Comparative kinetic analyses of interaction of inhibitors with Rauscher murine leukemia virus and human immunodeficiency virus reverse transcriptases

Cherrington, Julie M.; Fuller, M.; Mulato, Andrew; Allen, S. J. W.; Sandra, Koen; Ussery, Michael A.; Leśnikowski, Zbigniew J.; Schinazi, Raymond F.; Sommadossi, Jean Pierre; Chen, M S

doi:10.1128/aac.40.5.1270

Cited by 21 publications

(6 citation statements)

References 20 publications

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“…Therefore, the differences in potency may lie in part in the efficiency with which the triphosphate form of the analogs is incorporated into DNA by HIV‐1 RT. The steady‐state kinetic analysis of incorporation of 3TC‐TP, (−)‐FTC‐TP, and their (+) isomers by HIV‐1 RT has been reported (8, 10, 29), and a pre‐steady‐state kinetic analysis has examined the incorporation of the (−) isomer 3TC‐TP into DNA by wild‐type and mutant HIV‐1 RT (23).…”

Section: Methodsmentioning

confidence: 99%

Mechanistic studies show that (−)‐FTC‐TP is a better inhibitor of HIV‐1 reverse transcriptase than 3TC‐TP

et al. 1999

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Of all of the nucleoside inhibitors approved by the FDA for treatment of AIDS, (-)-beta-2',3'-dideoxy-3'-thiacytidine (3TC, lamivudine) is the only one with the unnatural (-)-beta-L configuration. The fluorinated derivative (-)-beta-2', 3'-dideoxy-5-fluoro-3'-thiacytidine [(-)-FTC] and its triphosphate form have also been reported to have excellent antiretroviral activity against HIV-1 reverse transcriptase (RT). Preliminary results of clinical trials suggest that (-)-FTC is 6- to 10-fold more potent than 3TC. However, the molecular mechanism for the observed enhanced clinical potency of (-)-FTC to inhibit viral replication is not understood. The present mechanistic studies used a transient kinetic approach and were designed to compare the incorporation of 3TC-TP and (-)-FTC-TP into DNA by HIV-1 RT and illuminate key features that may play a role in the differential potency. Here we show that (-)-FTC-TP is incorporated 10-fold more efficiently than 3TC-TP during HIV-1 RT-catalyzed RNA-dependent DNA synthesis. The enhanced incorporation efficiency of (-)-FTC-TP may be a key mechanistic feature that, in part, is responsible for the enhanced potency of (-)-FTC observed in ongoing clinical trials.

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Section: Methodsmentioning

confidence: 99%

Mechanistic studies show that (−)‐FTC‐TP is a better inhibitor of HIV‐1 reverse transcriptase than 3TC‐TP

et al. 1999

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“…Wild-type (WT) HXB2D, K65R, 4Y (M41L+D67N+L210W+T215Y; 4-TAM) and 5R (M41L+K65R+D67N+L210W+T215Y; 4-TAM+K65R) mutant HIV-1 viruses were constructed as previously described [8,18,19]. Susceptibilities of the WT molecular clone HXB2D and mutant viruses to TFV and AZT were evaluated using an XTT-based viability assay in MT-2 cells as previously described [20].…”

Section: Antiviral Susceptibility Assaymentioning

confidence: 99%

The K65R Reverse Transcriptase Mutation in HIV-1 Reverses the Excision Phenotype of Zidovudine Resistance Mutations

