennessee Health Science Center, Memphis, 38163, Tennessee, United States
| | - Yuxi Wang
- Targeted Tracer Research and Development Laboratory, Institute of Respiratory Health, Frontiers Science Center for Disease-related Molecular Network, National Clinical Research Center for Geriatrics, West China Hospital, Sichuan University, Chengdu, 610041, Sichuan, China; State Key Laboratory of Biotherapy and Cancer Center, Department of Respiratory and Critical Care Medicine, National Clinical Research Center for Geriatrics, West China Hospital, Sichuan University, Chengdu, 610041, Sichuan, China; Precision Medicine Key Laboratory of Sichuan Province & Precision Medicine Research Center, West China Hospital, Sichuan University, Chengdu, 610041, Sichuan, China; Tianfu Jincheng Laboratory, Chengdu, 610041, Sichuan, China.
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13
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Ul Haq A, Sheikh A, Naeem S, Abidi SH. Molecular docking analysis of fluoroquinolones and other natural and synthetic compounds with the HCV NS3 helicase. Bioinformation 2022; 18:147-154. [PMID: 36518146 PMCID: PMC9722412 DOI: 10.6026/97320630018147] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/08/2021] [Revised: 03/07/2022] [Accepted: 03/31/2022] [Indexed: 08/10/2023] Open
Abstract
It is of an interest to document the molecular docking analysis of fluoroquinolones and other natural and synthetic compounds with the HCV NS3 helicase. Data shows that three fluoroquinolones interacted with the NS3 helicase in the catalytic region, targeting some of the amino acids known to play a crucial role in NS3 helicase activity. Similarly, binding energy shows that the fluoroquinolones were comparable to the thiazolpiperazinyl derivatives, while superior to several of the synthetic and natural derivatives. The results show three fluoroquinolones to be potent helicase inhibitors that can be repurposed as supplemental therapy against HCV especially in cases non-responsive to DAAAs.
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Affiliation(s)
- Ahtesham Ul Haq
- Department of Biochemistry, University of Karachi, Karachi-Pakistan
| | - Alisalman Sheikh
- Department of Biological and Biomedical Sciences, Aga Khan University, Karachi-Pakistan
| | - Sadaf Naeem
- Department of Biochemistry, University of Karachi, Karachi-Pakistan
| | - Syed Hani Abidi
- Department of Biological and Biomedical Sciences, Aga Khan University, Karachi-Pakistan
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14
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Cao W, Geng ZZ, Wang N, Pan Q, Guo S, Xu S, Zhou J, Liu WR. A Reversible Chemogenetic Switch for Chimeric Antigen Receptor T Cells**. Angew Chem Int Ed Engl 2022. [DOI: 10.1002/ange.202109550] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/05/2022]
Affiliation(s)
- Wenyue Cao
- Department of Hematology Tongji Hospital Tongji Medical College Huazhong University of Science and Technology Wuhan Hubei China
- The Texas A&M Drug Discovery Laboratory Department of Chemistry Texas A&M University College Station TX 77843-3255 USA
| | - Zhi Zachary Geng
- The Texas A&M Drug Discovery Laboratory Department of Chemistry Texas A&M University College Station TX 77843-3255 USA
| | - Na Wang
- Department of Hematology Tongji Hospital Tongji Medical College Huazhong University of Science and Technology Wuhan Hubei China
| | - Quan Pan
- The Texas A&M Drug Discovery Laboratory Department of Chemistry Texas A&M University College Station TX 77843-3255 USA
| | - Shaodong Guo
- The Texas A&M Drug Discovery Laboratory Department of Chemistry Texas A&M University College Station TX 77843-3255 USA
| | - Shiqing Xu
- The Texas A&M Drug Discovery Laboratory Department of Chemistry Texas A&M University College Station TX 77843-3255 USA
| | - Jianfeng Zhou
- Department of Hematology Tongji Hospital Tongji Medical College Huazhong University of Science and Technology Wuhan Hubei China
| | - Wenshe Ray Liu
- The Texas A&M Drug Discovery Laboratory Department of Chemistry Texas A&M University College Station TX 77843-3255 USA
- Institute of Biosciences and Technology and Department of Translational Medical Sciences College of Medicine Texas A&M University Houston TX 77030 USA
- Department of Biochemistry and Biophysics Texas A&M University Houston TX 77843 USA
- Department of Molecular and Cellular Medicine College of Medicine Texas A&M University Houston TX 77843 USA
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15
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Liu W, Cao W, Geng ZZ, Wang N, Pan Q, Guo S, Zhou J, Xu S. A Recurring Chemogenetic Switch for Chimeric Antigen Receptor T Cells. Angew Chem Int Ed Engl 2021; 61:e202109550. [PMID: 34783141 DOI: 10.1002/anie.202109550] [Citation(s) in RCA: 10] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/16/2021] [Revised: 10/08/2021] [Indexed: 11/11/2022]
Abstract
As a revolutionary cancer treatment, the chimeric antigen receptor (CAR) T cell therapy suffers from complications such as cytokine release syndromes and T cell exhaustion. Their mitigation desires controllable activation of CAR-T cells that is achievable through regulatory display of CARs. By embedding the hepatitis C virus NS3 protease (HCV-NS3) between the single-chain variable fragment (scFv) and the hinge domain, we showed that the display of anti-CD19 scFv on CAR-T cells was positively correlated to the presence of a clinical HCV-NS3 inhibitor asunaprevir (ASV). This novel CAR design that allows the display of anti-CD19 scFv in the presence of ASV and its removal in the absence of ASV creates a practically recurring chemical switch. We demonstrated that the intact CAR on T cells can be repeatedly turned on and off by controlling the presence of ASV in a dose dependent manner both in vitro and in vivo, which enables delicate modulation of CAR-T activation during cancer treatment.
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Affiliation(s)
- Wenshe Liu
- Texas A&M University, Department of Chemistry, Corner of Ross and Spence Streets, 77845, College Station, UNITED STATES
| | - Wenyue Cao
- Tongji Medical College of Huazhong University of Science and Technology: Huazhong University of Science and Technology Tongji Medical College, Hemotology, CHINA
| | - Zhi Z Geng
- Texas A&M University, Chemistry, Department of Chemistry, Corner of Spence and Ross Streets, 77843-3255, United States, College Station, UNITED STATES
| | - Na Wang
- Tongji Medical College of Huazhong University of Science and Technology: Huazhong University of Science and Technology Tongji Medical College, Hemotology, UNITED STATES
| | - Quan Pan
- Texas A&M University, Nutrition and food science, UNITED STATES
| | - Shaodong Guo
- Texas A&M University, Nutrition and food science, UNITED STATES
| | - Jianfeng Zhou
- Tongji Medical College of Huazhong University of Science and Technology: Huazhong University of Science and Technology Tongji Medical College, Hemotology, CHINA
| | - Shiqing Xu
- Texas A&M University, Chemistry, UNITED STATES
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16
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Zhou R, Liu L, Wang Y. Viral proteins recognized by different TLRs. J Med Virol 2021; 93:6116-6123. [PMID: 34375002 DOI: 10.1002/jmv.27265] [Citation(s) in RCA: 41] [Impact Index Per Article: 10.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/02/2021] [Revised: 07/08/2021] [Accepted: 08/08/2021] [Indexed: 12/24/2022]
Abstract
Virus invasion activates the host's innate immune response, inducing the production of numerous cytokines and interferons to eliminate pathogens. Except for viral DNA/RNA, viral proteins are also targets of pattern recognition receptors. Membrane-bound receptors such as Toll-like receptor (TLR)1, TLR2, TLR4, TLR6, and TLR10 relate to the recognition of viral proteins. Distinct TLRs perform both protective and detrimental roles for a specific virus. Here, we review viral proteins serving as pathogen-associated molecular patterns and their corresponding TLRs. These viruses are all enveloped, including respiratory syncytial virus, hepatitis C virus, measles virus, herpesvirus human immunodeficiency virus, and coronavirus, and can encode proteins to activate innate immunity in a TLR-dependent way. The TLR-viral protein relationship plays an important role in innate immunity activation. A detailed understanding of their pathways contributes to a novel direction for vaccine development.
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Affiliation(s)
- Rui Zhou
- Department of Microbiology, Institute of Basic Medical Sciences, Zunyi Medical University, Zunyi, China
| | - Li Liu
- Department of Microbiology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and School of Basic Medicine, Peking Union Medical College, Beijing, China
| | - Yu Wang
- Department of Microbiology, Institute of Basic Medical Sciences, Zunyi Medical University, Zunyi, China
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17
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Weber R, McCullagh M. Role of ATP in the RNA Translocation Mechanism of SARS-CoV-2 NSP13 Helicase. J Phys Chem B 2021; 125:8787-8796. [PMID: 34328740 PMCID: PMC8353885 DOI: 10.1021/acs.jpcb.1c04528] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/23/2021] [Revised: 07/21/2021] [Indexed: 11/29/2022]
Abstract
The COVID-19 pandemic has demonstrated the need to develop potent and transferable therapeutics to treat coronavirus infections. Numerous antiviral targets are being investigated, but nonstructural protein 13 (nsp13) stands out as a highly conserved and yet understudied target. Nsp13 is a superfamily 1 (SF1) helicase that translocates along and unwinds viral RNA in an ATP-dependent manner. Currently, there are no available structures of nsp13 from SARS-CoV-1 or SARS-CoV-2 with either ATP or RNA bound, which presents a significant hurdle to the rational design of therapeutics. To address this knowledge gap, we have built models of SARS-CoV-2 nsp13 in Apo, ATP, ssRNA and ssRNA+ATP substrate states. Using 30 μs of a Gaussian-accelerated molecular dynamics simulation (at least 6 μs per substrate state), these models were confirmed to maintain substrate binding poses that are similar to other SF1 helicases. A Gaussian mixture model and linear discriminant analysis structural clustering protocol was used to identify key structural states of the ATP-dependent RNA translocation mechanism. Namely, four RNA-nsp13 structures are identified that exhibit ATP-dependent populations and support the inchworm mechanism for translocation. These four states are characterized by different RNA-binding poses for motifs Ia, IV, and V and suggest a power stroke-like motion of domain 2A relative to domain 1A. This structural and mechanistic insight of nsp13 RNA translocation presents novel targets for the further development of antivirals.
