Steady-state kinetic studies with the non-nucleoside HIV-1 reverse transcriptase inhibitor U-87201E.

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.