title: Steady-state kinetic studies with the non-nucleoside HIV-1 reverse transcriptase inhibitor U-87201E.
abstract: The multifunctional HIV-1 RT (human immunodeficiency virus type 1-reverse transcriptase) enzyme possesses three main functions including the RNA- and DNA-directed DNA polymerases and the RNase H. The bisheteroarylpiperazine U-87201E inhibits the two polymerase functions but not the RNase H. Enzymatic kinetic studies of the HIV-1 RT-catalyzed RNA- and DNA-directed DNA polymerase activities were carried out in order to determine if the inhibitor interferes with either the template:primer or the deoxyribonucleotide triphosphate (dNTP)-binding sites of the enzyme. The data were analyzed using steady-state kinetics, considering that the polymerase reaction is ordered in that the template:primer is added first, followed by the dNTP and that the enzyme functions processively. The data were consistent with the model. The steady-state rate constants for the forward and backward reactions were of similar magnitude for both the RNA- and DNA-catalyzed DNA polymerases and suggest that both functions share the same substrate-binding sites. The dissociation constants for the enzyme-inhibitor and enzyme-substrate-inhibitor complexes were somewhat higher for the DNA-directed DNA polymerase function as compared to the RNA directed one. This indicates that U-87201E is a more potent inhibitor for the RNA-directed DNA polymerase than the DNA-directed DNA polymerase. The pattern of inhibition exerted by U-87201E was noncompetitive with respect to both the nucleic acid and nucleotide-binding sites of the RT enzyme for both the RNA- and DNA-directed DNA polymerases. Hence, U-87201E inhibits these functions by interacting with a site distinct from the template:primer and dNTP-binding sites. HIV-2 RT was insensitive to U-87201E, demonstrating the unique sensitivity of HIV-1 RT to this inhibitor.
authors.affiliation: Upjohn Company, Kalamazoo, Michigan 49001.
authors.forename: I W
authors.initials: IW
authors.lastname: Althaus
authors.forename: J J
authors.initials: JJ
authors.lastname: Chou
authors.forename: A J
authors.initials: AJ
authors.lastname: Gonzales
authors.forename: M R
authors.initials: MR
authors.lastname: Deibel
authors.forename: K C
authors.initials: KC
authors.lastname: Chou
authors.forename: F J
authors.initials: FJ
authors.lastname: Kezdy
authors.forename: D L
authors.initials: DL
authors.lastname: Romero
authors.forename: P A
authors.initials: PA
authors.lastname: Aristoff
authors.forename: W G
authors.initials: WG
authors.lastname: Tarpley
authors.forename: F
authors.initials: F
authors.lastname: Reusser
mesh_terms: D004279:DNA, Viral / Q000378:metabolism; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D054303:HIV Reverse Transcriptase; D015497:HIV-1 / Q000201:enzymology*; D007700:Kinetics; D015394:Molecular Structure; D019384:Nucleic Acid Synthesis Inhibitors; D010879:Piperazines / Q000378:metabolism / Q000494:pharmacology*; D012367:RNA, Viral / Q000378:metabolism; D018894:Reverse Transcriptase Inhibitors; D013379:Substrate Specificity
chemical_list: D004279:DNA, Viral; D019384:Nucleic Acid Synthesis Inhibitors; D010879:Piperazines; D012367:RNA, Viral; D018894:Reverse Transcriptase Inhibitors; D054303:HIV Reverse Transcriptase; D004259:DNA-Directed DNA Polymerase; C076632:atevirdine