title: Inhibition of hepatitis B virus replication by targeting ribonucleotide reductase M2 protein.
abstract: Chronic hepatitis B virus (HBV) infection is a key factor for hepatocellular carcinoma worldwide. Ribonucleotide reductase (RR) regulates the deoxyribonucleoside triphosphates biosynthesis and serves as a target for anti-cancer therapy. Here, we demonstrate that RR is essential for HBV replication and the viral covalently-closed-circular DNA (cccDNA) synthesis in host liver cells. By performing computer-assisted virtual screening against the crystal structure of RR small subunit M2 (RRM2), osalmid, was identified as a potential RRM2-targeting compound. Osalmid was shown to be 10-fold more active in inhibiting RR activity than hydroxyurea, and significantly inhibited HBV DNA and cccDNA synthesis in HepG2.2.15 cells. In contrast, hydroxyurea and the RR large subunit (RRM1)-inhibitory drug gemcitabine showed little selective activity against HBV replication. In addition, osalmid also was shown to possess potent activity against a 3TC-resistant HBV strain, suggesting utility in treating drug-resistant HBV infections. Interestingly, osalmid showed synergistic effects with lamivudine (3TC) in vitro and in vivo without significant toxicity, and was shown to inhibit RR activity in vivo, thus verifying its in vivo function. Furthermore, 4-cyclopropyl-2-fluoro-N-(4-hydroxyphenyl) benzamide (YZ51), a novel derivative of osalmid, showed higher efficacy than osalmid with more potent RR inhibitory activity. These results suggest that RRM2 might be targeted for HBV inhibition, and the RRM2-targeting compound osalmid and its derivative YZ51 could be a novel class of anti-HBV candidates with potential use for hepatitis B and HBV-related HCC treatment.
authors.affiliation: Department of Pathology and Pathophysiology, Key Laboratory of Disease Proteomics of Zhejiang Province, Research Center for Air Pollution and Health, Zhejiang University School of Medicine, Hangzhou 310058, China; Central Laboratory, The Second Clinical Medical College, Zhejiang Chinese Medical University, Hangzhou, China.
authors.forename: Xia
authors.initials: X
authors.lastname: Liu
authors.affiliation: Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China.
authors.forename: Zhijian
authors.initials: Z
authors.lastname: Xu
authors.affiliation: Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China.
authors.forename: Chuanwei
authors.initials: C
authors.lastname: Hou
authors.affiliation: Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China.
authors.forename: Meng
authors.initials: M
authors.lastname: Wang
authors.affiliation: Department of Pathology and Pathophysiology, Key Laboratory of Disease Proteomics of Zhejiang Province, Research Center for Air Pollution and Health, Zhejiang University School of Medicine, Hangzhou 310058, China; Department of Pathology and Pathophysiology, Zhengzhou University School of Medicine, Zhengzhou 450001, China.
authors.forename: Xinhuan
authors.initials: X
authors.lastname: Chen
authors.affiliation: Department of Pathology and Pathophysiology, Key Laboratory of Disease Proteomics of Zhejiang Province, Research Center for Air Pollution and Health, Zhejiang University School of Medicine, Hangzhou 310058, China.
authors.forename: Qinghui
authors.initials: Q
authors.lastname: Lin
authors.affiliation: Department of Pathology and Pathophysiology, Key Laboratory of Disease Proteomics of Zhejiang Province, Research Center for Air Pollution and Health, Zhejiang University School of Medicine, Hangzhou 310058, China.
authors.forename: Rui
authors.initials: R
authors.lastname: Song
authors.affiliation: Department of Pathology and Pathophysiology, Key Laboratory of Disease Proteomics of Zhejiang Province, Research Center for Air Pollution and Health, Zhejiang University School of Medicine, Hangzhou 310058, China.
authors.forename: Meng
authors.initials: M
authors.lastname: Lou
authors.affiliation: Department of Pathology and Pathophysiology, Key Laboratory of Disease Proteomics of Zhejiang Province, Research Center for Air Pollution and Health, Zhejiang University School of Medicine, Hangzhou 310058, China.
authors.forename: Lijun
authors.initials: L
authors.lastname: Zhu
authors.affiliation: State Key Laboratory of Infectious Disease Diagnosis and Treatment, The First Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou 310003, China.
authors.forename: Yunqing
authors.initials: Y
authors.lastname: Qiu
authors.affiliation: State Key Laboratory of Infectious Disease Diagnosis and Treatment, The First Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou 310003, China.
authors.forename: Zhi
authors.initials: Z
authors.lastname: Chen
authors.affiliation: Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China. Electronic address: chyang@simm.ac.cn.
authors.forename: Chunhao
authors.initials: C
authors.lastname: Yang
authors.affiliation: Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China. Electronic address: wlzhu@mail.shcnc.ac.cn.
authors.forename: Weiliang
authors.initials: W
authors.lastname: Zhu
authors.affiliation: Department of Pathology and Pathophysiology, Key Laboratory of Disease Proteomics of Zhejiang Province, Research Center for Air Pollution and Health, Zhejiang University School of Medicine, Hangzhou 310058, China. Electronic address: shaojimin@zju.edu.cn.
authors.forename: Jimin
authors.initials: J
authors.lastname: Shao
mesh_terms: D000818:Animals; D000998:Antiviral Agents / Q000494:pharmacology*; D045744:Cell Line, Tumor; D004261:DNA Replication; D004270:DNA, Circular / Q000096:biosynthesis; D004279:DNA, Viral / Q000096:biosynthesis; D024882:Drug Resistance, Viral; D004357:Drug Synergism; D016679:Genome, Viral; D006515:Hepatitis B virus / Q000187:drug effects* / Q000502:physiology; D006801:Humans; D006918:Hydroxyurea / Q000494:pharmacology; D019259:Lamivudine / Q000494:pharmacology; D008099:Liver / Q000821:virology*; D051379:Mice; D009154:Mutation; D012262:Ribonucleoside Diphosphate Reductase / Q000037:antagonists & inhibitors*; D012458:Salicylanilides / Q000494:pharmacology; D014779:Virus Replication
chemical_list: C000609925:4-cyclopropyl-2-fluoro-N-(4-hydroxyphenyl)benzamide; D000998:Antiviral Agents; D004270:DNA, Circular; D004279:DNA, Viral; D012458:Salicylanilides; D019259:Lamivudine; C005414:osalmide; C093713:ribonucleotide reductase M2; D012262:Ribonucleoside Diphosphate Reductase; D006918:Hydroxyurea
keywords: Drug synergism and drug resistance; Hepatitis B virus; Human ribonucleotide reductase small subunit M2 (RRM2); Small molecule compounds; Viral genomic DNA and cccDNA synthesis