In this study, we first examined 13 FDA-approved NRTIs as references, which are or were in clinical use or under clinical trials as anti-HIV-1 and/or anti-HBV NRTIs for antiviral activity against HIV-1. We also tested approximately 150 mostly newly designed and synthesized nucleoside analogs. When examined with the MTT assay using MT4 cells for anti-HIV-1 activity, 12 of the 13 reference compounds exerted moderate to highly potent activity against a wild-type strain HIV-1NL4-3. While ETV and ddI were moderately active against HIV-1NL4-3 with IC50 values of 1 microM, 3TC, FTC, TDF, ADV, ABC, d4T, Ed4T, and ddC were substantially active. AZT also proved to be substantially active. EFdA showed the most potent activity, while telbivudine (LdT), an FDA-approved anti-HBV therapeutic, showed no detectable anti-HIV-1 activity when examined at concentrations of up to 100 microM. Of note, two novel compounds, YMS-01144 and YMS-01145, were identified among the 150 nucleoside analogss to be extremely active against HIV-1NL4-3. We then attempted to evaluate anti-HBV activity of all the compounds described above. In general, no in vitro inhibition assay of de novo HBV infection using hepatocyte-derived cells and infectious HBV particles has been established. In the present study, in order to evaluate whether a given test compound reduced the amount of intracellular HBV DNA in Hep2.2.15 cells, DNA isolated from HepG2 2.2.15 cells cultured in the presence of each test compound over 12 days was subjected to real-time PCR. A representative standard curve for HBV quantification was generated and the standard curve for HBV-DNA amplification was given using diluted HBV-plasmid, and the CT value of each DNA sample was converted to an HBV copy number. The HepG2 2.2.15 cells intracellularly contain HBV DNA as both integrated HBV DNA and episomal DNA. The amount of integrated HBV DNA does not change with the addition of anti-HBV compounds but episomal DNA amounts decrease if reverse transcription of HBV RNA is blocked. When cultured alone, HepG2 2.2.15 cells proved to contain 106 copies/well;however, in the presence of ETV, the numbers of HBV copy continued to decrease down to 106 copies/well in a dose-response fashion at up to 100 nM and no further decrease in the copy number was seen at and beyond 100 nM of ETV. The S.D. values were mostly within 5% of the mean values as determined in triplicate. These data indicate that the assay system used in the present study is appropriately reproducible and quantitative and that each cell contains 2 copies of HBV, in line with the initial report that an HepG2 2.2.15 cell has two copies integrated in cellular DNA in a head-to-head tandem. We consequently examined all the compounds for activity against HBV in the real-time HBV-PCR assay. The order of the potency of the five analogs examined was: ETV LdT 3TC TDF ddC. YMS-01144 and -01145 also proved to be highly active against HBV. In order to corroborate the anti-HBV activity observed using the real-time HBV-PCR assay, we also conducted Southern blotting assay and determined whether ETV, YMS-01144, and YMS-01145 would blocked HBV replication. Huh-7 cells were first transfected with a plasmid containing 1.24-fold HBV genome of a wild-type HBV (HBVWT) or a plasmid containing an AB246345-based HBV genome carrying three ETV-resistance conferring amino acid substitutions (L180M, S202G, M204V)(ETVL180M/S202G/M204V). In 5-6 hours following transfection, the cells were exposed to various concentrations of an agent to be tested, further incubated for 72 hours, lysed, and subsequently the cell lysates were subjected to DNA extraction. Thus obtained DNA samples were subjected to Southern blot assay as previously published. The results revealed the presence of a single-stranded DNA signal as cultured in the absence of agents. The signal was more clearly seen in the cells treated with a low concentration of ETV (1 nM). However, the signal weakened in a dose-dependent manner and was virtually not seen in the cells exposed to 100 nM ETV. The IC50 value calculated by measuring the changes in the signal density was 2.2 nM. As examined under the same conditions, the IC50 values of YMS-01144 and -01145 against HBVWT were 6.5 and 6.4 nM, respectively. We subsequently asked whether YMS-01144 and YMS-01145 blocked the replication of HBVL180M/S202G/M204V. When the cells were cultured without drug exposure, a strong and clear signal was seen. As expected, ETV at 1 to 1,000 nM concentrations failed to block the replication of HBVL180M/S202G/M204V;even the highest concentration (1,000 nM) of ETV failed to achieve 50% signal reduction and IC50 value could not be determined in the assay shown in Figure 4. By contrast, both YMS-01144 and YMS-01145 significantly blocked HBVL180M/S202G/M204V replication in a dose response fashion. The average fold-reductions of the potency of YMS-01144 and YMS-01145 against HBVL180M/S202G/M204V were only 6.6 and 3.3, respectively, comparing their IC50 values against HBVWT and HBVL180M/S202G/M204V. The data clearly indicate that HBVL180M/S202G/M204V is highly susceptible to both YMS-01144 and YMS-01145. We then asked whether YMS-01144 and YMS-01145 blocked the replication of HBV in severe combined immunodeficiency transgenic mice (uPA+/+/SCID+/+ mice) with their liver replaced with human hepatocytes (hu-liver-chimeric-uPA+/+/SCID+/+ mice). In 8 days following inoculation of these mice with a wild-type HBV (HBVC2/CeWT), ETV, YMS-01144 or YMS-01145 was administered orally in drinking water containing each agent so that the dose resulted in 0.02 mg/kg/day. Just before the administration of ETV, the HBV copy numbers in their plasma were sufficiently high;however, the viremia levels significantly went down with ETV administration (p0.01) and the viremia reduction further went down even after the termination of ETV administration to the lowest (day 21). After day 21, the viremia went up slowly, but the viremia level on day 42 was still significantly lower than the day 1 viremia level (p0.01). Both YMS-01144 and YMS-01145 also significantly blocked the replication of HBV under the same conditions. The magnitudes of the greatest viremia reduction with YMS-01144 and YMS-01145 were larger and comparable, respectively, as compared to that with ETV. With both YMS-01144 and YMS-01145, the viremia levels continued to be significantly lower (p0.01) when examined after the conclusion of administration of each agent. On day 8 after the same hu-liver-chimeric-uPA+/+/SCID+/+ mice were inoculation with HBVC2/CeL180M/S202G/M204V, the viremia levels had reached sufficiently high. In a preliminary study, ETV at a dose of 0.02 mg/kg/day showed essentially no reduction in the viremia levels. Therefore, a 10-fold greater dose (0.2 mg/kg/day) was chosen. However, even with such a high dose, no significant reduction of HBVC2/CeL180M/S202G/M204V viremia was seen. By contrast, YMS-01144 at a dose of 0.02 mg/kg/day brought about a significant level of viremia reduction by day 7 of administration. The viremia reduction continued even after the conclusion f the administration and and the significant reduction was seen throughout the 35-day period of observation. The present data that ETV is predominantly active against HBV while EFdA is predominantly active against HIV-1, and that EFdA-related YMS-01144 and -01145 are active against both HIV-1 and HBV shed lights in the structural and functional similarities of HBV and HIV-1 RTs and should aid in the development of more potent agents against HBV and/or HIV-1.
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