Partial selective inhibition of HIV-1 reverse transcriptase and human DNA polymerases γ and β by thiated 3'-fluorothymidine analogue 5'-triphosphates.
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 γ, 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 γ (pol γ) and β (pol β) 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 K(i)(app) values of 0.091 and 0.022 μM 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 γ with K(i)(app) of 0.174 μM, while 4-SFLTTP in anti conformation inhibited this enzyme similarly to FLTTP, i.e., non-competitively, with K(i)(app) of 0.055 μM. Both 4-SFLTTP and 2-SFLTTP were competitive inhibitors of human DNA pol β, with K(i)(app) values of 16.84 and 4.04 μM, 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 γ 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.
Wińska
Patrycja
P
Institute of Biochemistry and Biophysics, Polish Academy of Sciences, 5A Pawińskiego St., 02-106 Warszawa, Poland.
Miazga
Agnieszka
A
Poznański
Jarosław
J
Kulikowski
Tadeusz
T
D019380:Anti-HIV Agents / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology*; D019951:DNA Polymerase beta / Q000037:antagonists & inhibitors*; D000074002:DNA Polymerase gamma; D004259:DNA-Directed DNA Polymerase; D015224:Dideoxynucleosides / Q000494:pharmacology; D054306:Dideoxynucleotides / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology*; D054303:HIV Reverse Transcriptase / Q000037:antagonists & inhibitors*; D015497:HIV-1 / Q000187:drug effects* / Q000201:enzymology; D006801:Humans; D008279:Magnetic Resonance Imaging; D008968:Molecular Conformation; D019384:Nucleic Acid Synthesis Inhibitors; D011131:Polyribonucleotides / Q000378:metabolism; D018894:Reverse Transcriptase Inhibitors / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology*; D013329:Structure-Activity Relationship; D013942:Thymine Nucleotides / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology*
C554525:3'-deoxy-3'-fluoro-2-thiothymidine 5'-triphosphate; C554524:3'-deoxy-3'-fluoro-4-thiothymidine 5'-triphosphate; D019380:Anti-HIV Agents; D015224:Dideoxynucleosides; D054306:Dideoxynucleotides; D019384:Nucleic Acid Synthesis Inhibitors; D011131:Polyribonucleotides; D018894:Reverse Transcriptase Inhibitors; D013942:Thymine Nucleotides; C514824:reverse transcriptase, Human immunodeficiency virus 1; D054303:HIV Reverse Transcriptase; D019951:DNA Polymerase beta; D000074002:DNA Polymerase gamma; D004259:DNA-Directed DNA Polymerase; C002854:alovudine