Intracellular Metabolism and Persistence of the Anti-Human Immunodeficiency Virus Activity of 2′,3′-Didehydro-3′-Deoxy-4′-Ethynylthymidine, a Novel Thymidine Analog

Paintsil, Elijah; Dutschman, Ginger E.; Hu, Rong; Grill, Susan P.; Lam, Wing; Baba, Masanori; Tanaka, Hiromichi; Cheng, Yung‐Chi

doi:10.1128/aac.00692-07

Cited by 16 publications

(34 citation statements)

References 54 publications

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“…We also observed variability in the phosphorylation of different thymidine analogs (AZT and 4Ј-Ed4T) in some individuals. This may be due to the fact that the substrate specificity of the kinases at each phosphorylation step may be different or that more than one enzyme may be responsible for phosphorylation at each step (24,39). Turriziani et al also observed individual variability in TK-1 activity, though the variability was more marked in HIV-infected individuals than in healthy individuals (45).…”

Section: Downloaded Frommentioning

confidence: 91%

“…Five days postinfection, culture supernatant was harvested for HIV-1 RNA extraction using a QIAmp viral RNA extraction kit (Qiagen, Valencia, CA), according to the manufacturer's instructions. HIV-1 RNA extracted from culture supernatant was quantified using real-time RT-PCR with previously published primer set, probes, and cycling parameters (13 H]3TC for 24 h, and the intracellular metabolites were quantified using HPLC as previously described (39). In brief, after 24 h of incubation, the cells were harvested by centrifugation and washed twice in ice-cold phosphatebuffered saline (PBS) containing 20 M dipyridamole (Sigma).…”

Section: Methodsmentioning

confidence: 99%

“…Expression of the cellular kinases involved in the phosphorylation of nucleoside analogs (i.e., TK-1, dCK, CMP kinase [CMPK], dTMP kinase [TMPK], and PGK) was determined by standard Western blot analysis as previously described using ␤-actin as a control (39). In brief, whole-cell lysate containing 40 g of protein from each donor was electrophoresed through a 12% SDS-polyacrylamide gel and transferred to a nitrocellulose membrane (Amersham).…”

Section: Methodsmentioning

confidence: 99%

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Determinants of Individual Variation in Intracellular Accumulation of Anti-HIV Nucleoside Analog Metabolites

Paintsil¹,

Dutschman²,

Hu³

et al. 2011

Antimicrob Agents Chemother

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Individual variation in response to antiretroviral therapy is well-known, but it is not clear if demographic characteristics such as gender, age, and ethnicity are responsible for the variation. To optimize anti-HIV therapy and guide antiretroviral drug discovery, determinants that cause variable responses to therapy need to be evaluated. We investigated the determinants of intracellular concentrations of nucleoside analogs using peripheral blood mononuclear cells from 40 healthy donors. We observed individual differences in the concentrations of the intracellular nucleoside analogs; the mean concentrations of the triphosphate metabolite of ethynylstavudine (4-Ed4T), zidovudine (AZT), and lamivudine (3TC) were 0.71 pmol/10 6 cells (minimum and maximum, 0.10 and 3.00 pmol/10 6 cells, respectively), 0.88 pmol/10 6 cells (minimum and maximum, 0.10 and 15.18 pmol/10 6 cells, respectively), and 1.70 pmol/10 6 cells (minimum and maximum, 0.20 and 7.73 pmol/10 6 cells, respectively). Gender and ethnicity had no effect on the concentration of 4-Ed4T and 3TC metabolites. There was a trend for moderation of the concentrations of AZT metabolites by gender (P ‫؍‬ 0.17 for gender ⅐ metabolite concentration). We observed variability in the activity and expression of cellular kinases. There was no statistically significant correlation between thymidine kinase 1 (TK-1) activity or expression and thymidine analog metabolite concentrations. The correlation between the activity of deoxycytidine kinase (dCK) and the 3TC monophosphate metabolite concentration showed a trend toward significance (P ‫؍‬ 0.1). We observed an inverse correlation between the multidrug-resistant protein 2 (MRP2) expression index and the concentrations of AZT monophosphate, AZT triphosphate, and total AZT metabolites. Our findings suggest that the observed variation in clinical response to nucleoside analogs may be due partly to the individual differences in the intracellular concentrations, which in turn may be affected by the cellular kinases involved in the phosphorylation pathway and ATP-binding cassette (ABC) transport proteins.Individual variation in response, such as viral suppression and adverse effects, to antiretroviral therapy is a well-described phenomenon (19, 49). Epidemiological and limited clinical studies suggest that demographic characteristics (e.g., gender, age, and ethnicity) and the HIV disease state of an individual may be partly responsible for the variation in efficacy and toxicity observed with treatment by nucleoside analog reverse transcriptase inhibitors (NRTIs). For example, published studies show that women experienced a 4-fold lower rate of disease progression than did men while they were on zidovudine (AZT) monotherapy; however, women experienced exaggerated toxicities during NRTI therapy compared to those of men (15-17, 20, 37). Gender and ethnicity have been suggested to be possible variables that explain the observed differences in treatment response to NRTIs (1,16,43).In a cohort of 4 HIV-1-infected women and 29 HIV-1-in...

