（論文

要様式３旨Studies on novel antiviral agents against bovine viral diarrhea virus(BVDV) and hepatitis C virus (HCV)
新規抗ウシウイルス性下痢症ウイルス（BVDV）薬および抗 C 型肝炎ウイルス（HCV）薬に関する研究
Mohammed Taha Ahmed Salim

Introduction and Objectives
Hepatitis C virus (HCV) infection represents a major public health problem. Nearly 3%of the population is chronically infected with this virus worldwide. The HCV vaccine is notavailable so far, and current available treatments are unsatisfactory. These facts mandate theurgent need for novel anti-HCV agents. Due to several characteristics shared between bovine
viral diarrhea virus (BVDV) and HCV with respect to virion structure, genome organization,and replication cycle, BVDV is considered as a good surrogate model for HCV and has beenused for investigating anti-HCV agents. In addition, the establishment of subgenomic andfull-genomic HCV replicon cells as well as the infectious strain JFH1 is also accelerates thediscovery of novel anti-HCV agents. The objectives of our study are 1) to find selective
inhibitors of BVDV and/or HCV, 2) to examine a variety of compounds for their inhibitory
effect on HCV replication in replicon cells, and 3) to elucidate the mechanism of action of theactive compounds.
Materials and Methods
1. Anti-BVDV study
Anti-BVDV activity and cytotoxicity of test compounds were examined in MDBK cells
by LDH and MTT colorimetric assays, respectively. The activity was confirmed by real-timereverse transcription (RT)-PCR and virus yield reduction assay. The time of drug-addition
experiment and docking study for active compounds were also performed.
2. Anti-HCV study
Anti-HCV activity and cytotoxicity of test compounds were examined in subgenomic
replicon cells by luciferase-based reporter assay and MTT assays, respectively. The activity
was confirmed in different subgenomic and full-genomic replicon cells by real-time RT-PCR.
The active compounds were also examined for their inhibitory effects on NS3 protease andNS5B RNA polymerase activity by biochemical assays.

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Results
Some of the γ-carboline derivatives were found to be potent and selective inhibitors ofBVDV replication. Among the compounds, 3,4,5-trimethyl-γ-carboline was found to be themost active against BVDV (Nose strain) in MDBK cells, with a 50% effective concentration(EC50) of 0.017 ± 0.005 µM and 50% cytotoxic concentration (CC50) of 7.4 ± 0.9 µM. The
compound inhibited viral RNA synthesis in a dose-dependent fashion. A time ofdrug-addition experiment and antiviral assay of the γ-carboline derivatives against the mutant
strains resistant to some classes of BVDV RNA-dependent RNA polymerase inhibitors
suggest that the compounds may target the RNA-dependent RNA polymerase.
A number of compounds were examined for their inhibitory effect on HCV replication in
Huh-7 cells harboring subgenomic viral RNA replicons with a luciferase reporter(LucNeo#2). Among the compounds, some phenanthridinone derivatives were found to be
active. The EC50 of the most active derivative was 0.063 ± 0.010 µM. This compound did notshow apparent cytotoxicity to the host cells at concentrations up to 40 µM. Its potent andselective anti-HCV activity was confirmed by real-time RT-PCR in different replicon cells.
Interestingly, the phenanthridinone derivatives were less potent inhibitors of genotype 2athan genotype 1b of HCV. Since the phenanthridinone derivatives did not inhibit NS3
protease or NS5B RNA polymerase activity in biochemical assays, their molecular target(mechanism of inhibition) remains unknown
Discussion and Conclusion
In initial studies, we used BVDV as a surrogate model to HCV and tested compounds fortheir anti-BVDV activity. Although some compounds including the γ-carboline derivatives
were potent and selective inhibitors of BVDV, they could not inhibit HCV replication.
Therefore, we also examined directly the anti-HCV activity of compounds in HCV repliconcells. After screening a number of compounds, we could identify novel phenanthridinone
derivatives as highly potent and selective inhibitors of HCV. The compounds were unique intheir mechanism of action, since they did not inhibit HCV polymerase or protease, both ofwhich are major targets for inhibition of HCV replication by antiviral agents currently underdevelopment.
In conclusion, compounds active against BVDV are not always good inhibitors of HCV
due to the genomic variability between the two viruses. However, these compounds may be
useful in the field of veterinary medicine and/or should be subjected to chemical modification
to confer anti-HCV activity.

（Antiviral Research Vol. 88，No. 1 2010 掲載）（Biochemical and Biophysical Research Communication Vol. 415，No. 4 2011 掲載）