Kinetics of inhibition of endogenous human immunodeficiency virus type 1 reverse transcription by 2',3'-dideoxynucleoside 5'-triphosphate, tetrahydroimidazo-[4,5,1-jk][1,4]-benzodiazepin-2(1H)-thion e, and 1-[(2-hydroxyethoxy)methyl]-6-(phenylthio)thymine derivatives.
Recently, tetrahydroimidazo-[4,5,1-jk][1,4]-benzodiazepin-2(1H)-one and -thione (TIBO) and 1-[(2-hydroxyethoxy)methyl]-6-(phenylthio)thymine (HEPT) compounds have been shown to be potent, selective, and specific inhibitors of human immunodeficiency virus type 1 (HIV-1) replication in vitro. They interact with the reverse transcriptase of HIV-1 in a way different from that of previously studied reverse transcriptase (RT) inhibitors. We established an endogenous RT assay, starting from intact HIV-1 virions. This assay mimics the reverse transcription process in the HIV-infected cell more closely than RT assays with artificial templates. We investigated the inhibition of endogenous HIV-1 reverse transcription by the TIBO derivative (+)-(S)-4,5,6,7-tetrahydro-5-methyl-6-(3-methyl-2-butenyl)imidazo [4,5,1-jk][1,4]-benzodiazepin-2(1H)-thione (R-82150) in comparison with the HEPT derivative 5-ethyl-1-ethoxymethyl-6-(phenylthio)uracil (E-EPU) and 2',3'-dideoxyguanosine 5'-triphosphate. The kinetics and characteristics of RT inhibition by TIBO in the endogenous RT assay were similar to those found previously for the exogenous RT assay (following addition of exogenous template/primer); thus, RT inhibition by TIBO was specific for HIV-1 and the extent of RT inhibition was dependent on which of the four substrates (dATP, dTTP, dGTP, and dCTP) was present in limited concentrations. Of the three enzymatic activities, RNA-dependent DNA polymerization was preferentially inhibited, and inhibition was not competitive with respect to the natural substrates. HIV-1 RT behaved as an allosteric enzyme, which means that positive cooperativity for binding of the substrate was observed. TIBO behaved as an allosteric inhibitor by causing a concentration-dependent decrease in this cooperativity.
Debyser
Z
Z
Rega Institute for Medical Research, Katholieke Universiteit Leuven, Belgium.
Vandamme
A M
AM
Pauwels
R
R
Baba
M
M
Desmyter
J
J
De Clercq
E
E
D000494:Allosteric Regulation; D000998:Antiviral Agents / Q000494:pharmacology*; D001483:Base Sequence; D001569:Benzodiazepines / Q000494:pharmacology*; D002460:Cell Line; D003848:Deoxyguanine Nucleotides / Q000494:pharmacology*; D054306:Dideoxynucleotides; D004587:Electrophoresis, Agar Gel; D054303:HIV Reverse Transcriptase; D015497:HIV-1 / Q000187:drug effects / Q000201:enzymology* / Q000302:isolation & purification / Q000502:physiology; D007093:Imidazoles / Q000494:pharmacology*; D007700:Kinetics; D008969:Molecular Sequence Data; D009000:Monocytes / Q000382:microbiology; D012194:RNA-Directed DNA Polymerase / Q000235:genetics; D018894:Reverse Transcriptase Inhibitors; D013941:Thymine / Q000031:analogs & derivatives* / Q000494:pharmacology; D014158:Transcription, Genetic; D014779:Virus Replication / Q000187:drug effects
D000998:Antiviral Agents; D003848:Deoxyguanine Nucleotides; D054306:Dideoxynucleotides; D007093:Imidazoles; D018894:Reverse Transcriptase Inhibitors; C061818:1-((2-hydroxyethoxy)methyl)-6-(phenylthio)thymine; C062687:R 82150; D001569:Benzodiazepines; C031709:2',3'-dideoxyguanosine 5'-triphosphate; D054303:HIV Reverse Transcriptase; D012194:RNA-Directed DNA Polymerase; D013941:Thymine