Intracellular metabolism and persistence of the anti-human immunodeficiency virus activity of 2',3'-didehydro-3'-deoxy-4'-ethynylthymidine, a novel thymidine analog

Abstract

The therapeutic benefits of current antiretroviral therapy are limited by the evolution of drug-resistant virus and long-term toxicity. Novel antiretroviral compounds with activity against drug-resistant viruses are needed. 2',3'-didehydro-3'-deoxy-4'-ethynylthymidine (4'-Ed4T), a novel thymidine analog, has potent anti-human immunodeficiency virus (HIV) activity, maintains considerable activity against multidrug-resistant HIV strains, and is less inhibitory to mitochondrial DNA synthesis in cell culture than its progenitor stavudine (D4T). We investigated the intracellular metabolism and anti-HIV activity of 4'-Ed4T. The profile of 4'-Ed4T metabolites was qualitatively similar to that for zidovudine (AZT), with the monophosphate metabolite as the major metabolite, in contrast to that for D4T, with relatively poor formation of total metabolites. The first phosphorylation step for 4'-Ed4T in cells was more efficient than that for D4T but less than that for AZT. The amount of 4'-Ed4T triphosphate (4'-Ed4TTP) was higher than that of AZTTP at 24 h in culture. There was a dose-dependent accumulation of 4'-Ed4T diphosphate and 4'-Ed4TTP on up-regulation of thymidylate kinase and 3-phosphoglycerate kinase expression in Tet-On RKO cells, respectively. The anti-HIV activity of 4'-Ed4T in cells persisted even after 48 h of drug removal from culture in comparison with AZT, D4T, and nevirapine (NVP). The order of increasing persistence of anti-HIV activity of these compounds after drug removal was 4'-Ed4T > D4T > AZT > NVP. In conclusion, with the persistence of 4'-Ed4TTP and persistent anti-HIV activity in cells, we anticipate less frequent dosing and fewer patient compliance issues than for D4T. 4'-Ed4T is a promising antiviral candidate for HIV type 1 chemotherapy.

FIG. 1.

(A) Time-dependent intracellular accumulation of radioactive metabolites in CEM cells during incubation with 2 μM (75 mCi/mmol) of radiolabeled nucleotide. Panels: I and II, [³H]AZT (all metabolites and AZTTP only, respectively); III and IV, [³H]D4T (all metabolites and D4TTP only, respectively); V and VI, [³H]4′-Ed4T (all metabolites and 4′-Ed4TTP only, respectively). At different time intervals (2, 4, 8, 12, 16, and 24 h), 2 × 10⁷ of the cells were harvested for acid extraction. The extracted samples containing radioactive metabolites of the nucleoside analogs were subjected to HPLC analyses. The detected radioactivity was calculated as picomoles of each type of metabolite for each nucleoside analog/10⁶ cells. Each value is the mean ± standard deviation from three independent experiments. (B) Dose-dependent intracellular accumulation of radioactive metabolites of 4′-Ed4T in CEM cells. CEM cells were cultured with various concentrations of [³H]4′-Ed4T (0.25, 0.5, 1, and 2 μM) for 12 h, with doubling of the radiospecificity to 150 mCi/mmol. Cell extracts were prepared and subjected to HPLC analyses. The detected radioactivity was calculated as picomoles of each type of metabolite of 4′-Ed4T analog/10⁶ cells. Each value is the mean ± standard deviation from three independent experiments.

FIG. 2.

Intracellular TMPK activity in the metabolism of 4′-Ed4T in Tet-On TMPK RKO cells. (A) Expression of TMPK in Tet-On TMPK RKO cells, as detected by Western blotting. Actin was used as the internal control. (B) Doxycycline-induced TMPK RKO cells (5 × 10⁵) were incubated with 2 μM [5′-³H]4′-Ed4T (478 mCi/mmol) for 16 h. Cell extracts were prepared and subjected to HPLC analyses. The detected radioactivity was calculated as picomoles of each type of metabolite of 4′-Ed4T analog/10⁶ cells for each doxycycline concentration. (C) Total amounts of di- and triphosphate metabolites formed. Each value is the mean ± standard deviation from three independent experiments.

FIG. 3.

Intracellular PGK activity in the metabolism of 4′-Ed4T in Tet-On PGK RKO cells. (A) Expression of PGK in Tet-On PGK RKO cells, as detected by Western blotting. APE-1 was used as the internal control. (B) Doxycycline-induced PGK RKO cells (5 × 10⁵) were incubated with 2 μM [5′-³H]4′-Ed4T (478 mCi/mmol) for 16 h. Cell extracts were prepared and subjected to HPLC analyses. The detected radioactivity was calculated as picomoles of each type of metabolite of 4′-Ed4T analog/10⁶ cells for each doxycycline concentration. (C) Total amounts of di- and triphosphate metabolites formed. Each value is the mean ± standard deviation from three independent experiments.

FIG. 4.

Intracellular persistence of radioactive metabolites in CEM cells during incubation of 10⁶ cells with 2 μM (75 mCi/mmol) of [³H]AZT, [³H]D4T, or [³H]4′-Ed4T for 24 h. The cells were then washed and resuspended in fresh medium without nucleoside analogs at 0, 2, and 4 h after 24 h of incubation. The intracellular metabolites of each analog were determined by HPLC at 0, 2, 4, 8, 12, and 24 h. (A) The detected total radioactive metabolites were calculated as picomoles of each analog/10⁶ cells. (B) Concentrations of triphosphates formed. *, confidence limit of detection. Each value is the mean ± standard deviation from three independent experiments.

FIG. 5.

Persistence of anti-HIV-1 activity after removal of AZT, D4T, 4′-Ed4T, or NVP from culture medium. The persistence of nucleoside analog activity was determined using a novel TZM-bl indicator cell line-based assay described in Materials and Methods. TZM-bl cells were cultured in the presence of various concentrations of 4′-Ed4T, D4T, AZT, or NVP for 24 h. The cells were then washed to remove extracellular drug and infected with HIV-1 IIIB at an MOI of 0.1 at 0, 24, and 48 h of drug removal. The percent inhibition of HIV-1 replication was determined by measuring the luciferase activity. (A) Percent inhibition of HIV-1 replication when cells were infected at the time of drug treatment and incubated together for 24 h (solid lines) and percent inhibition when cells were infected at 0 h after drug removal (dotted lines) and incubated for 24 h. (B) Percent inhibition when cells were infected at 0 h after drug removal (dotted lines) and incubated for 24 h and percent inhibition when cells were infected at 24 h after drug removal (dashed lines) and incubated for 24 h (the infection was after two consecutive 24-h medium changes without drug replacement). The percent inhibition at 0 h after drug removal was plotted for ease of comparison. Error bars indicate standard deviations.