KINETIC-STUDIES WITH THE NONNUCLEOSIDE HUMAN-IMMUNODEFICIENCY-VIRUS TYPE-1 REVERSE-TRANSCRIPTASE INHIBITOR U-90152E

The bisheteroarylpiperazine U-90152E is a potent inhibitor of human im munodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) and pos sesses excellent anti-HIV activity in HIV-1-infected lymphocytes grown in tissue culture. The compound inhibits both the RNA- and DNA-direct ed DNA polymerase functions of HIV-1 RT. Kinetic studies were carried out to elucidate the mechanism of RT inhibition by U-90152E. Michaelis -Menten kinetics, which are based on the establishment of a rapid equi librium between the enzyme and its substrates, proved inadequate for t he analysis of the experimental data. The data were thus analyzed usin g Briggs-Haldane kinetics, assuming that the reaction is ordered in th at the template:primer binds to the enzyme first, followed by the addi tion of dNTP and that the polymerase is a processive enzyme. Based on these assumptions, a velocity equation was derived, which allows the c alculation of all the essential forward and backward rate constants fo r the reactions occurring between the enzyme, its substrates and the i nhibitor. The results obtained indicate that U-90152E acts exclusively as a mixed inhibitor with respect to the template:primer and dNTP bin ding sites for both the RNA- and DNA-directed DNA polymerase domains o f the enzyme. The inhibitor shows a significantly higher binding affin ity for the enzyme-substrate complexes than for the free enzyme and co nsequently does not directly impair the functions of the substrate bin ding sites. Therefore, U-90152E appears to impair an event occurring a fter the formation of the enzyme-substrate complexes, which involves e ither inhibition of the phosphoester bond formation or translocation o f the enzyme relative to its template:primer following the formation o f the ester bond.