1. Anti-HIV and anti-HBV activity of tested compounds Since published and our own data indicate that certain NRTIs are potent against HIV but not potent against HBV, and other NRTIs are, conversely, not potent against HIV-1 but highly potent against HBV, we surmised that we might find NRTIs that are not potent against HIV (therefore, they were put aside, frozen in the freezer, and """"""""untouched'since then) but potent against HBV in our inventory that has been accumulated from 1980s to present. It is hoped that some of such NRTIs might serve as """"""""lead compounds"""""""" to synthesize and identify anti-HBV candidates. In the present study, we comparatively determined anti-HIV and anti-HBV activity of selected nucleoside/nucleotide reverse transcriptase inhibitors (NRTI/NtRTIs). First, we chose 15 drugs, which are nucleoside/nucleotide reverse transcriptase inhibitors and are in clinical use or under clinical trials as anti-HIV-1 and/or anti-HBV NRTIs. They include zidovudine (AZT), emtricitabine (FTC), didanosine (ddI), sanilvudine (d4T) and abacavir (ABC), which are approved drugs as anti-HIV drug. Entecavir (ETV), lamivudine (3TC), tenofovir (TDF), adefovir pivoxil (ADV), and telbivudine (LdT), which are FDA-approved anti-HBV drugs, were laso examined. 4'-Ethynyl-2-fluoro-2'-deoxyadenosine (EFdA), 2',3'-didehydro-3'-deoxy-4'-ethynylthymidine (Ed4T), zalcitabine (ddC), 2', 3'-dideoxyguanosine (ddG), 2', 3'-dideoxythymidine (ddT), are under or had been clinical trials as anti-HIV drugs. Among 15 drugs, 13 drugs had detectable activity against wild type HIV in MTT assay. Among such active drugs, EFdA exerted most potent activity, against HIV and Ed4T, a recently reported new generation anti-HIV NRTI, also exerted potent activity against HIV. TDF, ADV, and AZT also showed substantial activity against HIV with IC50 values of less than 100 nM. On the other hand, telbivudine (LdT), an FDA-approved HBV therapeutic, had no anti-HIV activity when we tested up to 10 microM. However, it is of note that the ETV had relatively poor anti-HIV activity with IC50 value of 375 nM. When we further conducted multiple anti-HIV assays using p24 assay, MAGI assay, and MTT assay with higher TCID50. In all assays, EFdA revealed the most potent anti-HIV activity with IC50 values ranging 0.8 to 3.3 microM, but ETV showed only poor anti-HIV activity in MAGI assay and MTT assay when high titer HIV was used. We then evaluated anti-HBV activity of selected inhibitors using 2.2.15 cell line by measuring changes of intracellular HBV-DNA level. We employed 2.2.15 cells for the assay and the real-time PCR to detect the level of viral DNA in the assay samples. Intracellular DNA containing HBV-DNA was isolated from cells after 12-days of assay, and CT (cycle threshold) values for each reaction was obtained. Standard reaction using diluted HBV-plasmid (C_AT) was also obtained simultaneously and CT values of each sample were converted to an HBV copy number using the standard curve. The well without the drug (control) had 1,670,000 copies of HBV, but in the presence of different concentration of ETV, the copy numbers decreased in a dose-response manner;with 1000 nM of ETV, the copy number decreased to 162,000 per well which is more than 1-log and significant decrease compared to that of control with 1,000 nM, 100 nM, 10 nM (p 0.005) and 1 nM (p 0.01). Most of the tested NRTI/NtRTIs gave dose response reductions in the viral DNA. For TDF, in the presence of 100 nM, viral DNA level in the cell decreased by more than 50% compared to that for no drug control. However, residual viral DNA was detected (515% compared to that for no drug control) even at the concentration of 10 microM and this finding was seen in all drugs tested. This residual DNA seems to represent the integrated HBV DNA. Lamivudine (3TC) had IC50 of 22.4 nM. TDF showed potent activity against both HIV and HBV with IC50 numbers less than 100 nM although its activity against HBV is 65-fold lower than that of ETV. ddI and ddC had anti-HBV activity in our assay system, but we could not detect activity of AZT against HBV in our system in which we employed drug concentration up to 10 microM. Ed4T showed essentially no activity against HBV, however, EFdA had moderate anti-HBV activity. 