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Affiliation(s)
- Ryan Weber
- Department
of Chemistry, Colorado State University, Fort Collins, Colorado 80523, United States
| | - Martin McCullagh
- Department
of Chemistry, Oklahoma State University, Stillwater, Oklahoma 74074, United States
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18
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In Vitro Antiviral Activity of Tyrosinase from Mushroom Agaricus bisporus against Hepatitis C Virus. Pharmaceuticals (Basel) 2021; 14:ph14080759. [PMID: 34451856 PMCID: PMC8399381 DOI: 10.3390/ph14080759] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/29/2021] [Revised: 07/29/2021] [Accepted: 07/30/2021] [Indexed: 11/16/2022] Open
Abstract
Tyrosinases from a commercial Agaricus bisporus protein extract and directly isolated from white mushrooms were purified in order to obtaining the well-known tyrosinase from A. bisporus (TyrAB) of 45 kDa and a newly discovered 50 kDa tyrosinase isoform (Tyr50 kDa), and tested showing high antiviral activity against the hepatitis C virus for the first time. Cell toxicity and antiviral activity of tyrosinases were determined in cultured Huh 5-2 liver tumor cells transfected with a replicon system (a plasmid that includes all non-structural hepatitis C virus proteins and replicates autonomously). TyrAB was able to inhibit the replication of the hepatitis C virus without inducing toxicity in liver cells. In addition, the post-translational isoform Tyr50 kDa showed higher antiviral capacity than the former (up to 10 times greater), also exhibiting 10 times higher activity than the commercial drug Ribavirin®. This antiviral activity was directly proportional to the enzymatic activity of tyrosinases, as no antiviral capacity was observed in the inactive form of the enzymes. The tyrosinases approach could represent a new antiviral inhibition mechanism, through a plausible catalytic mechanism of selective hydroxylation of the key role of tyrosine residues in viral proteases.
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19
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Intra-host evolutionary dynamics of the hepatitis C virus among people who inject drugs. Sci Rep 2021; 11:9986. [PMID: 33976241 PMCID: PMC8113533 DOI: 10.1038/s41598-021-88132-8] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/09/2020] [Accepted: 03/31/2021] [Indexed: 02/03/2023] Open
Abstract
Most individuals chronically infected with hepatitis C virus (HCV) are asymptomatic during the initial stages of infection and therefore the precise timing of infection is often unknown. Retrospective estimation of infection duration would improve existing surveillance data and help guide treatment. While intra-host viral diversity quantifications such as Shannon entropy have previously been utilized for estimating duration of infection, these studies characterize the viral population from only a relatively short segment of the HCV genome. In this study intra-host diversities were examined across the HCV genome in order to identify the region most reflective of time and the degree to which these estimates are influenced by high-risk activities including those associated with HCV acquisition. Shannon diversities were calculated for all regions of HCV from 78 longitudinally sampled individuals with known seroconversion timeframes. While the region of the HCV genome most accurately reflecting time resided within the NS3 gene, the gene region with the highest capacity to differentiate acute from chronic infections was identified within the NS5b region. Multivariate models predicting duration of infection from viral diversity significantly improved upon incorporation of variables associated with recent public, unsupervised drug use. These results could assist the development of strategic population treatment guidelines for high-risk individuals infected with HCV and offer insights into variables associated with a likelihood of transmission.
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20
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Tarannum H, Chauhan B, Samadder A, Roy H, Nandi S. To Explore the Potential Targets and Current Structure-based Design Strategies Utilizing Co-crystallized Ligand to Combat HCV. Curr Drug Targets 2021; 22:590-604. [PMID: 32720601 DOI: 10.2174/1389450121999200727215020] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/06/2020] [Revised: 05/05/2020] [Accepted: 05/11/2020] [Indexed: 11/22/2022]
Abstract
BACKGROUND Hepatitis C Virus (HCV) belongs to the Hepacivirus family. HCV has been designated as a very dreadful virus as it can attack the liver, causing inflammation and even may lead to cancer in chronic conditions. It was estimated that 71 million people around the world have chronic HCV infection. World Health Organization (WHO) reported that about 399000 people died because of chronic cirrhosis and liver cancer globally. In spite of the abundance of availability of drugs for the treatment of HCV, however, the issue of drug resistance surpasses all the possibilities of therapeutic management of HCV. Therefore, to address this issue of 'drug-resistance', various HCV targets were explored to quest the evaluation of the mechanism of the disease progression. METHODS An attempt has been made in the present study to explore the various targets of HCV involved in the mechanism(s) of the disease initiation and progression and to focus on the mode of binding of ligands, which are co-crystallized at the active cavity of different HCV targets. CONCLUSION The present study could predict some crucial features of these ligands, which possibly interacted with various amino acid residues responsible for their biological activity and molecular signaling pathway(s). Such binding mode may be considered as a template for the high throughput screening and designing of active congeneric ligands to combat HCV.
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Affiliation(s)
- Heena Tarannum
- Department of Pharmaceutical Chemistry, Global Institute of Pharmaceutical Education and Research (Affiliated to Uttarakhand Technical University), Kashipur-244713, India
| | - Bhumika Chauhan
- Department of Pharmaceutical Chemistry, Global Institute of Pharmaceutical Education and Research (Affiliated to Uttarakhand Technical University), Kashipur-244713, India
| | - Asmita Samadder
- Cytogenetics and Molecular Biology Lab., Department of Zoology, University of Kalyani, Kalyani, Nadia, 741235, India
| | - Harekrishna Roy
- Nirmala College of Pharmacy, Mangalagiri, Guntur, Andhra Pradesh, 522503, India
| | - Sisir Nandi
- Department of Pharmaceutical Chemistry, Global Institute of Pharmaceutical Education and Research (Affiliated to Uttarakhand Technical University), Kashipur-244713, India
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21
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An in silico approach to analyze HCV genotype-specific binding-site variation and its effect on drug-protein interaction. Sci Rep 2020; 10:20885. [PMID: 33257748 PMCID: PMC7705671 DOI: 10.1038/s41598-020-77720-9] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/17/2019] [Accepted: 09/18/2020] [Indexed: 01/06/2023] Open
Abstract
Genotype variation in viruses can affect the response of antiviral treatment. Several studies have established approaches to determine genotype-specific variations; however, analyses to determine the effect of these variations on drug–protein interactions remain unraveled. We present an in-silico approach to explore genotype-specific variations and their effect on drug–protein interaction. We have used HCV NS3 helicase and fluoroquinolones as a model for drug–protein interaction and have investigated the effect of amino acid variations in HCV NS3 of genotype 1a, 1b, 2b and 3a on NS3-fluoroquinolone interaction. We retrieved 687, 667, 101 and 248 nucleotide sequences of HCV NS3 genotypes 1a, 1b, 2b, and 3a, respectively, and translated these into amino acid sequences and used for genotype variation analysis, and also to construct 3D protein models for 2b and 3a genotypes. For 1a and 1b, crystal structures were used. Drug–protein interactions were determined using molecular docking analyses. Our results revealed that individual genotype-specific HCV NS3 showed substantial sequence heterogeneity that resulted in variations in docking interactions. We believe that our approach can be extrapolated to include other viruses to study the clinical significance of genotype-specific variations in drug–protein interactions.
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22
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Asiri YI, Alsayari A, Muhsinah AB, Mabkhot YN, Hassan MZ. Benzothiazoles as potential antiviral agents. J Pharm Pharmacol 2020; 72:1459-1480. [PMID: 32705690 PMCID: PMC7405065 DOI: 10.1111/jphp.13331] [Citation(s) in RCA: 42] [Impact Index Per Article: 8.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/24/2020] [Revised: 06/03/2020] [Accepted: 06/13/2020] [Indexed: 12/11/2022]
Abstract
OBJECTIVES The recent viral pandemic poses a unique challenge for healthcare providers. Despite the remarkable progress, the number of novel antiviral agents in the pipeline is woefully inadequate against the evolving virulence and drug resistance of current viruses. This highlights the urgent need for new and improved vaccines, diagnostics and therapeutic agents to obviate the viral pandemic. KEY FINDINGS Benzothiazole plays a pivotal role in the design and development of antiviral drugs. This is evident from the fact that it comprises many clinically useful agents. The current review is aimed to provide an insight into the recent development of benzothiazole-based antiviral agents, with a special focus on their structure-activity relationships and lead optimisation. One hundred and five articles were initially identified, and from these studies, 64 potential novel lead molecules and main findings were highlighted in this review. SUMMARY We hope this review will provide a logical perspective on the importance of improving the future designs of novel broad-spectrum benzothiazole-based antiviral agents to be used against emerging viral diseases.
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Affiliation(s)
- Yahya I Asiri
- Department of Pharmacology, College of Pharmacy, King Khalid University, Abha, Saudi Arabia
| | - Abdulrhman Alsayari
- Department of Pharmacognosy, College of Pharmacy, King Khalid University, Abha, Saudi Arabia
| | - Abdullatif B Muhsinah
- Department of Pharmacognosy, College of Pharmacy, King Khalid University, Abha, Saudi Arabia
| | - Yahia N Mabkhot
- Department of Pharmaceutical Chemistry, College of Pharmacy, King Khalid University, Abha, Saudi Arabia
| | - Mohd Z Hassan
- Department of Pharmaceutical Chemistry, College of Pharmacy, King Khalid University, Abha, Saudi Arabia
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23
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Sies H, Parnham MJ. Potential therapeutic use of ebselen for COVID-19 and other respiratory viral infections. Free Radic Biol Med 2020; 156:107-112. [PMID: 32598985 PMCID: PMC7319625 DOI: 10.1016/j.freeradbiomed.2020.06.032] [Citation(s) in RCA: 122] [Impact Index Per Article: 24.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 06/03/2020] [Revised: 06/23/2020] [Accepted: 06/24/2020] [Indexed: 12/21/2022]
Abstract
Ebselen is an organoselenium compound exhibiting hydroperoxide- and peroxynitrite-reducing activity, acting as a glutathione peroxidase and peroxiredoxin enzyme mimetic. Ebselen reacts with a multitude of protein thiols, forming a selenosulfide bond, which results in pleiotropic effects of antiviral, antibacterial and anti-inflammatory nature. The main protease (Mpro) of the corona virus SARS-CoV-2 is a potential drug target, and a screen with over 10,000 compounds identified ebselen as a particularly promising inhibitor of Mpro (Jin, Z. et al. (2020) Nature 582, 289-293). We discuss here the reaction of ebselen with cysteine proteases, the role of ebselen in infections with viruses and with other microorganisms. We also discuss effects of ebselen in lung inflammation. In further research on the inhibition of Mpro in SARS-CoV-2, ebselen can serve as a promising lead compound, if the inhibitory effect is confirmed in intact cells in vivo. Independently of this action, potential beneficial effects of ebselen in COVID-19 are ascribed to a number of targets critical to pathogenesis, such as attenuation of inflammatory oxidants and cytokines.
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Affiliation(s)
- Helmut Sies
- Institute of Biochemistry and Molecular Biology I, Heinrich-Heine-University Düsseldorf, Düsseldorf, Germany; Leibniz Research Institute for Environmental Medicine, Heinrich-Heine-University Düsseldorf, Düsseldorf, Germany.
| | - Michael J Parnham
- Faculty of Biochemistry, Chemistry and Pharmacy, JW Goethe University Frankfurt, Frankfurt am Main, Germany; Pharmacology Consultant, Bad Soden am Taunus, Germany.
