Kinetics of Inhibition of HIV Type 1 Reverse Transcriptase-Bearing NRTI-associated Mutations by Apricitabine Triphosphate

Abstract

We wished to investigate the effects of various mutations in HIV-1 reverse transcriptase (RT) on biochemical inhibition by the active form of a novel nucleoside termed apricitabine. Accordingly, we studied the efficiency of chain-termination mediated by apricitabine triphosphate (TP) in cellfree assays that used either recombinant wild-type or mutated RTs. We also performed steady-statekinetics and primer-unblocking assays. Subtype C RTs were also analysed. The results showed that the K65R mutation in RT caused reductions in the efficiency of chain-termination of apricitabine-TP by increasing its K i . However, K65R did not affect rates of primer unblocking for apricitabine-TP. No significant differences were found between subtype C and subtype B RTs with regard to any of the parameters studied. Other mutations such as M184V, L74V and K103N had no effect on the efficiency of chain termination by apricitabine-TP. Thus, the mechanism of reduced susceptibility to apricitabine of viruses containing K65R in RT seems to be mediated exclusively through a reduction in binding or incorporation of apricitabine-TP. Unlike some other nucleoside analogues, increased excision of incorporated apricitabine does not seem to be a cause of resistance to apricitabine.

A Template-Dependent Dislocation Mechanism Potentiates K65R Reverse Transcriptase Mutation Development in Subtype C Variants of HIV-1

“…Because a proportion of nt polymorphisms that distinguish the two subtypes do not result in changes of the amino acid sequence, it is not surprising that RTs derived from either subtype B or C are similar enzymatically [46], [47], [48], [50].…”

“…K65R-associated treatment failures are uncommon and usually develop at rates between 0 and 3% [42], [43], [44], [45]. To explore this discrepancy, biochemical analysis of the RT enzymes derived from both subtypes were performed and showed that they are very similar; the subtype origin of RT cannot explain the discrepancies observed with respect to K65R development [46], [47].…”

HIV cell-to-cell transmission: effects on pathogenesis and antiretroviral therapy

“…These experimental observations favor the development of NRTIs that resist excision, such as apricitabine, that are currently in clinical trials [83–85]. Moreover, we predict that allosteric drug inhibitors, with similar mechanism of action as NNRTIs and PIs, will be effective against HIV cell-to-cell transmission on their own.…”

Effects of the K65R and K65R/M184V reverse transcriptase mutations in subtype C HIV on enzyme function and drug resistance

“…The Km for dATP and the K i for TFV-DP were determined by filter binding assays as described previously [ 36 ]. In brief, 200 nM dPR were heat-annealed to 300 nM subtype C HIV-1 PBS RNA in a buffer containing 50 mM Tris-HCl pH 7.8 and 50 mM NaCl.…”

“…Incorporated radioactivity was measured by liquid scintillation counting. Kinetic constants were determined using Graphpad Prism 4.0 software as described [ 36 ].…”