Original Article Effect of Nucleoside and Nucleotide Reverse Transcriptase Inhibitors of HIV on Endogenous Nucleotide Pools
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Antiviral Therapy 13:789–797 Original article Effect of nucleoside and nucleotide reverse transcriptase inhibitors of HIV on endogenous nucleotide pools Jennifer E Vela1, Michael D Miller2, Gerald R Rhodes1 and Adrian S Ray1* 1Department of Preclinical Drug Metabolism, Gilead Sciences, Inc., Foster City, CA, USA 2Department of Clinical Virology, Gilead Sciences, Inc., Foster City, CA, USA *Corresponding author: E-mail: [email protected] Background: Alterations in endogenous nucleotide pools Incubation of 10 µM AZT, either alone or in combination as a result of HIV therapy with nucleoside and nucleotide with other N(t)RTIs, increased 2′-deoxyadenosine triphos- reverse transcriptase inhibitors (N[t]RTIs) is a proposed phate, 2′-deoxyguanosine triphosphate and thymidine mechanism for therapy-related adverse events and drug triphosphate levels by up to 1.44-fold the concentrations interactions resulting in treatment failure. In vitro stud- observed in untreated cells. At higher than pharmaco- ies were performed in order to understand the effect of logical concentrations of AZT, evidence for inhibition of N(t)RTIs on endogenous nucleotide pools. 2′-deoxycytidylate deaminase and enzymes involved in the Methods: The T-cell line CEM-CCRF was treated with control salvage of thymidine was also observed. Phosphorylated antimetabolites or the N(t)RTIs abacavir, didanosine, lami- metabolites of TFV are known to inhibit purine nucleoside vudine, tenofovir (TFV) and zidovudine (AZT), either alone phosphorylase (PNP). However, in contrast to a potent PNP or in combination. The levels of natural 2′-deoxynucleoside inhibitor, TFV was unable to alter intracellular dNTP pools triphosphates (dNTP) and ribonucleoside triphophosphates upon addition of exogenous 2′-deoxyguanosine. were determined by liquid chromatography coupled with Conclusions: N(t)RTIs have the potential to alter nucleotide triple quadrupole mass spectrometry. pools; however, at the pharmacologically relevant concen- Results: Antimetabolites altered nucleotide pools in a trations, tested N(t)RTI or their combinations did not have manner consistent with their known mechanisms of action. an effect on nucleotide pools with the notable exception AZT was the only N(t)RTI that significantly altered dNTP pools. of AZT. Introduction Nucleoside and nucleotide reverse transcriptase triphosphate levels or endogenous dNTP pools could inhibitors (N[t]RTIs), used for the treatment of HIV alter the activity and toxicity of N(t)RTIs. infection, are activated by the cellular machinery respon- Treatment with zidovudine (AZT) is associated with sible for regulating endogenous 2′-deoxynucleoside adverse events including lipodystrophy and anaemia triphosphate (dNTP) and ribonucleoside triphosphate [2,3]. Different non-mutually exclusive mechanisms (rNTP) pools (summarized in Figure 1). Once formed, for toxicities associated with AZT therapy have been the active N(t)RTI triphosphate competes with its cor- proposed including a decrease in intracellular thymidine responding natural dNTP for incorporation by the viral triphosphate (TTP) because of inhibition of thymidine reverse transcriptase or host DNA polymerases caus- salvage [4–6], direct inhibition of cellular polymerases ing suppression of viral replication or toxicity, respec- through incorporation and chain termination [7,8], tively. N(t)RTIs could cause perturbations in endog- inhibition of 3′ to 5′ exonuclease proofreading by host enous nucleotide metabolism by causing competitive DNA polymerases by AZT monophosphate (AZTMP) inhibition, allosteric modulation or altered expression [9] and formation of a highly toxic metabolite through of nucleotide metabolizing enzymes (reviewed previ- the metabolism of the 3′-azido group to an amine by ously [1]). Due to the competitive nature of their incor- cytochrome P450 and their reductases [10]. A direct poration by polymerases, changes in either N(t)RTI role for AZTMP in toxicity observed in some cell types © 2008 International Medical Press 1359-6535 789 Ray.indd 789 5/9/08 17:35:29 JE Vela et al. has been suggested on the basis of the correlation of Purine nucleoside phosphorylase (PNP) is a crucial AZTMP levels with cytotoxicity in cultured cells [11]. enzyme in the regulation of purine nucleotide pools AZT treatment might affect TTP levels by inhibition [13]. Results supporting inhibition of PNP by phos- of cytosolic thymidine kinase (TK1), mitochondrial phorylated metabolites of tenofovir (TFV) as the TK (TK2) and thymidylate kinase (TMPK). AZT and mechanism for increased circulating levels of didanos- AZTMP have been observed to bind with similar affinity ine (ddI) when it is coadministered