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24
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Lim SK, Othman R, Yusof R, Heh CH. Rational drug discovery: Ellagic acid as a potent dual-target inhibitor against hepatitis C virus genotype 3 (HCV G3) NS3 enzymes. Chem Biol Drug Des 2020; 97:28-40. [PMID: 32657543 DOI: 10.1111/cbdd.13756] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/15/2019] [Revised: 06/15/2020] [Accepted: 06/28/2020] [Indexed: 12/19/2022]
Abstract
Structure-based virtual screening (SBVS) has served as a popular strategy for rational drug discovery. In this study, we aimed to discover novel benzopyran-based inhibitors that targeted the NS3 enzymes (NS3/4A protease and NS3 helicase) of HCV G3 using a combination of in silico and in vitro approaches. With the aid of SBVS, six novel compounds were discovered to inhibit HCV G3 NS3/4A protease and two phytochemicals (ellagic acid and myricetin) were identified as dual-target inhibitors that inhibited both NS3/4A protease and NS3 helicase in vitro (IC50 = 40.37 ± 5.47 nm and 6.58 ± 0.99 µm, respectively). Inhibitory activities against the replication of HCV G3 replicons were further assessed in a cell-based system with four compounds showed dose-dependent inhibition. Compound P8 was determined to be the most potent compound from the cell-based assay with an EC50 of 19.05 µm. The dual-target inhibitor, ellagic acid, was determined as the second most potent (EC50 = 32.37 µm) and the most selective in its inhibitory activity against the replication of HCV replicons, without severely affecting the viability of the host cells (selectivity index > 6.18).
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Affiliation(s)
- See Khai Lim
- Drug Design and Development Research Group, University of Malaya, Kuala Lumpur, Malaysia.,Department of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Malaya, Kuala Lumpur, Malaysia
| | - Rozana Othman
- Drug Design and Development Research Group, University of Malaya, Kuala Lumpur, Malaysia.,Department of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Malaya, Kuala Lumpur, Malaysia.,Center for Natural Products Research and Drug Discovery (CENAR), University of Malaya, Kuala Lumpur, Malaysia
| | - Rohana Yusof
- Drug Design and Development Research Group, University of Malaya, Kuala Lumpur, Malaysia.,Department of Molecular Medicine, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia
| | - Choon Han Heh
- Drug Design and Development Research Group, University of Malaya, Kuala Lumpur, Malaysia.,Department of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Malaya, Kuala Lumpur, Malaysia
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25
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Tallorin L, Villareal VA, Hsia CY, Rodgers MA, Burri DJ, Pfeil MP, Llopis PM, Lindenbach BD, Yang PL. Hepatitis C virus NS3-4A protease regulates the lipid environment for RNA replication by cleaving host enzyme 24-dehydrocholesterol reductase. J Biol Chem 2020; 295:12426-12436. [PMID: 32641492 PMCID: PMC7458815 DOI: 10.1074/jbc.ra120.013455] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/13/2020] [Revised: 06/29/2020] [Indexed: 12/12/2022] Open
Abstract
Many RNA viruses create specialized membranes for genome replication by manipulating host lipid metabolism and trafficking, but in most cases, we do not know the molecular mechanisms responsible or how specific lipids may impact the associated membrane and viral process. For example, hepatitis C virus (HCV) causes a specific, large-fold increase in the steady-state abundance of intracellular desmosterol, an immediate precursor of cholesterol, resulting in increased fluidity of the membrane where HCV RNA replication occurs. Here, we establish the mechanism responsible for HCV's effect on intracellular desmosterol, whereby the HCV NS3-4A protease controls activity of 24-dehydrocholesterol reductase (DHCR24), the enzyme that catalyzes conversion of desmosterol to cholesterol. Our cumulative evidence for the proposed mechanism includes immunofluorescence microscopy experiments showing co-occurrence of DHCR24 and HCV NS3-4A protease; formation of an additional, faster-migrating DHCR24 species (DHCR24*) in cells harboring a HCV subgenomic replicon RNA or ectopically expressing NS3-4A; and biochemical evidence that NS3-4A cleaves DHCR24 to produce DHCR24* in vitro and in vivo. We further demonstrate that NS3-4A cleaves DHCR24 between residues Cys91 and Thr92 and show that this reduces the intracellular conversion of desmosterol to cholesterol. Together, these studies demonstrate that NS3-4A directly cleaves DHCR24 and that this results in the enrichment of desmosterol in the membranes where NS3-4A and DHCR24 co-occur. Overall, this suggests a model in which HCV directly regulates the lipid environment for RNA replication through direct effects on the host lipid metabolism.
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Affiliation(s)
- Lorillee Tallorin
- Department of Microbiology and Blavatnik Institute, Harvard Medical School, Boston, Massachusetts, USA
| | - Valerie A Villareal
- Department of Microbiology and Blavatnik Institute, Harvard Medical School, Boston, Massachusetts, USA
| | - Chih-Yun Hsia
- Department of Microbiology and Blavatnik Institute, Harvard Medical School, Boston, Massachusetts, USA
| | - Mary A Rodgers
- Department of Microbiology and Blavatnik Institute, Harvard Medical School, Boston, Massachusetts, USA
| | - Dominique J Burri
- Department of Microbiology and Blavatnik Institute, Harvard Medical School, Boston, Massachusetts, USA
| | - Marc-Philipp Pfeil
- Department of Microbiology and Blavatnik Institute, Harvard Medical School, Boston, Massachusetts, USA
| | - Paula Montero Llopis
- Department of Microbiology and Blavatnik Institute, Harvard Medical School, Boston, Massachusetts, USA
| | - Brett D Lindenbach
- Department of Microbial Pathogenesis, Yale Medical School, New Haven, Connecticut, USA
| | - Priscilla L Yang
- Department of Microbiology and Blavatnik Institute, Harvard Medical School, Boston, Massachusetts, USA
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26
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Davidson RB, Hendrix J, Geiss BJ, McCullagh M. RNA-Dependent Structures of the RNA-Binding Loop in the Flavivirus NS3 Helicase. J Phys Chem B 2020; 124:2371-2381. [PMID: 32105483 DOI: 10.1021/acs.jpcb.0c00457] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/28/2022]
Abstract
The flavivirus NS3 protein is a helicase that has pivotal functions during the viral genome replication process, where it unwinds double-stranded RNA and translocates along the nucleic acid polymer in a nucleoside triphosphate hydrolysis-dependent mechanism. Crystallographic and computational studies of the flavivirus NS3 helicase have identified the RNA-binding loop as an interesting structural element that may function as a component of the RNA-enhanced NTPase activity observed for this family of helicases. Microsecond-long unbiased molecular dynamics and extensive replica exchange umbrella sampling simulations of the Zika NS3 helicase have been performed to investigate the RNA dependence of this loop's structural conformations. Specifically, the effect of the bound single-stranded RNA (ssRNA) oligomer on the putative "open" and "closed" conformations of this loop is studied. In the Apo substrate state, the two loop conformations are nearly isoergonic (ΔAO→C = -0.22 kcal mol-1), explaining the structural ambiguity observed in Apo NS3h crystal structures. The bound ssRNA is seen to stabilize the "open" conformation (ΔAO→C = 1.97 kcal mol-1) through direct protein-RNA interactions at the top of the loop. Interestingly, a small ssRNA oligomer bound over 13 Å away from the loop is seen to affect the free energy surface to favor the "open" structure, while minimizing barriers between the two states. Both the mechanism of the "open" to "closed" transition and important residues of the RNA-binding loop structures are characterized. From these results, point mutations that are hypothesized to stabilize the "closed" RNA-binding loop and negatively impact RNA-binding and the RNA-enhanced NTPase activity are posited.
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Affiliation(s)
- Russell B Davidson
- Department of Chemistry, Colorado State University, Fort Collins 80523, Colorado, United States
| | - Josie Hendrix
- Department of Chemistry, Colorado State University, Fort Collins 80523, Colorado, United States
| | - Brian J Geiss
- Department of Microbiology, Immunology, and Pathology, Colorado State University, Fort Collins 80523, Colorado, United States
- School of Biomedical Engineering, Colorado State University, Fort Collins 80523, Colorado, United States
| | - Martin McCullagh
- Department of Chemistry, Oklahoma State University, Stillwater, Oklahoma 74074, United States
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Ablenas CJ, Gidi Y, Powdrill MH, Ahmed N, Shaw TA, Mesko M, Götte M, Cosa G, Pezacki JP. Hepatitis C Virus Helicase Binding Activity Monitored through Site-Specific Labeling Using an Expanded Genetic Code. ACS Infect Dis 2019; 5:2118-2126. [PMID: 31640339 DOI: 10.1021/acsinfecdis.9b00220] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/28/2022]
Abstract
The mechanism of unwinding catalyzed by the hepatitis C virus nonstructural protein 3 helicase (NS3h) has been a subject of considerable interest, with NS3h serving as a prototypical enzyme in the study of helicase function. Recent studies support an ATP-fueled, inchworm-like stepping of NS3h on the nucleic acid that would result in the displacement of the complementary strand of the duplex during unwinding. Here, we describe the screening of a site of incorporation of an unnatural amino acid in NS3h for fluorescent labeling of the enzyme to be used in single-molecule Förster resonance energy transfer (FRET) experiments. From the nine potential sites identified in NS3h for incorporation of the unnatural amino acid, only one allowed for expression and fluorescent labeling of the recombinant protein. Incorporation of the unnatural amino acid was confirmed via bulk assays to not interfere with unwinding activity of the helicase. Binding to four different dsDNA sequences bearing a ssDNA overhang segment of varying length (either minimal 6 or 7 base length overhang to ensure binding or a long 24 base overhang) and sequence was recorded with the new NS3h construct at the single-molecule level. Single-molecule fluorescence displayed time intervals with anticorrelated donor and acceptor emission fluctuations associated with protein binding to the substrates. An apparent FRET value was estimated from the binding events showing a single FRET value of ∼0.8 for the 6-7 base overhangs. A smaller mean value and a broad distribution was in turn recorded for the long ssDNA overhang, consistent with NS3h exploring a larger physical space while bound to the DNA construct. Notably, intervals where NS3h binding was recorded were exhibited at time periods where the acceptor dye reversibly bleached. Protein induced fluorescence intensity enhancement in the donor channel became apparent at these intervals. Overall, the site-specific fluorescent labeling of NS3h reported here provides a powerful tool for future studies to monitor the dynamics of enzyme translocation during unwinding by single-molecule FRET.
