Mechanism of Inhibition of HIV-1 Reverse Transcriptase by 4 '-Ethynyl-2-fluoro-2 '-deoxyadenosine Triphosphate, a Translocation-defective Reverse Transcriptase Inhibitor

Christopher Bond Life Sciences Center, Department of Molecular Microbiology and Immunology, University of Missouri, Columbia, Missouri 65211, USA.
Journal of Biological Chemistry (Impact Factor: 4.57). 10/2009; 284(51):35681-91. DOI: 10.1074/jbc.M109.036616
Source: PubMed


Nucleoside reverse transcriptase inhibitors (NRTIs) are employed in first line therapies for the treatment of human immunodeficiency virus (HIV) infection. They generally lack a 3'-hydroxyl group, and thus when incorporated into the nascent DNA they prevent further elongation. In this report we show that 4'-ethynyl-2-fluoro-2'-deoxyadenosine (EFdA), a nucleoside analog that retains a 3'-hydroxyl moiety, inhibited HIV-1 replication in activated peripheral blood mononuclear cells with an EC(50) of 0.05 nm, a potency several orders of magnitude better than any of the current clinically used NRTIs. This exceptional antiviral activity stems in part from a mechanism of action that is different from approved NRTIs. Reverse transcriptase (RT) can use EFdA-5'-triphosphate (EFdA-TP) as a substrate more efficiently than the natural substrate, dATP. Importantly, despite the presence of a 3'-hydroxyl, the incorporated EFdA monophosphate (EFdA-MP) acted mainly as a de facto terminator of further RT-catalyzed DNA synthesis because of the difficulty of RT translocation on the nucleic acid primer possessing 3'-terminal EFdA-MP. EFdA-TP is thus a translocation-defective RT inhibitor (TDRTI). This diminished translocation kept the primer 3'-terminal EFdA-MP ideally located to undergo phosphorolytic excision. However, net phosphorolysis was not substantially increased, because of the apparently facile reincorporation of the newly excised EFdA-TP. Our molecular modeling studies suggest that the 4'-ethynyl fits into a hydrophobic pocket defined by RT residues Ala-114, Tyr-115, Phe-160, and Met-184 and the aliphatic chain of Asp-185. These interactions, which contribute to both enhanced RT utilization of EFdA-TP and difficulty in the translocation of 3'-terminal EFdA-MP primers, underlie the mechanism of action of this potent antiviral nucleoside.

