Abstract
A major mechanism for human immunodeficiency virus 1 (HIV-1) reverse transcriptase (RT) resistance to nucleoside analogs involves the phosphorolytical removal of the chain-terminating nucleotide from the 3'-end of the primer. In this work, we analyzed the effect of phosphonoformate (PFA) and other pyrophosphate (PP(i)) analogs on PP(i)- and ATP-dependent phosphorolysis catalyzed by HIV-1 RT. Our experimental data demonstrated that PFA did not behave as a linear inhibitor but as an alternative substrate, allowing RT to remove AZT from a terminated primer through a PFA-dependent mechanism. Interestingly, in non-terminated primers, PFA was not a substrate for this reaction and competitively inhibited PP(i)- and ATP-dependent phosphorolysis. In fact, binding of PFA to the RT.template/primer complex was hindered by the presence of a chain terminator at the 3'-end of the primer. Other pyrophosphate analogs, such as phosphonoacetate, were substrates for the excision reaction with both terminated and nonterminated primers, whereas pamidronate, a bisphosphonate that prevents bone resorption, was not a substrate for these reactions and competitively inhibited the phosphorolytic activity of RT. As expected from their mechanisms of action, pamidronate (but not PFA) synergistically inhibits HIV-1 RT in combination with AZT-triphosphate in the presence of PP(i) or ATP. These results provide new clues about the mechanism of action of PFA and demonstrate that only certain pyrophosphate analogs can enhance the effect of nucleosidic inhibitors by blocking the excision of chain-terminating nucleotides catalyzed by HIV-1 RT. The relevance of these findings in combined chemotherapy is discussed.
Highlights
Of RT inhibitors have been identified, nucleoside analogs and non-nucleoside reverse transcriptase inhibitors (NNRTIs) [1, 2]
NNRTIs, such as nevirapine or efavirenz, are a diverse set of compounds that bind to a hydrophobic pocket in close proximity to the active site, inhibiting DNA polymerization
Thereafter, it was shown that NNRTIs inhibit the excision of other nucleoside analogs [11] and that this inhibition is responsible for the synergy found in antiviral combinations between AZT and NNRTIs [12]
Summary
Of RT inhibitors have been identified, nucleoside analogs and non-nucleoside reverse transcriptase inhibitors (NNRTIs) [1, 2]. Using combinations of various mutations, it is likely that some mutants of RT will be able to excise most nucleoside analogs with varying efficiencies [8]. For this reason, there is an urgent need for new drugs that can inhibit the excision reaction. We and others have described that NNRTIs inhibit the excision of the chain-terminating AZTMP catalyzed by HIV-1 RT, resensitizing the AZT-resistant enzyme to this compound [9, 10]. Phosphonoformate (foscarnet or PFA) is a classic PPi analog that has been shown to inhibit a broad spectrum of DNA polymerases, including herpes viruses and HIV-1. The combination of AZT with foscarnet in antiviral assays, shows low synergy or is merely additive [17,18,19,20], and clinical data indicate that even this additive effect is transient [21]
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