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Affiliation(s)
- Christopher J. Ablenas
- Department of Biochemistry, McGill University, Montreal, Quebec H3G1Y6, Canada
- Department of Chemistry and Biomolecular Sciences, University of Ottawa, Ottawa, Ontario K1N6N5, Canada
| | - Yasser Gidi
- Department of Chemistry, McGill University, Montreal, Quebec H3A0B8, Canada
| | - Megan H. Powdrill
- Department of Chemistry and Biomolecular Sciences, University of Ottawa, Ottawa, Ontario K1N6N5, Canada
| | - Noreen Ahmed
- Department of Chemistry and Biomolecular Sciences, University of Ottawa, Ottawa, Ontario K1N6N5, Canada
| | - Tyler A. Shaw
- Department of Chemistry and Biomolecular Sciences, University of Ottawa, Ottawa, Ontario K1N6N5, Canada
| | - Mihai Mesko
- Department of Chemistry, McGill University, Montreal, Quebec H3A0B8, Canada
| | - Matthias Götte
- Department of Medical Microbiology and Immunology, University of Alberta, Edmonton, Alberta T6G2R7, Canada
| | - Gonzalo Cosa
- Department of Chemistry, McGill University, Montreal, Quebec H3A0B8, Canada
| | - John Paul Pezacki
- Department of Chemistry and Biomolecular Sciences, University of Ottawa, Ottawa, Ontario K1N6N5, Canada
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Mohapatra S, Lin CT, Feng XA, Basu A, Ha T. Single-Molecule Analysis and Engineering of DNA Motors. Chem Rev 2019; 120:36-78. [DOI: 10.1021/acs.chemrev.9b00361] [Citation(s) in RCA: 37] [Impact Index Per Article: 6.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/28/2022]
Affiliation(s)
| | | | | | | | - Taekjip Ha
- Howard Hughes Medical Institute, Baltimore, Maryland 21205, United States
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29
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Nazario de Moraes L, Tommasini Grotto RM, Targino Valente G, de Carvalho Sampaio H, Magro AJ, Fogaça L, Wolf IR, Perahia D, Faria Silva G, Plana Simões R. A novel molecular mechanism to explain mutations of the HCV protease associated with resistance against covalently bound inhibitors. Virus Res 2019; 274:197778. [PMID: 31618615 DOI: 10.1016/j.virusres.2019.197778] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/29/2019] [Revised: 10/07/2019] [Accepted: 10/08/2019] [Indexed: 02/07/2023]
Abstract
NS3 is an important therapeutic target for direct-acting antiviral (DAA) drugs. However, many patients treated with DAAs have unsustained virologic response (UVR) due to the high mutation rate of HCV. The aim of this work was to shed some light on the puzzling molecular mechanisms of the virus's of patients who showed high viral loads even under treatment with DAA. Bioinformatics tools, molecular modelling analyses were employed to identify mutations associated with HCV resistance to boceprevir and possible structural features related to this phenomenon. We identified two mutations of NS3 that may be associated with HCV resistance: D168N and L153I. The substitution D168N was previously reported in the literature as related with drug failure. Additionally, we identified that its molecular resistance mechanism can be explained by the destabilization of receptor-ligand hydrogen bonds. For the L153I mutation, the resistance mechanism is different from previous models reported in the literature. The L153I substitution decreases the S139 deprotonation susceptibility, and consequently, this mutation impairs the covalent binding between the residue S139 from NS3 and the electrophilic trap on boceprevir, which can induce drug failure. These results were supported by the time course analysis of the mutations of the NS3 protease, which showed that boceprevir was designed for enzymes with an L residue at position 153; however, the sequences with I153 are predominant nowadays. The results presented here could be used to infer about resistance in others DAA, mainly protease inhibitors.
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Affiliation(s)
- Leonardo Nazario de Moraes
- Sao Paulo State University (UNESP), School of Agriculture, Department of Bioprocess and Biotechnology, Avenue Universitária, 3780, Botucatu, SP, Brazil
| | - Rejane Maria Tommasini Grotto
- Sao Paulo State University (UNESP), School of Agriculture, Department of Bioprocess and Biotechnology, Avenue Universitária, 3780, Botucatu, SP, Brazil; Sao Paulo State University (UNESP), Medical School, Blood Center, Avenue Prof. Mário Rubens Guimarães Montenegro, s/n, Botucatu, SP, Brazil
| | - Guilherme Targino Valente
- Sao Paulo State University (UNESP), School of Agriculture, Department of Bioprocess and Biotechnology, Avenue Universitária, 3780, Botucatu, SP, Brazil; Max Planck Institut for Heart and Lung Research, Ludwigstraße 43, 61231, Bad Nauheim, Germany
| | - Heloisa de Carvalho Sampaio
- Sao Paulo State University (UNESP), Medical School, Blood Center, Avenue Prof. Mário Rubens Guimarães Montenegro, s/n, Botucatu, SP, Brazil
| | - Angelo José Magro
- Sao Paulo State University (UNESP), School of Agriculture, Department of Bioprocess and Biotechnology, Avenue Universitária, 3780, Botucatu, SP, Brazil; Sao Paulo State University (UNESP), Medical School, Blood Center, Avenue Prof. Mário Rubens Guimarães Montenegro, s/n, Botucatu, SP, Brazil; Sao Paulo State University (UNESP), Institute of Biosciences, Street Prof. Dr. Antônio Celso Wagner Zanin, 250, Botucatu, SP, Brazil
| | - Lauana Fogaça
- Sao Paulo State University (UNESP), School of Agriculture, Department of Bioprocess and Biotechnology, Avenue Universitária, 3780, Botucatu, SP, Brazil; Sao Paulo State University (UNESP), Institute of Biosciences, Street Prof. Dr. Antônio Celso Wagner Zanin, 250, Botucatu, SP, Brazil
| | - Ivan Rodrigo Wolf
- Sao Paulo State University (UNESP), Institute of Biosciences, Street Prof. Dr. Antônio Celso Wagner Zanin, 250, Botucatu, SP, Brazil
| | - David Perahia
- École Normale Supérieure Paris-Saclay, Laboratory of Biology and Applied Pharmacology, Cachan, 94235, France
| | - Giovanni Faria Silva
- Sao Paulo State University (UNESP), Medical School, Blood Center, Avenue Prof. Mário Rubens Guimarães Montenegro, s/n, Botucatu, SP, Brazil
| | - Rafael Plana Simões
- Sao Paulo State University (UNESP), School of Agriculture, Department of Bioprocess and Biotechnology, Avenue Universitária, 3780, Botucatu, SP, Brazil; Sao Paulo State University (UNESP), Medical School, Blood Center, Avenue Prof. Mário Rubens Guimarães Montenegro, s/n, Botucatu, SP, Brazil.
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30
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Ren J, Ojeda I, Patel M, Johnson ME, Lee H. Exploring small molecules with pan-genotypic inhibitory activities against hepatitis C virus NS3/4A serine protease. Bioorg Med Chem Lett 2019; 29:2349-2353. [PMID: 31201062 DOI: 10.1016/j.bmcl.2019.06.009] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/02/2019] [Revised: 06/05/2019] [Accepted: 06/06/2019] [Indexed: 01/03/2023]
Abstract
Among the many Hepatitis C virus (HCV) genotypes and subtypes, genotypes 1b and 3a are most prevalent in United States and Asia, respectively. A total of 132 commercially available analogs of a previous lead compound were initially investigated against wild-type HCV genotype 1b NS3/4A protease. Ten compounds showed inhibitory activities (IC50 values) below 10 µM with comparable direct binding affinities (KD values) determined by surface plasmon resonance (SPR). To identify pan-genotypic inhibitors, these ten selected compounds were tested against four additional genotypes (1a, 2a, 3a, and 4) and three drug-resistant mutants (A156S, R155K, and V36M). Four new analogs have been identified with better activities against all five tested genotypes than the prior lead compound. Further, the original lead compound did not show activity against genotype 3a NS3/4A, whereas four newly identified compounds exhibited IC50 values below 33 µM against genotype 3a NS3/4A. Encouragingly, the best new compound F1813-0710 possessed promising activity toward genotype 3a, which is a huge improvement over the previous lead compound that had no effect on genotype 3a. This intriguing observation was further analyzed by molecular docking and molecular dynamics (MD) simulations to understand their different binding interactions, which should benefit future pan-genotypic inhibitor design and drug discovery.
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Affiliation(s)
- Jinhong Ren
- Center for Biomolecular Sciences, University of Illinois at Chicago, 900 S. Ashland Ave, Chicago, IL 60607, USA
| | - Isabel Ojeda
- Center for Biomolecular Sciences, University of Illinois at Chicago, 900 S. Ashland Ave, Chicago, IL 60607, USA
| | - Maulik Patel
- Center for Biomolecular Sciences, University of Illinois at Chicago, 900 S. Ashland Ave, Chicago, IL 60607, USA
| | - Michael E Johnson
- Center for Biomolecular Sciences, University of Illinois at Chicago, 900 S. Ashland Ave, Chicago, IL 60607, USA; Department of Medicinal Chemistry and Pharmacognosy, University of Illinois at Chicago, 833 S. Wood Street, IL 60612, USA.
| | - Hyun Lee
- Center for Biomolecular Sciences, University of Illinois at Chicago, 900 S. Ashland Ave, Chicago, IL 60607, USA; Biophysics Core at Research Resource Center, University of Illinois at Chicago, Chicago, IL 60612, USA; Department of Medicinal Chemistry and Pharmacognosy, University of Illinois at Chicago, 833 S. Wood Street, IL 60612, USA.
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31
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Alizadeh S, Irani S, Bolhassani A, Sadat SM. Simultaneous use of natural adjuvants and cell penetrating peptides improves HCV NS3 antigen-specific immune responses. Immunol Lett 2019; 212:70-80. [PMID: 31254535 DOI: 10.1016/j.imlet.2019.06.011] [Citation(s) in RCA: 15] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/12/2019] [Revised: 06/11/2019] [Accepted: 06/25/2019] [Indexed: 02/07/2023]
Abstract
To improve an effective hepatitis C virus (HCV) therapeutic vaccine, induction of a strong and long term HCV antigen-specific immune response is an important parameter. HCV non-structural protein 3 (NS3) has antigenic properties and plays a major role in viral clearance. In this study, DNA constructs encoding HCV NS3 and heat shock protein 27 (Hsp27)-NS3 genes, and the recombinant (r) NS3 and rHsp27-NS3 proteins complexed with HR9 and Cady-2 cell penetrating peptides (CPPs) were utilized to evaluate antibody, cytokine and Granzyme B secretion in mice. Herein, the formation of NS3 and Hsp27-NS3 DNA/ HR9 CPP complexes were revealed by gel retardation assay and protection against DNase and protease. Cady-2 peptide was used to form the nanoparticles with rNS3 and rHsp27-NS3 proteins. The size and charge of the nanoparticles were confirmed by SEM and Zetasizer instruments. Next, in vitro transfection of the nanoparticles was assessed by flow cytometry and western blotting. Finally, humoral and cellular immune responses were evaluated using different modalities in mice. Our data showed that HR9 and Cady-2 could form stable nanoparticles with DNA and proteins, respectively and enhance their delivery into HEK-293 T cells in a non-covalent approach. Furthermore, the heterologous Hsp27-NS3 DNA + HR9 prime/rHsp27-NS3+Cady-2 protein boost elicited a higher Th1 cellular immune response with a predominant IgG2a, IgG2b, IFN-γ profile and strong Granzyme B secretion than those induced by other groups. Briefly, the combination of a natural adjuvant (Hsp27) and CPPs (HR9 and Cady-2) could significantly stimulate effective immune responses as a promising approach for development of HCV therapeutic vaccines.
