Inhibition of human immunodeficiency virus type 1 reverse transcriptase by the 5'-triphosphate beta enantiomers of cytidine analogs

Faraj, Abdesslem; Agrofoglio, Luigi A.; Wakefield, John K.; McPherson, Sylvia A.; Morrow, Casey D.; Gosselin, Gilles; Mathé, Christophe; Imbach, J.‐L.; Schinazi, R. F.; Sommadossi, Jean Pierre

doi:10.1128/aac.38.10.2300

Cited by 65 publications

(47 citation statements)

References 29 publications

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“…This difference results from variation in a single amino acid in the conserved YXDD region found in their active sites (YMDD of HIV-1 RT and YVDD of MuLV RT). Consistently, the Val and Ile mutations at the Met 184 residue of the HIV-1 YMDD sequence are selected during the 3TC treatment (31,32). In addition, these two 3TC-resistant HIV-1 RT mutants, M184V and M184I, also show increased enzyme fidelity (33)(34)(35).…”

mentioning

confidence: 72%

“…For studying misinsertion and mismatched primer extension fidelity, either a matched 32 P-labeled T primer (matched T primer, 5Ј-CGCGC-CGAATTCCCGC-3Ј) or a mismatched 32 P-labeled G/T primer (5Ј-CGCGCCGAATTCCCGT-3Ј) was annealed onto the 38-mer RNA template (see above). This generated a T/P that was identical, except that the 3Ј nucleotide on the primer strand of the mismatched T/P was not complementary to the corresponding template nucleotide, which is representative of a mismatched T/P product formed after a misincorporation event.…”

Section: Methodsmentioning

confidence: 99%

“…In addition, a mismatched DNA T/P was prepared with a 38-mer DNA template encoding the exact nucleotide sequence as the 38-mer RNA template annealed to a mismatched C/A primer (5Ј-CGCGCCGAATTCCCGCTAA-3Ј). All primers used in this assay were 32 P-labeled at their 5Ј end (template:primer, 2.5:1). Assay mixtures (20 l) contained 10 nM T/P, the RT protein concentrations showing the primer extension activities described in the individual figure legends, and four dNTPs at the concentrations indicated in the figure legends under the condition described in the presteady-state kinetics assay described above.…”

Section: Determination Of T/p Binding Affinity (K D )-mentioning

confidence: 99%

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Mechanistic Differences in RNA-dependent DNA Polymerization and Fidelity between Murine Leukemia Virus and HIV-1 Reverse Transcriptases

Skasko

Weiss

Reynolds

et al. 2005

Journal of Biological Chemistry

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We compared the mechanistic and kinetic properties of murine leukemia virus (MuLV) and human immunodeficiency virus type 1 (HIV-1) reverse transcriptases (RTs) during RNA-dependent DNA polymerization and mutation synthesis using pre-steady-state kinetic analysis. First, MuLV RT showed 6.5-121.6-fold lower binding affinity (K d ) to deoxynucleotide triphosphate (dNTP) substrates than HIV-1 RT, although the two RTs have similar incorporation rates (k pol ). Second, compared with HIV-1 RT, MuLV RT showed dramatic reduction during multiple dNTP incorporations at low dNTP concentrations. Presumably, due to its low dNTP binding affinity, the dNTP binding step becomes rate-limiting in the multiple rounds of the dNTP incorporation by MuLV RT, especially at low dNTP concentrations. Third, similar fold differences between MuLV and HIV-1 RTs in the K d and k pol values to correct and incorrect dNTPs were observed. This indicates that these two RT proteins have similar misinsertion fidelities. Fourth, these two RT proteins have different mechanistic capabilities regarding mismatch extension. MuLV RT has a 3.1-fold lower mismatch extension fidelity, compared with HIV-1 RT. Finally, MuLV RT has a 3.8-fold lower binding affinity to mismatched template/primer (T/P) substrate compared with HIV-1 RT. Our data suggest that the active site of MuLV RT has an intrinsically low dNTP binding affinity, compared with HIV-1 RT. In addition, instead of the misinsertion step, the mismatch extension step, which varies between MuLV and HIV-1 RTs, contributes to their fidelity differences. The implications of these kinetic differences between MuLV and HIV-1 RTs on viral cell type specificity and mutagenesis are discussed.Retroviruses encode a versatile DNA polymerase called reverse transcriptase (RT).1 The function of RT is to synthesize linear double-stranded proviral DNAs from single-stranded positive sense viral RNA genomes during viral replication. In order to catalyze this process, RTs perform several distinct enzymatic reactions including RNA-dependent DNA polymerization, DNA-dependent DNA polymerization, strand transfer, and RNase H cleavage (1). The DNA polymerase activity of RTs has been targeted, using various types of RT inhibitors such as nucleoside substrate-like compounds (i.e. azidothymidine (AZT) and didanosine (ddI)) (2), as a means to reduce viral replication in infected individuals. Lentiviruses such as human immunodeficiency virus type 1 (HIV-1) uniquely infect terminally differentiated/nondividing cells (i.e. macrophages) as well as dividing cells (i.e. activated CD4ϩ T cells), whereas oncoretroviruses such as murine leukemia virus (MuLV) productively replicate mainly in dividing cells (3,4). Numerous studies have reported that actively dividing cells have higher cellular deoxynucleotide triphosphate (dNTP) concentrations than nondividing cells (5). Recently, it was reported that the cellular dNTP concentration of human macrophages (ϳ40 nM) is ϳ100 times lower than that of dividing CD4 ϩ T cells (ϳ5 M) (6). Therefore, RTs ...