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Available from: Eleftherios Michailidis, Nov 08, 2015
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    • "We have previously shown that a series of NRTIs with 4’-substitutions and a 3’-OH group are very potent inhibitors of WT and multi-drug resistant HIV-1. The most effective of these compounds is the adenosine analog 4’-ethynyl-2-fluoro-2’-deoxyadenosine (EFdA) [45,46]. We have demonstrated that EFdA acts in a DNA-sequence specific manner, primarily inhibiting DNA synthesis as an immediate chain terminator, but less often, at some DNA sequences can also act as a delayed chain terminator [46]. "
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    ABSTRACT: The K65R substitution in human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) is the major resistance mutation selected in patients treated with first-line antiretroviral tenofovir disoproxil fumarate (TDF).4'-ethynyl-2-fluoro-2'-deoxyadenosine (EFdA), is the most potent nucleoside analog RT inhibitor (NRTI) that unlike all approved NRTIs retains a 3'-hydroxyl group and has remarkable potency against wild-type (WT) and drug-resistant HIVs. EFdA acts primarily as a chain terminator by blocking translocation following its incorporation into the nascent DNA chain. EFdA is in preclinical development and its effect on clinically relevant drug resistant HIV strains is critically important for the design of optimal regimens prior to initiation of clinical trials. Here we report that the K65R RT mutation causes hypersusceptibility to EFdA. Specifically, in single replication cycle experiments we found that EFdA blocks WT HIV ten times more efficiently than TDF. Under the same conditions K65R HIV was inhibited over 70 times more efficiently by EFdA than TDF. We determined the molecular mechanism of this hypersensitivity using enzymatic studies with WT and K65R RT. This substitution causes minor changes in the efficiency of EFdA incorporation with respect to the natural dATP substrate and also in the efficiency of RT translocation following incorporation of the inhibitor into the nascent DNA. However, a significant decrease in the excision efficiency of EFdA-MP from the 3' primer terminus appears to be the primary cause of increased susceptibility to the inhibitor. Notably, the effects of the mutation are DNA-sequence dependent. We have elucidated the mechanism of K65R HIV hypersusceptibility to EFdA. Our findings highlight the potential of EFdA to improve combination strategies against TDF-resistant HIV-1 strains.
    Retrovirology 06/2013; 10(1):65. DOI:10.1186/1742-4690-10-65 · 4.19 Impact Factor
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    • "In fact, on the one hand, the North (C2′-exo/C3′-endo) EFdA sugar ring conformation (which is the proper 3′-terminus position for in-line nucleophilic attack on the α-phosphate of the incoming dNTP) has been shown to be required for efficient binding at the primer-binding and RT polymerase active sites suggesting that, once incorporated into the DNA, the EFdA 3′-hydroxyl group is not likely to prevent by itself additional nucleotides incorporation, and, thus, it does not contribute to the mechanism of chain termination [94]. On the other hand, molecular modeling studies suggested that the 4′-ethynyl of EFdA may fit into a hydrophobic pocket defined by residues A114, Y215, F160, M184 and the aliphatic D185 chain [93]. Hence, it has been proposed that the presence of a 4′-ethynyl substitution on the ribose ring possibly hampers RT to translocate the 3′-EFdA-MP terminus DNA. "
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    ABSTRACT: During the retrotranscription process, characteristic of all retroviruses, the viral ssRNA genome is converted into integration-competent dsDNA. This process is accomplished by the virus-coded reverse transcriptase (RT) protein, which is a primary target in the current treatments for HIV-1 infection. In particular, in the approved therapeutic regimens two classes of drugs target RT, namely, nucleoside RT inhibitors (NRTIs) and nonnucleoside RT inhibitors (NNRTIs). Both classes inhibit the RT-associated polymerase activity: the NRTIs compete with the natural dNTP substrate and act as chain terminators, while the NNRTIs bind to an allosteric pocket and inhibit polymerization noncompetitively. In addition to these two classes, other RT inhibitors (RTIs) that target RT by distinct mechanisms have been identified and are currently under development. These include translocation-defective RTIs, delayed chain terminators RTIs, lethal mutagenesis RTIs, dinucleotide tetraphosphates, nucleotide-competing RTIs, pyrophosphate analogs, RT-associated RNase H function inhibitors, and dual activities inhibitors. This paper describes the HIV-1 RT function and molecular structure, illustrates the currently approved RTIs, and focuses on the mechanisms of action of the newer classes of RTIs.
    06/2012; 2012(4599):586401. DOI:10.1155/2012/586401
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    • "()-FTC-TP 290,000 16 Feng et al., 2004 CBV-TP 900,000 34 Johnson et al., 2001 N.D., no data; KP1212, 5-aza-5,6-dihydro-2'-deoxycytidine; 3TC, lamivudine; PMPApp, tenofovir diphosphate; FTC, emtricitabine; CBV, carbovir. a Steady-state studies indicated an efficiency of 0.5 (Michailidis et al., 2009). "
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    ABSTRACT: Two novel thymidine analogs, 3'-fluoro-3'-deoxythymidine (FLT) and 2',3'-didehydro-3'-deoxy-4'-ethynylthymidine (Ed4T), have been investigated as nucleoside reverse transcriptase inhibitors (NRTIs) for treatment of HIV infection. Ed4T seems very promising in phase II clinical trials, whereas toxicity halted FLT development during this phase. To understand these different molecular mechanisms of toxicity, pre-steady-state kinetic studies were used to examine the interactions of FLT and Ed4T with wild-type (WT) human mitochondrial DNA polymerase γ (pol γ), which is often associated with NRTI toxicity, as well as the viral target protein, WT HIV-1 reverse transcriptase (RT). We report that Ed4T-triphosphate (TP) is the first analog to be preferred over native nucleotides by RT but to experience negligible incorporation by WT pol γ, with an ideal balance between high antiretroviral efficacy and minimal host toxicity. WT pol γ could discriminate Ed4T-TP from dTTP 12,000-fold better than RT, with only an 8.3-fold difference in discrimination being seen for FLT-TP. A structurally related NRTI, 2',3'-didehydro-2',3'-dideoxythymidine, is the only other analog favored by RT over native nucleotides, but it exhibits only a 13-fold difference (compared with 12,000-fold for Ed4T) in discrimination between the two enzymes. We propose that the 4'-ethynyl group of Ed4T serves as an enzyme selectivity moiety, critical for discernment between RT and WT pol γ. We also show that the pol γ mutation R964C, which predisposes patients to mitochondrial toxicity when receiving 2',3'-didehydro-2',3'-dideoxythymidine to treat HIV, produced some loss of discrimination for FLT-TP and Ed4T-TP. These molecular mechanisms of analog incorporation, which are critical for understanding pol γ-related toxicity, shed light on the unique toxicity profiles observed during clinical trials.
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