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Affiliation(s)
- Sina Alizadeh
- Department of Biology, School of Basic Sciences, Science and Research Branch, Islamic Azad University, Tehran, Iran
| | - Shiva Irani
- Department of Biology, School of Basic Sciences, Science and Research Branch, Islamic Azad University, Tehran, Iran
| | - Azam Bolhassani
- Department of Hepatitis and AIDS, Pasteur Institute of Iran, Tehran, Iran.
| | - Seyed Mehdi Sadat
- Department of Hepatitis and AIDS, Pasteur Institute of Iran, Tehran, Iran
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32
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Venkatesan A, Prabhu Dass J F. Review on chemogenomic approaches towards hepatitis C viral targets. J Cell Biochem 2019; 120:12167-12181. [PMID: 30887580 DOI: 10.1002/jcb.28581] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/09/2018] [Revised: 01/09/2019] [Accepted: 01/14/2019] [Indexed: 12/18/2022]
Abstract
Hepatitis C virus (HCV) is the most prevalent viral pathogen that infects more than 185 million people worldwide. HCV infection leads to chronic liver diseases such as liver cirrhosis and hepatocellular carcinoma. Direct-acting antivirals (DAAs) are the recent combination therapy for HCV infection with reduced side effects than prior therapies. Sustained virological response (SVR) acts as a gold standard marker to monitor the success of antiviral treatment. Older treatment therapies attain 50-55% of SVR compared with DAAs which attain around 90-95%. The current review emphasizes the recent chemogenomic updates that have been unfolded through structure-based drug design of HCV drug target proteins (NS3/4A, NS5A, and NS5B) and ligand-based drug design of DAAs in achieving a stable HCV viral treatment strategies.
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Affiliation(s)
- Arthi Venkatesan
- Department of Integrative Biology, School of BioSciences and Technology (SBST), VIT, Vellore, Tamil Nadu, India
| | - Febin Prabhu Dass J
- Department of Integrative Biology, School of BioSciences and Technology (SBST), VIT, Vellore, Tamil Nadu, India
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Ezat AA, Elshemey WM. A comparative study of the efficiency of HCV NS3/4A protease drugs against different HCV genotypes using in silico approaches. Life Sci 2018; 217:176-184. [PMID: 30528183 DOI: 10.1016/j.lfs.2018.12.004] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/19/2018] [Revised: 11/21/2018] [Accepted: 12/03/2018] [Indexed: 02/06/2023]
Abstract
AIMS To investigate the efficacy of Direct Acting Antivirals (DAAs) in the treatment of different Hepatitis C Virus (HCV) genotypes. MAIN METHODS Homology modeling is used to predict the 3D structures of different genotypes while molecular docking is employed to predict genotype - drug interactions (Binding Mode) and binding free energy (Docking Score). KEY FINDINGS Simeprevir (TMC435) and to a lesser degree MK6325 are the best drugs among the studied drugs. The predicted affinity of drugs against genotype 1a is always better than other genotypes. P2-P4 macrocyclic drugs show better performance against genotypes 2, 3 and 5. Macrocyclic drugs are better than linear drugs. SIGNIFICANCE HCV is one of the major health problems worldwide. Until the discovery of DAAs, HCV treatment faced many failures. DAAs target key functional machines of the virus life cycle and shut it down. NS3/4A protease is an important target and several drugs have been designed to inhibit its functions. There are several NS3/4A protease drugs approved by Food and Drug Administration (FDA). Unfortunately, the virus exhibits resistance against these drugs. This study is significant in elucidating that no one drug is able to treat different genotypes with the same efficiency. Therefore, treatment should be prescribed based on the HCV genotype.
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Affiliation(s)
- Ahmed A Ezat
- Biophysics Department, Faculty of Science, Cairo University, 12613 Giza, Egypt.
| | - Wael M Elshemey
- Biophysics Department, Faculty of Science, Cairo University, 12613 Giza, Egypt
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Ren X, Zou L, Lu J, Holmgren A. Selenocysteine in mammalian thioredoxin reductase and application of ebselen as a therapeutic. Free Radic Biol Med 2018; 127:238-247. [PMID: 29807162 DOI: 10.1016/j.freeradbiomed.2018.05.081] [Citation(s) in RCA: 97] [Impact Index Per Article: 13.9] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 03/09/2018] [Revised: 04/27/2018] [Accepted: 05/23/2018] [Indexed: 12/29/2022]
Abstract
Thioredoxin system is a ubiquitous disulfide reductase system evolutionarily conserved through all living organisms. It contains thioredoxin (Trx), thioredoxin reductase (TrxR) and NADPH. TrxR can use NADPH to reduce Trx which passes the reducing equivalent to its downstream substrates involved in various biomedical events, such as ribonucleotide reductase for deoxyribonucleotide and DNA synthesis, or peroxiredoxins for counteracting oxidative stress. Obviously, TrxR stays in the center of the system to maintain the electron flow. Mammalian TrxR contains a selenocysteine (Sec) in its active site, which is not present in the low molecular weight prokaryotic TrxRs. Due to the special property of Sec, mammalian TrxR employs a different catalytic mechanism from prokaryotic TrxRs and has a broader substrate-spectrum. On the other hand, Sec is easily targeted by electrophilic compounds which inhibits the TrxR activity and may turn TrxR into an NADPH oxidase. Ebselen, a synthetic seleno-compound containing selenazol, has been tested in several clinical studies. In mammalian cells, ebselen works as a GSH peroxidase mimic and mainly as a peroxiredoxin mimic via Trx and TrxR to scavenge hydrogen peroxide and peroxynitrite. In prokaryotic cells, ebselen is an inhibitor of TrxR and leads to elevation of reactive oxygen species (ROS). Recent studies have made use of the difference and developed ebselen as a potential antibiotic, especially in combination with silver which enables ebselen to kill multi-drug resistant Gram-negative bacteria. Collectively, Sec is important for the biological functions of mammalian TrxR and distinguishes it from prokaryotic TrxRs, therefore it is a promising drug target.
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Affiliation(s)
- Xiaoyuan Ren
- Division of Biochemistry, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, SE-171 77 Stockholm, Sweden
| | - Lili Zou
- Division of Biochemistry, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, SE-171 77 Stockholm, Sweden; Translational Neuroscience & Neural Regeneration and Repair Institute/Institute of Cell Therapy, The First Hospital of Yichang, Three Gorges University, 443000 Yichang, China
| | - Jun Lu
- School of Pharmaceutical Sciences, Southwest University, 400715 Chongqing, China
| | - Arne Holmgren
- Division of Biochemistry, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, SE-171 77 Stockholm, Sweden.
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Xi Y, Zhang Y, Fang J, Whittaker K, Luo S, Huang RP. Prokaryotic Expression of Hepatitis C Virus-NS3 Protein and Preparation of a Monoclonal Antibody. Monoclon Antib Immunodiagn Immunother 2018; 36:251-258. [PMID: 29267148 DOI: 10.1089/mab.2017.0033] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/11/2022] Open
Abstract
Hepatitis C virus (HCV) is a significant health threat that has been extensively investigated worldwide. Improving the sensitivity and specificity of laboratory tests for screening and early diagnosis of HCV in a relevant population is an effective measure to control the spread of HCV. To build a more reliable diagnostic method for HCV, we expressed gene fragments of HCV-NS3 linked to a carrier, pET28a, and then transformed this vector into Escherichia. coli. The produced recombinant NS3 protein with a molecular weight of 38 kDa, which was purified through Ni-chelating affinity chromatography, was used to immunize BALB/C mice, which generated a serum antibody titer of 1:160,000 against the immunogen. Three positive monoclonal isolates (2A5, 2A6, and 5B12) were screened and established. Western blot and enzyme-linked immunosorbent assay (ELISA) results of these monoclonal cells show that each could specifically recognize the recombinant protein. Antibodies 2A5 and 2A6 were developed into an ELISA sandwich antibody pair for the recombinant protein. The detection sensitivity of our developed ELISA was 1.6 ng/mL, with a linear range of 2.5-80 ng/mL (R2 = 0.998). Serum NS3 ELISA results show that the average value in the healthy group, liver disease group, and hepatitis C group was 3.71, 7.28, and 13.11 ng/mL, respectively. The positive rates of HCV-NS3 protein in the liver disease group and hepatitis C group was 17.2% and 41.7%, respectively. Detection of HCV-NS3 antigen can be used as an auxiliary test for anti-HCV antibody detection, thus reducing leakage detection and providing a reliable basis for clinical practice.
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Affiliation(s)
- Yun Xi
- 1 Department of Laboratory Medicine, The Third Affiliated Hospital of Sun Yat-sen University , Guangzhou, China
| | | | - Jianmin Fang
- 2 Raybiotech, Inc. , Guangzhou, China .,3 RayBiotech, Inc. , Norcross, Georgia .,4 South China Biochip Research Center , Guangzhou, China
| | | | - Shuhong Luo
- 2 Raybiotech, Inc. , Guangzhou, China .,3 RayBiotech, Inc. , Norcross, Georgia .,4 South China Biochip Research Center , Guangzhou, China
| | - Ruo-Pan Huang
- 2 Raybiotech, Inc. , Guangzhou, China .,3 RayBiotech, Inc. , Norcross, Georgia .,4 South China Biochip Research Center , Guangzhou, China
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Vázquez-Ulloa E, Lizano M, Sjöqvist M, Olmedo-Nieva L, Contreras-Paredes A. Deregulation of the Notch pathway as a common road in viral carcinogenesis. Rev Med Virol 2018; 28:e1988. [PMID: 29956408 DOI: 10.1002/rmv.1988] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/28/2017] [Revised: 03/27/2018] [Accepted: 05/23/2018] [Indexed: 12/15/2022]
Abstract
The Notch pathway is a conserved signaling pathway and a form of direct cell-cell communication related to many biological processes during development and adulthood. Deregulation of the Notch pathway is involved in many diseases, including cancer. Almost 20% of all cancer cases have an infectious etiology, with viruses responsible for at least 1.5 million new cancer cases per year. Seven groups of viruses have been classified as oncogenic: hepatitis B and C viruses (HBV and HCV respectively), Epstein-Barr virus (EBV), Kaposi sarcoma-associated herpesvirus (KSHV), human T lymphotropic virus (HTLV-1), human papillomavirus (HPV), and Merkel cell polyomavirus (MCPyV). These viruses share the ability to manipulate a variety of cell pathways that are critical in proliferation and differentiation, leading to malignant transformation. Viral proteins interact directly or indirectly with different members of the Notch pathway, altering their normal function. This review focuses exclusively on the direct interactions of viral oncoproteins with Notch elements, providing a deeper understanding of the dual behavior of the Notch pathway as activator or suppressor of neoplasia in virus-related cancers.
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Affiliation(s)
- Elenaé Vázquez-Ulloa
- Programa de Maestría y Doctorado en Ciencias Bioquímicas, Facultad de Química, Universidad Nacional Autónoma de México, Mexico City, Mexico.,Tecnológico Nacional de México, Instituto Tecnológico de Gustavo A. Madero, Mexico City, Mexico
| | - Marcela Lizano
- Unidad de Investigación Biomédica en Cáncer, Instituto Nacional de Cancerología-Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, Mexico City, Mexico
| | - Marika Sjöqvist
- Faculty of Science and Engineering, Biosciences, Åbo Akademi University, Turku, Finland
| | - Leslie Olmedo-Nieva
- Programa de Maestría y Doctorado en Ciencias Bioquímicas, Facultad de Química, Universidad Nacional Autónoma de México, Mexico City, Mexico
| | - Adriana Contreras-Paredes
- Unidad de Investigación Biomédica en Cáncer, Instituto Nacional de Cancerología-Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, Mexico City, Mexico
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Lemos MAN, Patiño SFS, Bernardino TC, Coroadinha AS, Soares H, Astray RM, Pereira CA, Jorge SAC. Intracellular Delivery of HCV NS3p gene using vectored particles. J Biotechnol 2018; 274:33-39. [PMID: 29577966 DOI: 10.1016/j.jbiotec.2018.03.010] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/08/2018] [Accepted: 03/18/2018] [Indexed: 02/07/2023]
Abstract
Viral hepatitis caused by the hepatitis C virus (HCV) affects millions of people worldwide. The non-structural protein 3 (NS3), one of the most conserved proteins in HCV, is the target of many therapeutic studies. The NS3 protease domain (NS3p) has a range of cytotoxic T lymphocyte (CTL) epitopes, and synthesizing the protein inside the cells is the most appropriate way to present it to the immune system. We developed a tool to study this kind of presentation, using two vectored particle (VP) systems, one based on the Semliki Forest virus (SFV) and the other on HCV pseudoparticles (HCVpp), both carrying the protease domain of the NS3 gene. In addition to producing the particles, we developed a method to quantify these VPs using qRT-PCR. We produced batches of approximately 2.4 × 104 SFV-NS3p/μL and 4.0 × 102 HCVpp-NS3p/μL. BHK-21 and HuH-7 cells treated with the VPs expressed the NS3 protein, thus showing the functionality of this system.
