Molecular basis for the enantioselectivity of HIV-1 reverse transcriptase: role of the 3′-hydroxyl group of the L-(β)-ribose in chiral discrimination between D- and L-enantiomers of deoxy- and dideoxy-nucleoside triphosphate analogs

In order to identify the basis for the relaxed enantioselectivity of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) and to evaluate possible cross-resistance patterns between L-nucleoside-, D-nucleosideand non-nucleoside RT inhibitors, to be utilised in anti-HIV-1 combination therapy, we applied an in vitro approach based on the utilisation of six recombinant HIV-1 RT mutants containing single amino acid substitutions known to confer Nevirapine resistance in treated patients. The mutants were compared on different RNA/DNA and DNA/DNA substrates to the wild type (wt) enzyme for their sensitivity towards inhibition by the Dand L-enantiomers of 2′-deoxyand 2′,3′-dideoxynucleoside triphosphate analogs. The results showed that the 3′-hydroxyl group of the L-(β)-2′-deoxyribose moiety caused an unfavourable steric hindrance with critic residues in the HIV-1 RT active site and this steric barrier was increased by the Y181I mutation. Elimination of the 3′-hydroxyl group removed this hindrance and significantly improved binding to the HIV-1 RT wt and to the mutants. These results demonstrate the critical role of both the tyrosine 181 of RT and the 3′-position of the sugar ring, in chiral discrimination between Dand L-nucleoside triphosphates. Moreover, they provide an important rationale for the combination of Dand L-(β)-dideoxynucleoside analogs with non-nucleoside RT inhibitors in anti-HIV chemotherapy, since non-nucleoside inhibitors resistance mutations did not confer crossresistance to dideoxynucleoside analogs. INTRODUCTION Human immunodeficiency virus type 1 (HIV-1) high mutation rate constitutes a major obstacle in the development of effective drugs and vaccines. Error-prone DNA synthesis by HIV-1 reverse transcriptase (RT) and high viral turnover in vivo are responsible for the emergence of cross-resistance between and within classes of anti-retroviral drugs, either nucleoside inhibitors (NI) or non-nucleoside inhibitors (NNI) (1–6). Recently, the combination of two NI, the thymidine analog 3′-azido-2′,3′-dideoxythymidine (AZT) and the new compound L-(β)-2′,3′-dideoxy-3′-thiacytidine (3TC; Lamivudine), has proved to be particularly effective with respect to AZT monotherapy (7–9) since 3TC resistance not only did not confer cross-resistance to AZT, but was also able to restore AZT sensitivity (10–12). 3TC is an L-nucleoside, its sugar moiety having the unnatural L-configuration, opposite to the D-configuration of the natural nucleoside analogs such as AZT. D-(β)and L-(β)-nucleotides have almost superimposable nucleobase and α-phosphorus, differing only in the mutual orientation of the sugar ring (13). HIV-1 RT, contrary to cellular DNA polymerases, has been shown to be able to bind and incorporate L-2′-deoxythymidine triphosphate (L-dTTP) when acting on a homopolymeric template–primer (TP) (14,15), as well as the L-enantiomers of 2′,3′-dideoxynucleoside triphosphate analogs such as L-2′,3′-dideoxythymidine triphosphate (L-ddTTP), L-2′,3′-dideoxycytidine triphosphate (L-ddCTP) and L-2′,3′dideoxy-5-fluorocytidine triphosphate (L-FddCTP) (16,17). Moreover, L-2′,3′-dideoxycytidine (L-ddC) and L-2′,3′dideoxy-5-fluorocytidine (L-FddC) showed potent anti-HIV activity on infected cells (18). Clinical trials are currently underway which explore the efficacy of multiple drug therapy protocols, involving the combined use of NI such as AZT and 3TC together with NNI (19). In light of the potential use of L-nucleoside *To whom correspondence should be addressed. Tel: +39 382 546355; Fax: +39 382 422286; Email: [email protected]