2. Cytotoxicity and mitochondrial toxicity of NRTI/NtRTIs. It is known that mitochondrial DNA (mtDNA) defects can be acquired by toxic agents such as tobacco, alcohol and drugs. NRTIs used in chemotherapy or antiviral therapy have been shown to induce mitochondrial toxicity not only anti-HIV NRTIs but also cytarabine, vidarabine, aciclovir, and ribavirin. These nucleoside analogs elicit complete mtDNA replication deficits due to their ability to inhibit polymerase-gamma the enzyme responsible for replication of mtDNA . Clinical features can be regarded as a complication of those seen in the genetic mitochondriocytopathy, such as myopathy, cardiomyopathy, neuropathy, lactic acidosis, exocrine pancreas failure, liver failure, and bone marrow failure. Some of the early generation NRTIs, such as ddC and d4T are known to reduce the mtDNA in vitro, we tried to establish the system to assess the mitochondrial toxicity of tested NRTIs. We employed Hep-G2 cells for the assay and the real-time PCR to detect the level of mtDNA in the assay samples. Intracellular DNA was isolated from cells after 12-days of assay, served for real-time PCR, and reduction of mtDNA has been compared from the cells with no drugs added. 3. The novel agents against anti-HIV and anti-HBV derivatives of EFdA. We also identified some derivatives of EFdA, YMS-01144 and YMS-01145 . They had very potent anti-HIV and anti-HBV activity with IC50 values for 0.15 and 0.21 nM, 0.37 nM and 0.39 nM, respectively. These values were as potent as EFdA for HIV and as potent as ETV for HBV. It is expected that considering the similarity of the structures of these derivatives, we can generate compounds that are potent against both HIV and HBV through redesign.

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Hashimoto, Mai; Taguchi, Kazuaki; Ishiguro, Takako et al. (2018) Pharmacokinetic properties of a novel inosine analog, 4'-cyano-2'-deoxyinosine, after oral administration in rats. PLoS One 13:e0198636
Hashimoto, Mai; Taguchi, Kazuaki; Ishiguro, Takako et al. (2018) Pharmacokinetics studies of 4'-cyano-2'-deoxyguanosine, a potent inhibitor of the hepatitis B virus, in rats. J Pharm Pharmacol 70:723-731
Kumamoto, Hiroki; Fukano, Misato; Imoto, Shuhei et al. (2017) A novel entecavir analogue constructing with a spiro[2.4]heptane core structure in the aglycon moiety: Its synthesis and evaluation for anti-hepatitis B virus activity. Nucleosides Nucleotides Nucleic Acids 36:463-473
Huber, Andrew D; Michailidis, Eleftherios; Tang, Jing et al. (2017) 3-Hydroxypyrimidine-2,4-Diones as Novel Hepatitis B Virus Antivirals Targeting the Viral Ribonuclease H. Antimicrob Agents Chemother 61:
Kumamoto, Hiroki; Fukano, Misato; Nakano, Tomohiko et al. (2016) Diastereoselective Synthesis of 6?-(Z)- and 6?-(E)-Fluoro Analogues of Anti-hepatitis B Virus Agent Entecavir and Its Evaluation of the Activity and Toxicity Profile of the Diastereomers. J Org Chem 81:2827-36
Salie, Zhe Li; Kirby, Karen A; Michailidis, Eleftherios et al. (2016) Structural basis of HIV inhibition by translocation-defective RT inhibitor 4'-ethynyl-2-fluoro-2'-deoxyadenosine (EFdA). Proc Natl Acad Sci U S A 113:9274-9
Imoto, Shuhei; Kohgo, Satoru; Tokuda, Ryoh et al. (2015) Design, Synthesis, and Evaluation of Anti-HBV Activity of Hybrid Molecules of Entecavir and Adefovir: Exomethylene Acycloguanine Nucleosides and Their Monophosphate Derivatives. Nucleosides Nucleotides Nucleic Acids 34:590-602
Takamatsu, Yuki; Tanaka, Yasuhito; Kohgo, Satoru et al. (2015) 4'-modified nucleoside analogs: potent inhibitors active against entecavir-resistant hepatitis B virus. Hepatology 62:1024-36
Muftuoglu, Yagmur; Sohl, Christal D; Mislak, Andrea C et al. (2014) Probing the molecular mechanism of action of the HIV-1 reverse transcriptase inhibitor 4'-ethynyl-2-fluoro-2'-deoxyadenosine (EFdA) using pre-steady-state kinetics. Antiviral Res 106:1-4
Maeda, Kenji; Desai, Darshan V; Aoki, Manabu et al. (2014) Delayed emergence of HIV-1 variants resistant to 4'-ethynyl-2-fluoro-2'-deoxyadenosine: comparative sequential passage study with lamivudine, tenofovir, emtricitabine and BMS-986001. Antivir Ther 19:179-89

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