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Affiliation(s)
| | | | | | - Ana Sofia Coroadinha
- Cell Line Development and Molecular Biotechnology Laboratory, iBET - Instituto de Biologia Experimental e Tecnológica, Av. República, Qta. do Marquês, Oeiras Portugal
| | - Hugo Soares
- Cell Line Development and Molecular Biotechnology Laboratory, iBET - Instituto de Biologia Experimental e Tecnológica, Av. República, Qta. do Marquês, Oeiras Portugal
| | - Renato Mancini Astray
- Laboratório de Imunologia Viral, Instituto Butantan, Avenida Vital Brasil, 1500, São Paulo Brazil
| | - Carlos Augusto Pereira
- Laboratório de Imunologia Viral, Instituto Butantan, Avenida Vital Brasil, 1500, São Paulo Brazil
| | - Soraia Attie Calil Jorge
- Laboratório de Imunologia Viral, Instituto Butantan, Avenida Vital Brasil, 1500, São Paulo Brazil.
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Davidson RB, Hendrix J, Geiss BJ, McCullagh M. Allostery in the dengue virus NS3 helicase: Insights into the NTPase cycle from molecular simulations. PLoS Comput Biol 2018; 14:e1006103. [PMID: 29659571 PMCID: PMC5919694 DOI: 10.1371/journal.pcbi.1006103] [Citation(s) in RCA: 30] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/03/2017] [Revised: 04/26/2018] [Accepted: 03/22/2018] [Indexed: 12/29/2022] Open
Abstract
The C-terminus domain of non-structural 3 (NS3) protein of the Flaviviridae viruses (e.g. HCV, dengue, West Nile, Zika) is a nucleotide triphosphatase (NTPase) -dependent superfamily 2 (SF2) helicase that unwinds double-stranded RNA while translocating along the nucleic polymer. Due to these functions, NS3 is an important target for antiviral development yet the biophysics of this enzyme are poorly understood. Microsecond-long molecular dynamic simulations of the dengue NS3 helicase domain are reported from which allosteric effects of RNA and NTPase substrates are observed. The presence of a bound single-stranded RNA catalytically enhances the phosphate hydrolysis reaction by affecting the dynamics and positioning of waters within the hydrolysis active site. Coupled with results from the simulations, electronic structure calculations of the reaction are used to quantify this enhancement to be a 150-fold increase, in qualitative agreement with the experimental enhancement factor of 10–100. Additionally, protein-RNA interactions exhibit NTPase substrate-induced allostery, where the presence of a nucleotide (e.g. ATP or ADP) structurally perturbs residues in direct contact with the phosphodiester backbone of the RNA. Residue-residue network analyses highlight pathways of short ranged interactions that connect the two active sites. These analyses identify motif V as a highly connected region of protein structure through which energy released from either active site is hypothesized to move, thereby inducing the observed allosteric effects. These results lay the foundation for the design of novel allosteric inhibitors of NS3. Non-structural protein 3 (NS3) is a Flaviviridae (e.g. Hepatitis C, dengue, and Zika viruses) helicase that unwinds double stranded RNA while translocating along the nucleic polymer during viral genome replication. As a member of superfamily 2 (SF2) helicases, NS3 utilizes the free energy of nucleotide triphosphate (NTP) binding, hydrolysis, and product unbinding to perform its functions. While much is known about SF2 helicases, the pathways and mechanisms through which free energy is transduced between the NTP hydrolysis active site and RNA binding cleft remains elusive. Here we present a multiscale computational study to characterize the allosteric effects induced by the RNA and NTPase substrates (ATP, ADP, and Pi) as well as the pathways of short-range, residue-residue interactions that connect the two active sites. Results from this body of molecular dynamics simulations and electronic structure calculations are highlighted in context to the NTPase enzymatic cycle, allowing for development of testable hypotheses for validation of these simulations. Our insights, therefore, provide novel details about the biophysics of NS3 and guide the next generation of experimental studies.
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Affiliation(s)
- Russell B. Davidson
- Department of Chemistry, Colorado State University, Fort Collins, Colorado, United States of America
| | - Josie Hendrix
- Department of Chemistry, Colorado State University, Fort Collins, Colorado, United States of America
| | - Brian J. Geiss
- Department of Microbiology, Immunology, and Pathology, Colorado State University, Fort Collins, Colorado, United States of America
- School of Biomedical Engineering, Colorado State University, Fort Collins, Colorado, United States of America
| | - Martin McCullagh
- Department of Chemistry, Colorado State University, Fort Collins, Colorado, United States of America
- * E-mail:
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The discovery and optimization of naphthalene-linked P2-P4 Macrocycles as inhibitors of HCV NS3 protease. Bioorg Med Chem Lett 2018; 28:43-48. [DOI: 10.1016/j.bmcl.2017.11.005] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/02/2017] [Revised: 10/29/2017] [Accepted: 11/02/2017] [Indexed: 12/18/2022]
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Bessa LM, Launay H, Dujardin M, Cantrelle FX, Lippens G, Landrieu I, Schneider R, Hanoulle X. NMR reveals the intrinsically disordered domain 2 of NS5A protein as an allosteric regulator of the hepatitis C virus RNA polymerase NS5B. J Biol Chem 2017; 292:18024-18043. [PMID: 28912275 DOI: 10.1074/jbc.m117.813766] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/21/2017] [Revised: 09/06/2017] [Indexed: 12/13/2022] Open
Abstract
Non-structural protein 5B (NS5B) is the RNA-dependent RNA polymerase that catalyzes replication of the hepatitis C virus (HCV) RNA genome and therefore is central for its life cycle. NS5B interacts with the intrinsically disordered domain 2 of NS5A (NS5A-D2), another essential multifunctional HCV protein that is required for RNA replication. As a result, these two proteins represent important targets for anti-HCV chemotherapies. Despite this importance and the existence of NS5B crystal structures, our understanding of the conformational and dynamic behavior of NS5B in solution and its relationship with NS5A-D2 remains incomplete. To address these points, we report the first detailed NMR spectroscopic study of HCV NS5B lacking its membrane anchor (NS5BΔ21). Analysis of constructs with selective isotope labeling of the δ1 methyl groups of isoleucine side chains demonstrates that, in solution, NS5BΔ21 is highly dynamic but predominantly adopts a closed conformation. The addition of NS5A-D2 leads to spectral changes indicative of binding to both allosteric thumb sites I and II of NS5BΔ21 and induces long-range perturbations that affect the RNA-binding properties of the polymerase. We compared these modifications with the short- and long-range effects triggered in NS5BΔ21 upon binding of filibuvir, an allosteric inhibitor. We demonstrate that filibuvir-bound NS5BΔ21 is strongly impaired in the binding of both NS5A-D2 and RNA. NS5A-D2 induces conformational and functional perturbations in NS5B similar to those triggered by filibuvir. Thus, our work highlights NS5A-D2 as an allosteric regulator of the HCV polymerase and provides new insight into the dynamics of NS5B in solution.
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Affiliation(s)
- Luiza M Bessa
- From the University of Lille, CNRS, UMR 8576, UGSF, Unité de Glycobiologie Structurale et Fonctionnelle, 59000 Lille, France
| | - Hélène Launay
- From the University of Lille, CNRS, UMR 8576, UGSF, Unité de Glycobiologie Structurale et Fonctionnelle, 59000 Lille, France
| | - Marie Dujardin
- From the University of Lille, CNRS, UMR 8576, UGSF, Unité de Glycobiologie Structurale et Fonctionnelle, 59000 Lille, France
| | - François-Xavier Cantrelle
- From the University of Lille, CNRS, UMR 8576, UGSF, Unité de Glycobiologie Structurale et Fonctionnelle, 59000 Lille, France
| | - Guy Lippens
- From the University of Lille, CNRS, UMR 8576, UGSF, Unité de Glycobiologie Structurale et Fonctionnelle, 59000 Lille, France
| | - Isabelle Landrieu
- From the University of Lille, CNRS, UMR 8576, UGSF, Unité de Glycobiologie Structurale et Fonctionnelle, 59000 Lille, France
| | - Robert Schneider
- From the University of Lille, CNRS, UMR 8576, UGSF, Unité de Glycobiologie Structurale et Fonctionnelle, 59000 Lille, France
| | - Xavier Hanoulle
- From the University of Lille, CNRS, UMR 8576, UGSF, Unité de Glycobiologie Structurale et Fonctionnelle, 59000 Lille, France
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Jin G, Lee J, Lee K. Chemical genetics-based development of small molecules targeting hepatitis C virus. Arch Pharm Res 2017; 40:1021-1036. [PMID: 28856597 DOI: 10.1007/s12272-017-0949-3] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/19/2017] [Accepted: 08/20/2017] [Indexed: 12/21/2022]
Abstract
Hepatitis C virus (HCV) infection is a major worldwide problem that has emerged as one of the most significant diseases affecting humans. There are currently no vaccines or efficient therapies without side effects, despite today's advanced medical technology. Currently, the common therapy for most patients (i.e. genotype 1) is combination of HCV-specific direct-acting antivirals (DAAs). Up to 2011, the standard of care (SOC) was a combination of peg-IFNα with ribavirin (RBV). After approval of NS3/4A protease inhibitor, SOC was peg-IFNα and RBV with either the first-generation DAAs boceprevir or telaprevir. In the past several years, various novel small molecules have been discovered and some of them (i.e., HCV polymerase, protease, helicase and entry inhibitors) have undergone clinical trials. Between 2013 and 2016, the second-generation DAA drugs simeprevir, asunaprevir, daclatasvir, dasabuvir, sofosbuvir, and elbasvir were approved, as well as the combinational drugs Harvoni®, Zepatier®, Technivie®, and Epclusa®. A number of reviews have been recently published describing the structure-activity relationship (SAR) in the development of HCV inhibitors and outlining current therapeutic approaches to hepatitis C infection. Target identification involves studying a drug's mechanism of action (MOA), and a variety of target identification methods have been developed in the past few years. Chemical biology has emerged as a powerful tool for studying biological processes using small molecules. The use of chemical genetic methods is a valuable strategy for studying the molecular mechanisms of the viral lifecycle and screening for anti-viral agents. Two general screening approaches have been employed: forward and reverse chemical genetics. This review reveals information on the small molecules in HCV drug discovery by using chemical genetics for targeting the HCV protein and describes successful examples of targets identified with these methods.
