4th International Workshop on HIV Drug Resistance & Treatment Strategies Sitges, Spain, 12–16 June 2000

UNIFYING MOLECULAR MECHANISMS OF RT-MEDIATED DRUG RESISTANCE: SPECIFIC TARGETTING OF DRUG-RESISTANT RT IN VITRO

Antivir Ther. 2000 Jun 12-16; 5 (Suppl. 3):15 (Abstract 17

B Selmi¹, P Meyer³, B Schneider², S Sarfati², D Deville-Bonne², C Guerreiro², J Boretto¹, J Janin³, M Véron² and B Canard^1
1ESIL-AFMB-CNRS, Marseille; ²Institut Pasteur, Paris; and 3 CNRS-LEBS, Gif-sur-Yvette, France

At least three interrelated limitations impede efficient targetting of HIV RT: inadequate intracellular concentrations of nucleoside triphosphate analogues, incomplete inhibition of viral replication and the appearance of drug-resistant virus. Recent results on phenotype/genotype correlations of RT-mediated drug resistance suggest that two critical factors govern RT- mediated drug resistance: the loss of affinity of RT for the nucleotide analogue (group 1) and/or post-replicative repair through pyrophosphorolysis of the analogue chain-terminated viral DNA (group 2). We show that nearly all nucleoside drug resistance mutations in RT belong to either group, which can be readily assessed by biochemical characterization of purified mutant RT. For zidovudine resistance, the pattern of acquisition of zidovudine-resistance mutations (67/70/215/219) over time follows the optimal accommodation of both zidovudine triphosphate discrimination and increased pyrophosphorolysis. In this background, the Y181C suppressive mutation kills pyrophosphorolysis specifically. For stavudine and dideoxynucleoside resistance, pyrophosphorolysis is also increased with the V75T and K65R resistance substitutions, respectively. Using existing data on mutant RTs and our results, a clear correlation between processivity and pyrophosphorolysis is apparent on drug-resistant RTs. Therefore, we decided to target RT-mediated pyrophosphorolysis. Using structural data on both nucleoside diphosphate kinase (NDPK) and RT bound to their respective nucleotides, we found that the pro(Rp) oxygen of the nucleotide aphosphate might be substituted. Consequently, we synthesized novel nucleotide analogues of zidovudine and stavudine nucleotide bearing a boranophosphate group on the a-phosphate. X-ray structures of the analogues in complex with NDPK at 1.85 Å resolution showed the Rp stereoisomer bound to the active site. The same Rp stereoisomer 5′-triphosphate analogue of both zidovudine and stavudine exhibited a 2–10-fold better K_i for RT, and a 5–10-fold decrease in pyrophos phorolytic repair by several clinically relevant drug-resistant RTs. To our surprise, the diphosphate form of these analogues exhibited also a 10-fold better affinity for NDPK, indicating that they might potentially increase the intracellular nucleotide analogue pool. Our results call for an increasing effort to design pyrophosphorolysis inhibitors, as well as to vectorize nucleotide analogues into a bioavailable form. In that context, our novel analogues are excellent candidates to overcome the three limitations mentioned above, and might have a generic value in fighting viral drug resistance.

Download abstract

2000-06-12
17

Copyright © 2000 - International Medical Press Ltd. Reproduction of this abstract (other than one copy for personal reference) must be cleared through the International Medical Press Ltd. 2-4 Idol Lane, London EC3R 5DD UK.