Steady state kinetics and inhibition of HIV-1 reverse transcriptase by a non-nucleoside dipyridodiazepinone, BI-RG-587, using a heteropolymeric template.
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.
Kopp
E B
EB
Boehringer Ingelheim Pharmaceuticals Inc., Ridgefield, CT 06877.
Miglietta
J J
JJ
Shrutkowski
A G
AG
Shih
C K
CK
Grob
P M
PM
Skoog
M T
MT
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
D011725:Pyridines; D012367:RNA, Viral; D018894:Reverse Transcriptase Inhibitors; D019829:Nevirapine