Dissecting the effects of DNA polymerase and ribonuclease H inhibitor combinations on HIV-1 reverse-transcriptase activities.
Although HIV-1 reverse transcriptase (RT) DNA polymerase and ribonuclease H (RNase H) activities reside in spatially distinct domains of the enzyme, inhibitors that bind in the RT polymerase domain can affect RNase H activity. We used both gel assays and a real-time FRET assay to analyze the impact of three mechanistically distinct RT polymerase inhibitors on RNase H activity in vitro. The nucleoside analogue 3'-azido-3'-deoxythymidine triphosphate (AZT-TP) had no effect, whereas the pyrophosphate analogue phosphonoformate (PFA) inhibited RNase H activity in a concentration-dependent manner. Nonnucleoside RT inhibitors (NNRTIs) enhanced RNase H catalysis, but the cleavage products differed substantially for RNA/DNA hybrid substrates of different lengths. A comparison of 61 different RT crystal structures revealed that NNRTI binding opened the angle between the polymerase and RNase H domains of the p66 subunit and reduced the relative motion of the thumb and RNase H regions, suggesting that NNRTI enhancement of RNase H cleavage may result from increased accessibility of the RNase H active site to the RNA/DNA hybrid duplex. We also examined the effects of combining a diketo acid (DKA) RNase H inhibitor with various RT polymerase inhibitors on polymerase-independent RNase H cleavage, RNA-dependent DNA polymerization, and in reverse-transcription assays. Interestingly, although the NNRTI decreased DKA potency in polymerase-independent RNase H assays, NNRTI/DKA combinations were synergistic in inhibiting reverse transcription overall, indicating that regimens incorporating both NNRTI and RNase H inhibitors may be therapeutically beneficial.
Shaw-Reid
Cathryn A
CA
Department of Antiviral Research, Merck Research Laboratories, West Point, Pennsylvania 19486-0004, USA.
Feuston
Bradley
B
Munshi
Vandna
V
Getty
Krista
K
Krueger
Julie
J
Hazuda
Daria J
DJ
Parniak
Michael A
MA
Miller
Michael D
MD
Lewis
Dale
D
D019380:Anti-HIV Agents / Q000737:chemistry* / Q000494:pharmacology; D001665:Binding Sites; D002087:Butyrates / Q000737:chemistry / Q000494:pharmacology; D002384:Catalysis / Q000187:drug effects; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D054306:Dideoxynucleotides; D004338:Drug Combinations; D004357:Drug Synergism; D017245:Foscarnet / Q000737:chemistry / Q000494:pharmacology; D054303:HIV Reverse Transcriptase / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D006868:Hydrolysis; D007700:Kinetics; D019384:Nucleic Acid Synthesis Inhibitors; D010078:Oxazines / Q000737:chemistry / Q000494:pharmacology; D018894:Reverse Transcriptase Inhibitors / Q000737:chemistry* / Q000494:pharmacology; D016914:Ribonuclease H / Q000037:antagonists & inhibitors* / Q000378:metabolism; D013876:Thiophenes / Q000737:chemistry / Q000494:pharmacology; D013942:Thymine Nucleotides / Q000737:chemistry / Q000494:pharmacology; D015215:Zidovudine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000494:pharmacology
C472557:4-(5-(benzoylamino)thien-2-yl)-2,4-dioxobutanoic acid; D019380:Anti-HIV Agents; D002087:Butyrates; D054306:Dideoxynucleotides; D004338:Drug Combinations; D019384:Nucleic Acid Synthesis Inhibitors; D010078:Oxazines; D018894:Reverse Transcriptase Inhibitors; D013876:Thiophenes; D013942:Thymine Nucleotides; C105749:ZM 260384; D017245:Foscarnet; D015215:Zidovudine; C052873:zidovudine triphosphate; D054303:HIV Reverse Transcriptase; D004259:DNA-Directed DNA Polymerase; D016914:Ribonuclease H