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Affiliation(s)
- Guanghai Jin
- College of Pharmacy, Dongguk University-Seoul, Goyang, 10326, Republic of Korea
| | - Jisu Lee
- College of Pharmacy, Dongguk University-Seoul, Goyang, 10326, Republic of Korea
| | - Kyeong Lee
- College of Pharmacy, Dongguk University-Seoul, Goyang, 10326, Republic of Korea.
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Abstract
Hepatitis C virus (HCV) currently affects several million people across the globe. One of the major classes of drugs against HCV inhibits the NS3/4A protease of the polyprotein chain. Efficacy of these drugs is severely limited due to the high mutation rate that results in several genetically related quasispecies. The molecular mechanism of drug resistance is frequently deduced from structural studies and binding free energies. However, prediction of new mutations requires the evaluation of both binding free energy of the drug as well as the parameters (kcat and KM) for the natural substrate. The vitality values offer a good approach to investigate and predict mutations that render resistance to the inhibitor. A successful mutation should only affect the binding of the drug and not the catalytic activity and binding of the natural substrate. In this article, we have calculated the vitality values for four known drug inhibitors that are either currently in use or in clinical trials, evaluating binding free energies by the relevant PDLD/S-LRA method and activation barriers by the EVB method. The molecular details pertaining to resistance are also discussed. We show that our calculations are able to reproduce the catalytic effects and binding free energies in a good agreement with the corresponding observed values. Importantly, previous computational approaches have not been able to achieve this task. The trend for the vitality values is in accordance with experimental findings. Finally, we calculate the vitality values for mutations that have either not been studied experimentally or reported for some inhibitors.
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Affiliation(s)
- Garima Jindal
- Department of Chemistry, University of Southern California , 3620 McClintock Avenue, Los Angeles, California 90089, United States
| | - Dibyendu Mondal
- Department of Chemistry, University of Southern California , 3620 McClintock Avenue, Los Angeles, California 90089, United States
| | - Arieh Warshel
- Department of Chemistry, University of Southern California , 3620 McClintock Avenue, Los Angeles, California 90089, United States
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Campos GRF, Bittar C, Jardim ACG, Shimizu JF, Batista MN, Paganini ER, Ribeiro de Assis L, Bartlett C, Harris M, da Silva Bolzani V, Regasini LO, Rahal P. Hepatitis C virus in vitro replication is efficiently inhibited by acridone Fac4. J Gen Virol 2017; 98:1693-1701. [PMID: 28699869 PMCID: PMC7615702 DOI: 10.1099/jgv.0.000808] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022] Open
Abstract
Hepatitis C virus (HCV) affects about 170 million people worldwide. The current treatment has a high cost and variable response rates according to the virus genotype. Acridones, a group of compounds extracted from natural sources, showed potential antiviral actions against HCV. Thus, this study aimed to evaluate the effect of a panel of 14 synthetic acridones on the HCV life cycle. The compounds were screened using an Huh7.5 cell line stably harbouring the HCV genotype 2a subgenomic replicon SGR-Feo-JFH-1. Cells were incubated in the presence or absence of compounds for 72 h and cell viability and replication levels were assessed by MTT and luciferase assays, respectively. At a concentration of 5 µM the acridone Fac4 exhibited a >90 % inhibition of HCV replication with no effect on cell viability. The effects of Fac4 on virus replication, entry and release steps were evaluated in Huh7.5 cells infected with the JFH-1 isolate of HCV (HCVcc). Fac4 inhibited JFH-1 replication to approximately 70 %, while no effect was observed on virus entry. The antiviral activity of Fac4 was also observed on viral release, with almost 80 % of inhibition. No inhibitory effect was observed against genotype 3 replication. Fac4 was able to intercalate into dsRNA, however did not inhibit NS5B polymerase activity or translation driven by the HCV IRES. Although its mode of action is partly understood, Fac4 presents significant inhibition of HCV replication and can therefore be considered as a candidate for the development of a future anti-HCV treatment.
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Affiliation(s)
| | - Cíntia Bittar
- Institute of Bioscience, Language and Exact Science, IBILCE, UNESP – São Paulo State University, São José do Rio Preto, SP, Brazil
| | - Ana Carolina Gomes Jardim
- Institute of Biomedical Science, ICBIM, UFU – Federal University of Uberlândia, Uberlândia, MG, Brazil
| | - Jacqueline Farinha Shimizu
- Institute of Bioscience, Language and Exact Science, IBILCE, UNESP – São Paulo State University, São José do Rio Preto, SP, Brazil
| | - Mariana Nogueira Batista
- Institute of Bioscience, Language and Exact Science, IBILCE, UNESP – São Paulo State University, São José do Rio Preto, SP, Brazil
| | - Eder Ramos Paganini
- Institute of Bioscience, Language and Exact Science, IBILCE, UNESP – São Paulo State University, São José do Rio Preto, SP, Brazil
| | - Letícia Ribeiro de Assis
- Institute of Bioscience, Language and Exact Science, IBILCE, UNESP – São Paulo State University, São José do Rio Preto, SP, Brazil
| | - Christopher Bartlett
- School of Molecular and Cellular Biology, Faculty of Biological Sciences, and Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds, LS2 9JT, UK
| | - Mark Harris
- School of Molecular and Cellular Biology, Faculty of Biological Sciences, and Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds, LS2 9JT, UK
| | | | - Luis Octavio Regasini
- Institute of Bioscience, Language and Exact Science, IBILCE, UNESP – São Paulo State University, São José do Rio Preto, SP, Brazil
- Institute of Chemistry, São Paulo State University, Araraquara, SP, Brazil
| | - Paula Rahal
- Institute of Bioscience, Language and Exact Science, IBILCE, UNESP – São Paulo State University, São José do Rio Preto, SP, Brazil
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El-Hasab MAEM, El-Bastawissy EE, El-Moselhy TF. Identification of potential inhibitors for HCV NS3 genotype 4a by combining protein–ligand interaction fingerprint, 3D pharmacophore, docking, and dynamic simulation. J Biomol Struct Dyn 2017; 36:1713-1727. [DOI: 10.1080/07391102.2017.1332689] [Citation(s) in RCA: 17] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/06/2023]
Affiliation(s)
| | | | - Tarek Faathy El-Moselhy
- Faculty of Pharmacy, Department of Pharmaceutical Chemistry, Tanta University , Tanta, Egypt
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Cuypers L, Libin P, Schrooten Y, Theys K, Di Maio VC, Cento V, Lunar MM, Nevens F, Poljak M, Ceccherini-Silberstein F, Nowé A, Van Laethem K, Vandamme AM. Exploring resistance pathways for first-generation NS3/4A protease inhibitors boceprevir and telaprevir using Bayesian network learning. INFECTION GENETICS AND EVOLUTION 2017; 53:15-23. [PMID: 28499845 DOI: 10.1016/j.meegid.2017.05.007] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 01/27/2017] [Revised: 04/25/2017] [Accepted: 05/08/2017] [Indexed: 12/19/2022]
Abstract
Resistance-associated variants (RAVs) have been shown to influence treatment response to direct-acting antivirals (DAAs) and first generation NS3/4A protease inhibitors (PIs) in particular. Interpretation of hepatitis C virus (HCV) genotypic drug resistance remains a challenge, especially in patients who previously failed DAA therapy and need to be retreated with a second DAA based regimen. Bayesian network (BN) learning on HCV sequence data from patients treated with DAAs could provide insight in resistance pathways against PIs for HCV subtypes 1a and 1b, in a similar way as applied before for HIV. The publicly available 'Rega-BN' tool chain was developed to study associative analyses for various pathogens. Our first analysis, comparing sequences from PI-naïve and PI-experienced patients, determined that NS3 substitutions R155K and V36M arise with PI-exposure in HCV1a infected patients, and were defined as major and minor resistance-associated variants respectively. NS3 variant 174H was newly identified as potentially related to PI resistance. In a second analysis, NS3 sequences from PI-naïve patients who cleared the virus during PI therapy and from PI-naïve patients who failed PI therapy were compared, showing that NS3 baseline variant 67S predisposes to treatment-failure and variant 72I to treatment success. This approach has the potential to better characterize the role of more RAVs, if sufficient therapy annotated sequence data becomes available in curated public databases. In addition, polymorphisms present in baseline sequences that predispose patients to therapy failure can be identified using this approach.
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Affiliation(s)
- Lize Cuypers
- KU Leuven, University of Leuven, Department of Microbiology and Immunology, Rega Institute for Medical Research, Clinical and Epidemiological Virology, Herestraat 49, box 1040, 3000 Leuven, Belgium.
| | - Pieter Libin
- KU Leuven, University of Leuven, Department of Microbiology and Immunology, Rega Institute for Medical Research, Clinical and Epidemiological Virology, Herestraat 49, box 1040, 3000 Leuven, Belgium; Artificial Intelligence Lab, Vrije Universiteit Brussel, Pleinlaan 2, 1050 Brussels, Belgium.
| | - Yoeri Schrooten
- KU Leuven, University of Leuven, Department of Microbiology and Immunology, Rega Institute for Medical Research, Clinical and Epidemiological Virology, Herestraat 49, box 1040, 3000 Leuven, Belgium.
| | - Kristof Theys
- KU Leuven, University of Leuven, Department of Microbiology and Immunology, Rega Institute for Medical Research, Clinical and Epidemiological Virology, Herestraat 49, box 1040, 3000 Leuven, Belgium.
| | - Velia Chiara Di Maio
- Department of Experimental Medicine and Surgery, University of Rome "Tor Vergata", Rome, Italy.
| | - Valeria Cento
- Department of Experimental Medicine and Surgery, University of Rome "Tor Vergata", Rome, Italy.
| | - Maja M Lunar
- Institute of Microbiology and Immunology, Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia.
| | - Frederik Nevens
- University Hospitals Leuven, Department of Hepatology, Herestraat 49, 3000 Leuven, Belgium.
| | - Mario Poljak
- Institute of Microbiology and Immunology, Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia.
| | | | - Ann Nowé
- Artificial Intelligence Lab, Vrije Universiteit Brussel, Pleinlaan 2, 1050 Brussels, Belgium.
| | - Kristel Van Laethem
- KU Leuven, University of Leuven, Department of Microbiology and Immunology, Rega Institute for Medical Research, Clinical and Epidemiological Virology, Herestraat 49, box 1040, 3000 Leuven, Belgium.
| | - Anne-Mieke Vandamme
- KU Leuven, University of Leuven, Department of Microbiology and Immunology, Rega Institute for Medical Research, Clinical and Epidemiological Virology, Herestraat 49, box 1040, 3000 Leuven, Belgium; Center for Global Health and Tropical Medicine, Microbiology Unit, Institute for Hygiene and Tropical Medicine, University Nova de Lisboa, Rua da Junqueira 100, 1349-008 Lisbon, Portugal.
