18th International HIV Drug Resistance Workshop Basic Principles & Clinical Implications June 9–13 2009, Fort Myers, Florida, USA

MECHANISMS OF INHIBITION AND RESISTANCE TO TRANSLOCATION DEFICIENT REVERSE TRANSCRIPTASE INHIBITORS

Antivir Ther 2009; 14 Suppl 1:A21 (abstract no. 20)

B Marchand¹, L Michailidis¹, EI Kodama², E Ryan¹, R Do¹, M Matsuoka², N Ashida³, E Nagy⁴, H Mitsuya^5,6, MA Parniak⁴ and SG Sarafianos^1
1University of Missouri, Columbia, MO, USA; ²Kyoto University, Kyoto, Japan; ³Yamasa Corporation, Chiba, Japan; ⁴University of Pittsburgh, Pittsburgh, PA, USA; ⁵Kumamoto University, Kumamoto, Japan; ⁶NIH, Bethesda, MD, USA

BACKGROUND: 4´-Ethynyl-2-fluoro-2´-deoxyadenosine (EFdA) is a highly potent nucleoside analog reverse transcriptase inhibitor (NRTI) of wild-type (WT) and clinically relevant drug resistant mutants. Unlike other NRTIs, EFdA has a 3´OH group, which could facilitate activation of the inhibitor to the triphosphate (TP) form, improve binding by the reverse transcriptase (RT) and decrease resistance. Resistance to EFdA was selected by serial passages in cell culture and three mutations were found in RT: M184V, T165R and I142V. We studied and identified the mechanism of inhibition of RT by EFdA, as well as the mechanism of resistance to EFdA conferred by these mutations.

METHODS: We used biochemical tools, such as enzyme kinetics and gel-based characterization to study the behavior of purified WT and mutant RTs in the presence of EFdA and other inhibitors.

RESULTS: Incorporation of EFdA-monophosphate (MP) in the elongating DNA chain results in interruption of primer extension at the point of incorporation of the inhibitor. We found that the presence of EFdA-MP at the 3'end of a primer prevents RT translocation. The study of the EFdA-resistant enzymes revealed that mutations M184V and T165R have a decreased affinity for EFdA-TP compared with WT RT, resulting in discrimination. These two mutations also decrease the rate of excision of incorporated EFdA, which may explain the low level of resistance observed. When mutation I142V is added, the rate of excision is partially rescued, improving the resistance level of the mutant enzyme. Notably, the presence of the resistance mutations mentioned above comes at a cost to the replication capacity of the mutant viruses, which can be explained by a reduced efficiency of nucleotide incorporation by the mutant RT.

CONCLUSIONS: We found that EFdA inhibits RT by preventing translocation of the polymerase, introducing a novel mechanism of inhibition for a nucleoside analog. We named this type of inhibitors ‘translocation-deficient RT inhibitors’ (TDRTI). Mutations M184V, T165R and I142V confer resistance to TDRTIs by both discrimination and excision mechanisms. To our knowledge, this is the first example of a mutant RT (M184V/T165R/I142V) using these previously thought mutually exclusive mechanisms to evade inhibition by an NRTI.

2009-06-09
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