Partial selective inhibition of HIV-1 reverse transcriptase and human DNA polymerases gamma and beta by thiated 3'-fluorothymidine analogue 5'-triphosphates

Abstract

3'-Deoxy-3'-fluorothymidine (FLT, alovudine®) belongs to the most potent agents inhibiting HIV-1 replication. Its 5'-triphosphate (FLTTP) is a potent inhibitor of HIV-1 reverse transcriptase (HIV RT). Unfortunately, FLT exerts substantial hematologic toxicity both in vitro and in vivo. It was suggested that this toxicity may be related to inhibition of human DNA polymerases, especially mitochondrial DNA polymerase gamma, by nucleoside analogue 5'-triphosphates leading to termination of DNA synthesis and mitochondrial dysfunction. To decrease the toxicity of FLT, its thiated analogues, 4-SFLT and 2-SFLT, were previously synthesized and shown to be potent inhibitors of HIV-1 with low in vitro cytotoxicity. To explain this phenomenon in the present study the synthesis of 5'-triphosphates of thiated FLT analogues was undertaken and their interaction with recombinant HIV-1 RT and human DNA polymerases gamma (pol �) and beta (pol �) was investigated. It was shown that 3'-deoxy-3'-fluoro-4-thiothymidine 5'-triphosphate (4-SFLTTP) and 3'-deoxy-3'-fluoro-2-thiothymidine 5'-triphosphate (2-SFLTTP) were, similarly to FLTTP, potent competitive inhibitors of HIV-1 RT, with Kapp i values of 0.091 and 0.022uM respectively. It is of interest that 2-SFLTTP, a compound in an unusual syn conformation around the glycosidic bond was an uncompetitive inhibitor of human mitochondrial DNA pol gamma with Kappi of 0.174uM, while 4- SFLTTP in anti conformation inhibited this enzyme similarly to FLTTP, i.e., non-competitively, with Kappi of 0.055uM. Both 4-SFLTTP and 2-SFLTTP were competitive inhibitors of human DNA pol beta, with Kappi values of 16.84 and 4.04uM, respectively. The results point to partially selective inhibition of HIV RT by thiated 3'-fluorothymidine 5'-triphosphate analogues. Of special interest is that 2-SFLTTP, showing syn conformation, is a less potent inhibitor of human mitochondrial pol gamma than 4-SFLTTP and FLTTP, both in the anti conformation, and has a higher inhibitory activity against HIV-1 RT than 4-SFLTTP. Moreover, the parent nucleoside 2-SFLT possessing the syn conformation shows a more potent anti-HIV-1 activity and a better selectivity index than its 4-thio isomer in the anti conformation (Matthes et al., 1989; Poopeiko et al., 1995), 2-SFLT is a potent and selective anti-HIV-1 agent with the selectivity index 4-fold higher than that of FLT. Findings regarding the mechanisms of antiviral and cytotoxic activities of FLT and its thioanalogues are discussed.