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confidence: 91%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Determinants of Individual Variation in Intracellular Accumulation of Anti-HIV Nucleoside Analog Metabolites

Paintsil¹,

Dutschman²,

Hu³

et al. 2011

Antimicrob Agents Chemother

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“…The antiviral activity of 4Ј-Ed4T in cells persisted even 48 h after removal of the drug from culture, in contrast to the persistence of the antiviral activities of AZT and d4T (14). In this study, we found that no efflux of 4Ј-Ed4TMP and the less efficient efflux of 4Ј-Ed4T nucleoside from cells after drug removal might be one of the biochemical determinants for the longer retention of the 4Ј-Ed4T metabolites and the persistent antiviral activity of 4Ј-Ed4T in cell culture.…”

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confidence: 86%

Retention of Metabolites of 2′,3′-Didehydro-3′-Deoxy-4′-Ethynylthymidine, a Novel Anti-Human Immunodeficiency Virus Type 1 Thymidine Analog, in Cells

Wang

Tanaka

Baba

et al. 2009

Antimicrob Agents Chemother

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2,3-Didehydro-3-deoxy-4-ethynylthymidine (4-Ed4T), a novel thymidine analog, has more potent antihuman immunodeficiency virus type 1 (HIV-1) activity than its progenitor, stavudine (d4T). The profile of the intracellular metabolites of 4-Ed4T was qualitatively similar to that of zidovudine (AZT) but not to that of d4T, while after drug removal it showed more persistent anti-HIV activity than AZT or d4T in cell culture. When CEM cells were exposed to various concentrations of 4-Ed4T, 4-Ed4T was efficiently taken up by the cells and was readily phosphorylated to 4-Ed4T monophosphate (4-Ed4TMP), 4-Ed4T diphosphate (4-Ed4TDP), and 4-Ed4T triphosphate (4-Ed4TTP). Most importantly, 4-Ed4TTP, the active metabolite of 4-Ed4T, persisted significantly longer than 4-Ed4TDP and 4-Ed4TMP after drug removal. We further investigated the efflux profiles of 4-Ed4T in the comparison with those of AZT in CEM cells. After drug removal, both 4-Ed4T and AZT were effluxed from the cells in a time-and temperature-dependent manner. However, the efflux of 4-Ed4T from cells was much less efficient than that of AZT. 4-Ed4T was effluxed from cells only in its nucleoside form, while AZT was effluxed from cells in both its nucleoside and monophosphate forms. The mechanism-of-action study showed that the efflux of 4-Ed4T or AZT nucleoside might be due to unknown nucleoside transporters which were not related to the equilibrative nucleoside transporters, while the efflux of AZT monophosphate might be due to multidrug resistance protein 4 (MRP4/ABCC4). The results demonstrated that no detectable 4-Ed4TMP efflux and the less efficient efflux of 4-Ed4T nucleoside from cells might be one of the biochemical determinants of its persistent antiviral activity in cell culture.Significant progress in the treatment of human immunodeficiency virus type 1 (HIV-1) infection has been achieved by the advent of antiretroviral therapy, which targets different steps in the viral replication cycle with multiple inhibitors (25). Antiretroviral therapy with these inhibitors has brought about a significant decrease in plasma viremia to undetectable levels and has considerably improved the survival of infected individuals (18). Nucleoside reverse transcriptase (RT) inhibitors (NRTIs) were the first therapeutic agents to demonstrate a clinical role in the treatment of HIV-1 infection, and they continue to play a central role in the treatment of HIV infections (http://AIDSinfo.nih.gov). However, their therapeutic benefits are limited by the rapid emergence of drug-resistant viral mutants and delayed toxicity through inhibition of the host DNA polymerase (8, 9). Therefore, there is an urgent need to develop novel antivirals that can inhibit the replication of drug-resistant HIV-1 isolates while displaying favorable pharmacological and toxicity profiles.Our continuous efforts to search for novel NRTIs with potent anti-HIV-1 activity, low toxicity, and a lack of cross-resistance to existing anti-HIV-1 agents have recently identified the 4Ј-substituted nucleoside analog 2Ј,...

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“…Fusogenicity Assay-The fusogenicity assay was adapted from previously published studies (17)(18)(19)(20)(21). The day prior to the fusogenicity assay, 2 ϫ 10 4 TZMbl cells were plated in a 96-well plate.…”

Section: Methodsmentioning

confidence: 99%

Unique Functional Properties of Conserved Arginine Residues in the Lentivirus Lytic Peptide Domains of the C-terminal Tail of HIV-1 gp41

Kuhlmann

Steckbeck

Sturgeon

et al. 2014

Journal of Biological Chemistry

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Background: The C-terminal tail of the HIV-1 envelope preferentially incorporates arginine over lysine despite the similar physicochemical properties of these two residues. Results: Conservative arginine to lysine substitutions impair HIV-1 Env functions and virus replication. Conclusion: Arginine conservation provides unique functions that cannot be provided by lysines. Significance: These observations conclusively demonstrate unique functional properties for the conserved arginine residues in mediating Env functional properties.

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Intracellular Metabolism and Persistence of the Anti-Human Immunodeficiency Virus Activity of 2′,3′-Didehydro-3′-Deoxy-4′-Ethynylthymidine, a Novel Thymidine Analog

Cited by 16 publications

References 54 publications

Determinants of Individual Variation in Intracellular Accumulation of Anti-HIV Nucleoside Analog Metabolites

Determinants of Individual Variation in Intracellular Accumulation of Anti-HIV Nucleoside Analog Metabolites

Retention of Metabolites of 2′,3′-Didehydro-3′-Deoxy-4′-Ethynylthymidine, a Novel Anti-Human Immunodeficiency Virus Type 1 Thymidine Analog, in Cells

Unique Functional Properties of Conserved Arginine Residues in the Lentivirus Lytic Peptide Domains of the C-terminal Tail of HIV-1 gp41

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