HIV-1 REVERSE-TRANSCRIPTASE CAN DISCRIMINATE BETWEEN 2 CONFORMATIONALLY LOCKED CARBOCYCLIC AZT TRIPHOSPHATE ANALOGS

It has been proposed that the preference of 3'-azido-3'-deoxythymidine (AZT) for the extreme E-3 (south) conformation, as observed in its X- ray structure, is responsible for its potent anti-HIV activity. Howeve r, it has also been suggested that the antipodal north conformation ma y be required for the strong interaction of AZT 5'-triphosphate with i ts target enzyme, HIV reverse transcriptase (RT). To resolve this issu e, we have constructed two conformationally rigid carbocyclic analogue s of AZT which are locked permanently into opposite E-2 (north) and E- 3 (south) conformations in order to test the ability of the correspond ing 5'-triphosphates to inhibit RT. The two isomeric carbocyclic analo gues of AZT, (N)-methano-carba-AZT (1) and (S)-methano-carba-AZT (2), were constructed on a bicyclo[3.1.0]hexane template that exhibits a ri gid pseudoboat conformation, capable of mimicking the furanose pucker in the classical north and south conformations that are characteristic of standard nucleosides. The unique conformational properties of 1 an d 2 observed by both X-ray and solution NMR studies showed the existen ce of the same invariant conformations in solution and in the solid st ate. In addition, differences observed in the outcome of the Mitsunobu inversion of a secondary hydroxyl function attempted with both bicycl o[3.1.0]hexane nucleoside analogues could be explained by the rigid ps eudoboat nature of this system. In one case, the bicyclic system facil itated formation of an anhydronucleoside intermediate, whereas in the other it completely prevented its formation. The chemically synthesize d 5'-triphosphates of 1 and 2 were evaluated directly as RT inhibitors using both a recombinant enzyme and enzyme obtained and purified dire ctly from wild-type viruses. The results showed that inhibition of RT occurred only with the conformationally locked E-2 (N)-methano-carba-A ZT 5'-triphosphate. This inhibition was equipotent to and kinetically indistinguishable from that produced by AZT 5'-triphosphate. The antip odal E-3 (S)-methano-carba-AZT 5'-triphosphate, on the other hand, did not inhibit RT.