Non-nucleoside inhibitors of HIV-1 reverse transcriptase inhibit phosphorolysis and resensitize the 3'-azido-3'-deoxythymidine (AZT)-resistant polymerase to AZT-5'-triphosphate.
Removal of 3'-azido-3'deoxythymidine (AZT) 3'-azido-3'-deoxythymidine 5'-monophosphate (AZTMP) from the terminated primer mediated by the human HIV-1 reverse transcriptase (RT) has been proposed as a relevant mechanism for the resistance of HIV to AZT. Here we compared wild type and AZT-resistant (D67N/K70R/T215Y/K219Q) RTs for their ability to unblock the AZTMP-terminated primer by phosphorolysis in the presence of physiological concentrations of pyrophosphate or ATP. The AZT-resistant enzyme, as it has been previously described, showed an increased ability to unblock the AZTMP-terminated primer by an ATP-dependent mechanism. We found that only mutations in the p66 subunit were responsible for this ability. We also found that three structurally divergent non-nucleoside reverse transcriptase inhibitor (NNRTI), nevirapine, TIBO, and a 4-arylmethylpyridinone derivative, were able to inhibit the phosphorolytic activity of the enzyme, rendering the AZT-resistant RT sensitive to AZTTP. The 4-arylmethylpyridinone derivative proved to be about 1000-fold more potent in inhibiting phosphorolysis than nevirapine or TIBO. Moreover, combinations of AZTTP with NNRTIs exhibited an exceptionally high degree of synergy in the inhibition of AZT-resistant enzyme only when ATP or PPi were present, indicating that inhibition of phosphorolysis was responsible for the synergy found in the combination. Our results not only demonstrate the importance of phosphorolysis concerning HIV-1 RT resistance to AZT but also point to the implication of this activity in the strong synergy found in some combinations of NNRTIs with AZT.
Odriozola
Leticia
L
Department of Biochemistry and Molecular Biology, University of Navarre, calle Irunlarrea s/n, 31008 Pamplona, Spain.
Cruchaga
Carlos
C
Andréola
Marieline
M
Dollé
Valérie
V
Nguyen
Chi Hung
CH
Tarrago-Litvak
Laura
L
Pérez-Mediavilla
Alberto
A
Martínez-Irujo
Juan J
JJ
D000255:Adenosine Triphosphate / Q000494:pharmacology; D019380:Anti-HIV Agents / Q000494:pharmacology*; D054306:Dideoxynucleotides; D011756:Diphosphates / Q000494:pharmacology; D024882:Drug Resistance, Viral; D004357:Drug Synergism; D004359:Drug Therapy, Combination; D054303:HIV Reverse Transcriptase / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D020125:Mutation, Missense; D019829:Nevirapine / Q000494:pharmacology; D010766:Phosphorylation / Q000187:drug effects; D021122:Protein Subunits / Q000235:genetics; D018894:Reverse Transcriptase Inhibitors / Q000494:pharmacology*; D013942:Thymine Nucleotides / Q000494:pharmacology; D015215:Zidovudine / Q000031:analogs & derivatives* / Q000494:pharmacology
D019380:Anti-HIV Agents; D054306:Dideoxynucleotides; D011756:Diphosphates; D021122:Protein Subunits; D018894:Reverse Transcriptase Inhibitors; D013942:Thymine Nucleotides; C057287:3'-azido-3'-deoxythymidine 5'phosphate; D015215:Zidovudine; C052873:zidovudine triphosphate; D000255:Adenosine Triphosphate; D019829:Nevirapine; D054303:HIV Reverse Transcriptase