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Zhou Y, Zhao Y, Gao Y, Hu W, Qu Y, Lou N, Zhu Y, Zhang X, Yang H. Hepatitis C virus NS3 protein enhances hepatocellular carcinoma cell invasion by promoting PPM1A ubiquitination and degradation. JOURNAL OF EXPERIMENTAL & CLINICAL CANCER RESEARCH : CR 2017; 36:42. [PMID: 28283039 PMCID: PMC5345236 DOI: 10.1186/s13046-017-0510-8] [Citation(s) in RCA: 28] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 12/21/2016] [Accepted: 02/23/2017] [Indexed: 01/03/2023]
Abstract
Background Growing evidence suggests that hepatitis C virus (HCV) contributes to hepatocellular carcinoma (HCC) by directly modulating oncogenic signaling pathways. Protein phosphatase magnesium-dependent 1A (PPM1A) has recently emerged as an important tumor suppressor as it can block a range of tumor-centric signaling pathways through protein dephosphorylation. However, the role and regulatory mechanisms of PPM1A in HCV-infected cells have not been reported. Methods Total, cytoplasmic, and nuclear PPM1A protein after HCV infection or overexpression of HCV nonstructural protein 3 (NS3) were detected by western blotting. The expression of PPM1A in normal liver and HCV-related HCC tissues was quantified by immunohistochemistry. The effects of HCV infection and NS3 expression on the PPM1A protein level were systematically analyzed, and the ubiquitination level of PPM1A was determined by precipitation with anti-PPM1A and immunoblotting with either anti-ubiquitin or anti-PPM1A antibody. Finally, the roles of NS3 and PPM1A in hepatoma cell migration and invasion were assessed by wound healing and transwell assays, respectively. Results HCV infection and replication decreased PPM1A abundance, mediated by NS3, in hepatoma cells. Compared to normal liver tissues, the expression of PPM1A was significantly decreased in the HCC tumor tissues and adjacent non-tumor tissues. NS3 directly interacted with PPM1A to promote PPM1A ubiquitination and degradation, which was dependent on its protease domain. Blockade of PPM1A through small interfering RNA significantly promoted HCC cell migration, invasion, and epithelial mesenchymal transition (EMT), which were further intensified by TGF-β1 stimulation, in vitro. Furthermore, restoration of PPM1A abrogated the NS3-mediated promotion of HCC migration and invasion to a great extent, which was dependent on its protein phosphatase function. Conclusions Our findings demonstrate that the HCV protein NS3 can downregulate PPM1A by promoting its ubiquitination and proteasomal degradation, which might contribute to the migration and invasion of hepatoma cells and may represent a new strategy of HCV in carcinogenesis. Electronic supplementary material The online version of this article (doi:10.1186/s13046-017-0510-8) contains supplementary material, which is available to authorized users.
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Affiliation(s)
- Yali Zhou
- Department of Pathogenic Biology, School of Basic Medicine, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430030, Hubei Province, China
| | - Yan Zhao
- Department of Pathogenic Biology, School of Basic Medicine, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430030, Hubei Province, China
| | - Yaoying Gao
- Department of Pathogenic Biology, School of Basic Medicine, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430030, Hubei Province, China
| | - Wenjun Hu
- Department of Pathogenic Biology, School of Basic Medicine, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430030, Hubei Province, China
| | - Yan Qu
- Department of Pathogenic Biology, School of Basic Medicine, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430030, Hubei Province, China
| | - Ning Lou
- Department of Urology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430022, Hubei Province, China
| | - Ying Zhu
- State Key Laboratory of Virology and College of Life Sciences, Wuhan University, Wuhan, 430072, Hubei Province, China
| | - Xiaoping Zhang
- Department of Urology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430022, Hubei Province, China.
| | - Hongmei Yang
- Department of Pathogenic Biology, School of Basic Medicine, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430030, Hubei Province, China.
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Lin CT, Tritschler F, Lee KS, Gu M, Rice CM, Ha T. Single-molecule imaging reveals the translocation and DNA looping dynamics of hepatitis C virus NS3 helicase. Protein Sci 2017; 26:1391-1403. [PMID: 28176403 DOI: 10.1002/pro.3136] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/02/2016] [Revised: 02/02/2017] [Accepted: 02/03/2017] [Indexed: 01/23/2023]
Abstract
Non-structural protein 3 (NS3) is an essential enzyme and a therapeutic target of hepatitis C virus (HCV). Compared to NS3-catalyzed nucleic acids unwinding, its translation on single stranded nucleic acids have received relatively little attention. To investigate the NS3h translocation with single-stranded nucleic acids substrates directly, we have applied a hybrid platform of single-molecule fluorescence detection combined with optical trapping. With the aid of mechanical manipulation and fluorescence localization, we probed the translocase activity of NS3h on laterally stretched, kilobase-size single-stranded DNA and RNA. We observed that the translocation rate of NS3h on ssDNA at a rate of 24.4 nucleotides per second, and NS3h translocates about three time faster on ssRNA, 74 nucleotides per second. The translocation speed was minimally affected by the applied force. A subpopulation of NS3h underwent a novel translocation mode on ssDNA where the stretched DNA shortened gradually and then recovers its original length abruptly before repeating the cycle repetitively. The speed of this mode of translocation was reduced with increasing force. With corroborating data from single-molecule fluorescence resonance energy transfer (smFRET) experiments, we proposed that NS3h can cause repetitive looping of DNA. The smFRET dwell time analysis showed similar translocation time between sole translocation mode versus repetitive looping mode, suggesting that the motor domain exhibits indistinguishable enzymatic activities between the two translocation modes. We propose a potential secondary nucleic acids binding site at NS3h which might function as an anchor point for translocation-coupled looping.
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Affiliation(s)
- Chang-Ting Lin
- Department of Biophysics and Biophysical Chemistry, Johns Hopkins University, Baltimore, Maryland
| | - Felix Tritschler
- Department of Physics Education, Kongju National University, Kongju, 32588, Republic of Korea.,Center for Free-Electron Laser Science, DESY, 22607, Hamburg, Germany
| | - Kyung Suk Lee
- Department of Physics Education, Kongju National University, Kongju, 32588, Republic of Korea
| | - Meigang Gu
- Center for the Study of Hepatitis C, Laboratory of Virology and Infectious Disease, The Rockefeller University, New York, New York
| | - Charles M Rice
- Center for the Study of Hepatitis C, Laboratory of Virology and Infectious Disease, The Rockefeller University, New York, New York
| | - Taekjip Ha
- Department of Biophysics and Biophysical Chemistry, Johns Hopkins University, Baltimore, Maryland.,Thomas C. Jenkins Department of Biophysics, Johns Hopkins University, Baltimore, Maryland.,Department of Biomedical Engineering, Johns Hopkins University, Baltimore, Maryland.,Howard Hughes Medical Institute, Baltimore, Maryland
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Zhu Z, Tran H, Mathahs MM, Moninger TO, Schmidt WN. HCV Induces Telomerase Reverse Transcriptase, Increases Its Catalytic Activity, and Promotes Caspase Degradation in Infected Human Hepatocytes. PLoS One 2017; 12:e0166853. [PMID: 28056029 PMCID: PMC5215869 DOI: 10.1371/journal.pone.0166853] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/10/2016] [Accepted: 10/17/2016] [Indexed: 01/09/2023] Open
Abstract
Introduction Telomerase repairs the telomeric ends of chromosomes and is active in nearly all malignant cells. Hepatitis C virus (HCV) is known to be oncogenic and potential interactions with the telomerase system require further study. We determined the effects of HCV infection on human telomerase reverse transcriptase (TERT) expression and enzyme activity in primary human hepatocytes and continuous cell lines. Results Primary human hepatocytes and Huh-7.5 hepatoma cells showed early de novo TERT protein expression 2–4 days after infection and these events coincided with increased TERT promoter activation, TERT mRNA, and telomerase activity. Immunoprecipitation studies demonstrated that NS3-4A protease-helicase, in contrast to core or NS5A, specifically bound to the C-terminal region of TERT through interactions between helicase domain 2 and protease sequences. Increased telomerase activity was noted when NS3-4A was transfected into cells, when added to reconstituted mixtures of TERT and telomerase RNA, and when incubated with high molecular weight telomerase ‘holoenzyme’ complexes. The NS3-4A catalytic effect on telomerase was inhibited with primuline or danoprevir, agents that are known to inhibit NS3 helicase and protease activities respectively. In HCV infected cells, NS3-4A could be specifically recovered with telomerase holoenzyme complexes in contrast to NS5A or core protein. HCV infection also activated the effector caspase 7 which is known to target TERT. Activation coincided with the appearance of lower molecular weight carboxy-terminal fragment(s) of TERT, chiefly sized at 45 kD, which could be inhibited with pancaspase or caspase 7 inhibitors. Conclusions HCV infection induces TERT expression and stimulates telomerase activity in addition to triggering Caspase activity that leads to increased TERT degradation. These activities suggest multiple points whereby the virus can influence neoplasia. The NS3-4A protease-helicase can directly bind to TERT, increase telomerase activity, and thus potentially influence telomere repair and host cell neoplastic behavior.
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Affiliation(s)
- Zhaowen Zhu
- Department of Internal Medicine and Research Service, Veterans Affairs Medical Center, Iowa City, IA, United States of America
- Department of Internal Medicine Roy G. and Lucille A. Carver College of Medicine, University of Iowa Iowa City, IA, United States of America
| | - Huy Tran
- Department of Internal Medicine Roy G. and Lucille A. Carver College of Medicine, University of Iowa Iowa City, IA, United States of America
| | - M. Meleah Mathahs
- Department of Internal Medicine and Research Service, Veterans Affairs Medical Center, Iowa City, IA, United States of America
| | - Thomas O. Moninger
- Central Microscopy Research Facility Roy G. and Lucille A. Carver College of Medicine, University of Iowa Iowa City, IA, United States of America
| | - Warren N. Schmidt
- Department of Internal Medicine and Research Service, Veterans Affairs Medical Center, Iowa City, IA, United States of America
- Department of Internal Medicine Roy G. and Lucille A. Carver College of Medicine, University of Iowa Iowa City, IA, United States of America
- * E-mail:
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Abstract
Viruses are major pathogenic agents that can cause a variety of diseases, such as AIDS, hepatitis, respiratory diseases, and many more, in humans, plants, and animals. The most prominent of them have been adenoviruses, alphaviruses, flaviviruses, hepatitis C virus, herpesviruses, human immunodeficiency virus of type 1, and picornaviruses. This chapter presents an introductory remark on such viruses, mechanisms of their invasion, and diseases related to them. The inhibition of these viruses is of great concern to human beings. Each of these viruses encodes one or more proteases that play crucial roles in their replication, and thus they are important targets for the design and development of potent antiviral agents. The chapter, therefore, also introduces the readers to such proteases and their structures and functions. This chapter is thus a prelude to the remaining chapters in the book, which present in detail about the different viruses and their proteases.
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Affiliation(s)
- Anjana Sharma
- Meerut Institute of Engineering and Technology, Meerut, Uttar Pradesh, India
| | - Satya P. Gupta
- National Institute of Technical Teachers’ Training and Research, Bhopal, Madhya Pradesh, India
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