with TFV diso- to TK1 and TMPK as their respective natural substrates, proxil fumarate (TDF, the oral prodrug of TFV) [14] and the build-up of high levels of AZTMP might facilitate led to the suggestion of further clinical implications for inhibition of thymidine phosphorylation [12]. PNP inhibition [15]. Based on the effects of treatment Figure 1. Pyrimidine and purine nucleotide metabolism pathways in mammalian cells A dCTP CTP UTP dUTP TTP RR RR dCDP CDP UDP dUDP TDP TS & DHFR TMPK dCMP CMP UMP dUMP TMP dCK TK dCytidine Cytidine Uridine dUridine Thymidine dCMPDA UP TP dCDA Uracil Thymine DD β-Alanine and Dihydrouracil and β-Aminoisobutyrate dihydrothymine B dATP ATP GTP dGTP RR RR dADP ADP GDP dGDP IMP DH dAMP AMP IMP XMP GMP dGMP APRT ADA HGPRT dAdenosine Adenosine Inosine Guanosine dGuanosine PNP dInosine PNP ADA Hypoxanthine Guanine Adenine XOD Xanthine Urate XOD Schemes of (A) pyrimidine and (B) purine nucleotide metabolism pathways in mammalian cells (reviewed in a prior publication [28]). The enzymes adenine phosphoribosyltransferase (APRT), adenosine deaminase (ADA), 2′-deoxycytidine deaminase (dCDA), 2′-deoxycytidine kinase (dCK), 2-deoxycytidylate deaminase (dCMPDA), dihydrofolate reductase (DHFR), dihydropyrimidine dehydrogenase (DD), hypoxanthine-guanine phosphoribosyltransferase (HGPRT), inosine monophosphate dehydrogenase (IMPDH), purine nucleoside phosphorylase (PNP), ribonucleotide reductase (RR), thymidine kinase (TK), thymidine phosphorylase (TP), thymidylate kinase (TMPK), thymidylate synthase (TS), uridine phosphorylase (UP) and xanthine oxidase (XOD) are shown. The nucleosides 2′-deoxyadenosine (dA), 2′-deoxycytidine (dC), 2′-deoxyguanosine (dG), 2′-deoxyuridine (dU), thymidine (T) and xanthine (X) are shown. Their mono-, di- and triphosphorylated forms are abbreviated as -MP, -DP and -TP, respectively. 790 © 2008 International Medical Press Ray.indd 790 5/9/08 17:35:33 Effect of nucleoside and nucleotide analogues on nucleotide pools with highly potent PNP inhibitors, including increased and rNTP were obtained from Spectra Stable Isotopes intracellular 2′-deoxyadenosine triphosphate (dATP) (Columbia, MD, USA). The PNP inhibitor 2-amino- and 2′-deoxyguanosine triphosphate (dGTP) and inhi- 7-[1-(2-fluorophenyl)-2-(R)-hydroxyethyl]-3,5-dihy- bition of T-cell division [16], it was proposed that TFV dro-4H-pyrrolo[3,2-d]pyrimidin-4-one (PNP405) was might cause an increase in dNTP pools resulting in synthesized by NAEJA Pharmaceutical (Edmonton, antagonism of the activity of other N(t)RTIs and the AB, Canada) by methods similar to those previously observation of decreased CD4+ T-cells with the combi- reported [26]. nation of TDF and non-dose-adjusted ddI [17,18]. Triple N(t)RTI-only regimens including ddI/ Cells lamivudine (3TC)/stavudine (d4T), abacavir (ABC)/ Human T leukaemic CEM-CCRF cells were obtained ddI/d4T, ABC/3TC/AZT, ABC/ddI/TDF, ddI/3TC/TDF from the American Type Culture Collection (Manas- and ABC/3TC/TDF have shown high rates of virologi- sas, VA, USA) and were maintained in RPMI 1640 cal non-response, viral rebound and resistance muta- media supplemented with 10% heat-inactivated fetal tion selection [19–24]. Results from clinical studies bovine serum, 100 units/ml penicillin G and 100 µg/ml with triple N(t)RTI-only regimens have led to a deci- streptomycin sulfate. sion by a Department of Health and Human Services panel to not recommend therapy with ABC/3TC/AZT Effect of antimetabolites and N(t)RTIs on for treatment-naive patients and to state that other tri- nucleotide pools ple N(t)RTI-only regimens including ABC/3TC/TDF CEM-CCRF cells were seeded at 0.5 million cells/ml in and ddI/3TC/TDF should not be considered at any tissue culture flasks and treated for 24 h with the anti- time [25]. Different hypotheses have been proposed metabolites ribavirin, hydroxyurea or methotrexate at to explain the poor performance of triple N(t)RTI- concentrations of 20 µM, 3 mM or 5 µM, respectively, only therapies including antagonism of intracellular or with N(t)RTIs either alone or in combination, each N(t)RTI phosphorylation resulting in reduced triphos- incubated at 10 µM. phate analogue levels, decreased activity of N(t)RTIs Following the 24 h incubations, cells were counted caused by increases in competing dNTP pools, an using a haemocytometer and isolated from drug- intrinsic lack of potency in N(t)RTI-only regimens, containing media by spinning through a layer of oil as and the overlapping resistance profiles of N(t)RTIs described previously [14]. After removal of the oil layer, included in these regimens. cells were resuspended in 70% methanol and stored at In order to understand the effects of N(t)RTIs and -20ºC overnight to facilitate extraction of nucleotides. their combinations on nucleotide pools, including the Cellular debris was then removed by centrifugation and