title: Steady state kinetics and inhibition of HIV-1 reverse transcriptase by a non-nucleoside dipyridodiazepinone, BI-RG-587, using a heteropolymeric template.
abstract: Steady state kinetics and inhibition by a dipyridodiazepinone of the reverse transcriptase from human immunodeficiency virus type 1 (HIV) were studied using a heteropolymeric RNA template with a sequence from the authentic initiation site on the HIV genome. For addition of the first deoxynucleotide to primer, kcat/KM is 0.05 (nM-min)-1 and KM is 10 nM. When all 4 deoxynucleotide triphosphates are present and processive synthesis occurs, catalysis is less efficient; kcat/KM = .0077 (nM-min)-1 and KM = 100 nM for dATP. These results are consistent with a rate determining conformation change involved in translocation of the enzyme along the template. Inhibition by the dipyridodiazepinone BI-RG-587 is noncompetitive with respect to both nucleotide and template-primer; this compound decreases Vmax but does not affect KM. Thus, this inhibitor binds to a site distinct from the substrate binding sites with Ki of 220 nM. Inhibition by BI-RG-587 results in a uniform decrease in amount of products of all lengths rather than a shift from longer to shorter products, suggesting the inhibitor does not affect processivity of reverse transcriptase.
authors.affiliation: Boehringer Ingelheim Pharmaceuticals Inc., Ridgefield, CT 06877.
authors.forename: E B
authors.initials: EB
authors.lastname: Kopp
authors.forename: J J
authors.initials: JJ
authors.lastname: Miglietta
authors.forename: A G
authors.initials: AG
authors.lastname: Shrutkowski
authors.forename: C K
authors.initials: CK
authors.lastname: Shih
authors.forename: P M
authors.initials: PM
authors.lastname: Grob
authors.forename: M T
authors.initials: MT
authors.lastname: Skoog
mesh_terms: D001483:Base Sequence; D004591:Electrophoresis, Polyacrylamide Gel; D015497:HIV-1 / Q000201:enzymology*; D007700:Kinetics; D008969:Molecular Sequence Data; D019829:Nevirapine; D011725:Pyridines / Q000494:pharmacology*; D012367:RNA, Viral / Q000235:genetics; D018894:Reverse Transcriptase Inhibitors; D013698:Templates, Genetic
chemical_list: D011725:Pyridines; D012367:RNA, Viral; D018894:Reverse Transcriptase Inhibitors; D019829:Nevirapine