HIV-1 resistance to nucleoside reverse transcriptase inhibitors (NRTI) involves discrimination (selective alterations of RT binding of NRTI-triphosphate compared to the analogous dNTP), and chain terminator excision (phosphorolytic removal of the chain-terminating NRTI from the 3'-end of the primer). The latter is important in AZT resistance, and recent data suggest this phenotype may figure widely in NRTI resistance. Both pyrophosphate and ATP act as excision acceptors in vitro, but the intracellular acceptor has not been identified. Unique sets of mutations (thymidine analog mutations, or TAMS) correlate with the chain terminator excision phenotype. TAM-mediated excision is a complex process that is not well understood mechanistically, since most of the work in this area has been descriptive rather than quantitative and has been conducted under varying experimental conditions. Furthermore, two mutation sets are noted in clinical AZT resistance, M41 L/L210W/T215Y, and D67N/K70R/T215F/K219Q. Only the latter set has been studied to any significant extent. Do both sets of mutations provide equivalent biochemical phenotypes? The global aim of this project is to quantitatively clarify the complex excision phenotype. The project comprises four Specific Aims: (1) To conduct a detailed and quantitative in vitro characterization of the effect of TAMS (both mutation sets), using pre-steady state and steady state kinetics as well as direct binding methods (eg., isothermal titration calorimetry) to identify alterations in the RT mechanism due to TAMs, alone and in combination; (2) To assess the effect of mutations antagonistic to TAMs (K65R, M184V and Y181C), examining whether these mutations function in similar manners to restore sensitivity to AZT despite the continued presence of TAMs; (3) To characterize the effect of NRTI chemistry on susceptibility to TAM-mediated excision, examining the effect of alterations in base, sugar, and phosphoryl linkage on RT-catalyzed phosphorolytic excision; and (4) To identify the components involved in the intracellular excision reaction. The studies will use both site-specific mutants as well as material derived from clinical sources in a combined biochemistry/virology approach to exactly define the NRTI excision mechanism, and provide directions for development of antivirals refractory to this resistance phenotype.
|Song, Yongcheng; Chan, Julian M W; Tovian, Zev et al. (2008) Bisphosphonate inhibitors of ATP-mediated HIV-1 reverse transcriptase catalyzed excision of chain-terminating 3'-azido, 3'-deoxythymidine: a QSAR investigation. Bioorg Med Chem 16:8959-67|
|Ilina, Tatiana; Parniak, Michael A (2008) Inhibitors of HIV-1 reverse transcriptase. Adv Pharmacol 56:121-67|
|Dharmasena, Sanjeewa; Pongracz, Zita; Arnold, Eddy et al. (2007) 3'-Azido-3'-deoxythymidine-(5')-tetraphospho-(5')-adenosine, the product of ATP-mediated excision of chain-terminating AZTMP, is a potent chain-terminating substrate for HIV-1 reverse transcriptase. Biochemistry 46:828-36|
|Sluis-Cremer, Nicolas; Arion, Dominique; Parikh, Urvi et al. (2005) The 3'-azido group is not the primary determinant of 3'-azido-3'-deoxythymidine (AZT) responsible for the excision phenotype of AZT-resistant HIV-1. J Biol Chem 280:29047-52|