Hypersusceptibility mechanism of Tenofovir-resistant HIV to EFdA

Eleftherios Michailidis,Bruno Marchand,Eiichi N Kodama,Kamalendra Singh,Emily M Ryan,Maxwell D Leslie,Yee T Ong,Andrew D Huber,Atsuko Hachiya,Hiroaki Mitsuya,Karen A Kirby,Stefan G Sarafianos,Michael A Parniak,Jacob C Jackson

doi:10.1186/1742-4690-10-65

Abstract

BackgroundThe 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.ResultsHere 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.ConclusionWe 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.

Highlights

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 nucleos(t) ide RT inhibitors (NRTIs) retains a 3'-hydroxyl group and has remarkable potency against wild-type (WT) and drugresistant Human immunodeficiency virus (HIV)
The K65R RT mutation enhances susceptibility of HIV to EFdA In order to determine the susceptibility of HIV-1 to EFdA we performed single infectivity viral replication assays according to the experimental procedures described in Methods section
We have previously demonstrated that EFdA is highly efficient in suppressing viral replication of clinical isolates harboring signature mutations to other NRTIs and nonnucleoside RT inhibitors (NNRTIs), including isolates containing 3TC/FTC resistance mutation M184V; Thymidine Associated Mutations (TAMs) or Q151M complex mutations that confer resistance to AZT, d4T, and abacavir; and nevirapine and efavirenz resistance mutations K103N and Y181C [45]

Summary

Introduction

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 drugresistant HIVs. 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 drugresistant HIVs. EFdA acts primarily as a chain terminator by blocking translocation following its incorporation into the nascent DNA chain. Human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) is the major target of antiretroviral drug treatments. RT inhibitors constitute the largest class of HIV-1 drugs and are grouped in two separate categories. The efficacy of combination therapies is being challenged by the selection of drugresistant variants of HIV-1

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Discussion

Conclusion