et al. 2006

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The HIV-1 nucleoside reverse transcriptase inhibitors (NRTIs) tenofovir (TFV), abacavir, didanosine and stavudine can select for K65R, whereas zidovudine (AZT) and stavudine can select for thymidine analogue mutations (TAMs) in HIV-1 reverse transcriptase (RT). HIV-1 with TAMs shows reduced susceptibility to all NRTIs, most notably AZT, whereas HIV-1 with K65R shows reduced susceptibility to all NRTIs except AZT. K65R and TAMs rarely occur together in patients. However, when present together, K65R can restore susceptibility to AZT. This study characterizes the underlying mechanisms of resistance of these RT mutants to TFV and AZT. K65R mediated decreased binding/incorporation of TFV and AZT (increased Ki/Km of 7.1- and 4.3-fold, respectively), but also decreased excision of TFV and AZT (0.7- and 0.3-fold, respectively) when compared with wild-type RT. By contrast, TAMs mediated increased TFV and AZT excision (11- and 5.4-fold, respectively), and showed no changes in binding/incorporation. When these mutations were combined, K65R reversed TAM-mediated AZT resistance by strongly reducing AZT excision. Molecular modelling studies suggest that K65R creates additional hydrogen bonds that reduce the conformational mobility of RT, resulting in reduced polymerization and excision. Thus, consistent with clinical HIV-1 genotyping data, there appears to be no net NRTI resistance benefit for TAMs and K65R to develop together in patients taking AZT and TFV disoproxil fumarate, where the TAM pathway alone provides the greatest resistance for both drugs.

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“…Susceptibility of the recombinant mutant viruses and the wild-type HIV-1 molecular clone HXB2D to adefovir, tenofovir, zidovudine, lamivudine and ritonavir was assessed with a modified XTT-based assay in MT-2 cells as previously described [24]. All infections were done with 1.2×10 6 cells at a multiplicity of infection (m.o.i.)…”

Section: Methodsmentioning

confidence: 99%

Increased Drug Susceptibility of HIV-1 Reverse Transcriptase Mutants Containing M184V and Zidovudine-Associated Mutations: Analysis of Enzyme Processivity, Chain-Terminator Removal and Viral Replication

2000

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The presence of the HIV reverse transcriptase (RT) resistance mutation, M184V, induced by lamivudine and abacavir treatment results in increased tenofovir, adefovir and zidovudine susceptibility for HIV-1 with zidovudine-associated RT mutations in vitro. Treatment with oral prodrugs of tenofovir and adefovir has resulted in substantial HIV-1 RNA reductions in antiretroviral-experienced patient populations who have lamivudine-and zidovudine-resistant HIV-1. An enzymatic analysis was undertaken to elucidate the mechanisms of altered drug susceptibilities of HIV-1 containing zidovudine-associated mutations in the presence or absence of M184V. The inhibition constants (Ki) for the active metabolites of tenofovir, adefovir and zidovudine did not vary significantly between recombinant mutant and wild-type RT enzymes. Although increased removal of chain-terminating inhibitors by pyrophosphorolysis and ATP-dependent unblocking correlated with reduced susceptibility of viruses with zidovudine-associated mutations, a reduction in the removal of chain-terminators was not observed, which would explain the increased drug susceptibility of mutants containing M184V plus zidovudine-associated mutations. However, analyses of single-cycle processivity of the mutant RT enzymes on heteropolymeric RNA templates showed that all M184V-containing mutant RT enzymes were less processive than wild-type RT, most notably for mutants expressing both zidovudine-associated mutations and M184V. Similarly, the in vitro replication capacity of a mutant virus expressing a zidovudine-associated mutation and M184V was significantly reduced compared with wild-type virus. The observed decrease in enzymatic processivity of the M184V-expressing RT enzymes might result in decreased viral replication, which then might contribute to the increased drug susceptibility of HIV-1 expressing these RT mutations.

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Comparative kinetic analyses of interaction of inhibitors with Rauscher murine leukemia virus and human immunodeficiency virus reverse transcriptases

Cited by 21 publications

References 20 publications

Mechanistic studies show that (−)‐FTC‐TP is a better inhibitor of HIV‐1 reverse transcriptase than 3TC‐TP

Mechanistic studies show that (−)‐FTC‐TP is a better inhibitor of HIV‐1 reverse transcriptase than 3TC‐TP

The K65R Reverse Transcriptase Mutation in HIV-1 Reverses the Excision Phenotype of Zidovudine Resistance Mutations

Increased Drug Susceptibility of HIV-1 Reverse Transcriptase Mutants Containing M184V and Zidovudine-Associated Mutations: Analysis of Enzyme Processivity, Chain-Terminator Removal and Viral Replication

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