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mentioning

confidence: 72%

Section: Methodsmentioning

confidence: 99%

Section: Determination Of T/p Binding Affinity (K D )-mentioning

confidence: 99%

See 1 more Smart Citation

Mechanistic Differences in RNA-dependent DNA Polymerization and Fidelity between Murine Leukemia Virus and HIV-1 Reverse Transcriptases

Skasko

Weiss

Reynolds

et al. 2005

Journal of Biological Chemistry

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“…Kinetic studies using cloned and expressed HIV-1 RT containing the M184V mutation Were performed under steady-state conditions using the 5'-nucleoside triphosBal.zarini, J., Herdewijn, P., and De Clercq, E. (1989) Differenphates of various enantiomeric pairs (Faraj et al, 1994).…”

Section: Enantioselective Antiviral Activity Of Nucleosides 353mentioning

confidence: 99%

The Effect of Absolute Configuration on the Anti-HIV and Anti-HBV Activity of Nucleoside Analogues

Furman

Wilson

Reardon

et al. 1995

Antivir Chem Chemother

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SummaryThis review concerns the effect of stereoisomerism on the selective activity of anti-HIV and anti-HBV nucleoside analogues.The synthesis of a number of nucleoside analogues with anti-HIV and anti-HBV actiVity yields mixtures of 1-~-D and 1-~-L stereoisomers. Anti-HIV and anti-HBV activity is associated primarily with one of the two enantiomers and the more potent actiVity does not always reside with the 1-~-D configuration characteristic of natural nucleosides. In the case of HIV, the origin of this stereoselectivity appears to be the result of dlfferentlal metabolism of the analogues and not due to differential inhibition of the target enzyme; the HIV reverse transcriptase. However, mutations at position 184 of the HIV-RT does result in stereoselective inhibition of the enzyme. On the other hand, with HBV, there is also a stereoselective inhibition of the HBV DNA polymerase, where the 5 '-triphosphate of the 1-~-L enantiomer is the more potent inhibitor.

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“…The ninefold enhancement in incorporation efficiency during RNA-directed L-FTCMP incorporation is the largest change in response to the addition of a 5-fluorine noted to date. This is suggested to be, at least in part, responsible for L-FTC's increased activity over 3TC in anti-HIV potency in vitro (Table 3) (Schinazi et al, 1992b;Faraj et al, 1994;Feng et al, 1999 (Otto et al, 2002, Antiviral Research HIV DART 2002 Final Program andAbstract Book, Abstract 51).…”

Section: Kinetic Consequences Of 5-and 2′-fluorine Addition On Hiv-1 mentioning

confidence: 99%

Probing the Mechanistic Consequences of 5-Fluorine Substitution on Cytidine Nucleotide Analogue Incorporation by HIV-1 Reverse Transcriptase

Ray

Schinazi

Murakami

et al. 2003

Antivir Chem Chemother

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β-D and β-L-enantiomers of 2′,3′-dideoxycytidine analogues are potent chain-terminators and antimetabolites for viral and cellular replication. Seemingly small modifications markedly alter their antiviral and toxicity patterns. This review discusses previously published and recently obtained data on the effects of 5- and 2′-fluorine substitution on the pre-steady state incorporation of 2′-deoxycytidine-5′-monophosphate analogues by HIV-1 reverse transcriptase (RT) in light of their biological activity. The addition of fluorine at the 5-position of the pyrimidine ring altered the kinetic parameters for all nucleotides tested. Only the 5-fluorine substitution of the clinically relevant nucleosides (-)-β-L-2′,3′-dideoxy-3′-thia-5-fluoro-cytidine (L-FTC, Emtriva™), and (+)-β-D-2′,3′-dide-hydro-2′,3′-dideoxy-5-fluorocytidine (D-D4FC, Reverset™), caused a higher overall efficiency of nucleotide incorporation during both DNA- and RNA-directed synthesis. Enhanced incorporation by RT may in part explain the potency of these nucleosides against HIV-1. In other cases, a lack of correlation between RT incorporation in enzymatic assays and antiviral activity in cell culture illustrates the importance of other cellular factors in defining antiviral potency. The substitution of fluorine at the 2′ position of the deoxyribose ring negatively affects incorporation by RT indicating the steric gate of RT can detect electrostatic perturbations. Intriguing results pertaining to drug resistance have led to a better understanding of HIV-1 RT resistance mechanisms. These insights serve as a basis for understanding the mechanism of action for nucleoside analogues and, coupled with studies on other key enzymes, may lead to the more effective use of fluorine to enhance the potency and selectivity of antiviral agents.

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Inhibition of human immunodeficiency virus type 1 reverse transcriptase by the 5'-triphosphate beta enantiomers of cytidine analogs

Cited by 65 publications

References 29 publications

Mechanistic Differences in RNA-dependent DNA Polymerization and Fidelity between Murine Leukemia Virus and HIV-1 Reverse Transcriptases

Mechanistic Differences in RNA-dependent DNA Polymerization and Fidelity between Murine Leukemia Virus and HIV-1 Reverse Transcriptases

The Effect of Absolute Configuration on the Anti-HIV and Anti-HBV Activity of Nucleoside Analogues

Probing the Mechanistic Consequences of 5-Fluorine Substitution on Cytidine Nucleotide Analogue Incorporation by HIV-1 Reverse Transcriptase

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