Abstract
The quinoline U-78036 represents a new class of non-nucleoside human immunodeficiency virus (HIV)-1 reverse transcriptase inhibitors. The agent possesses excellent antiviral activity at nontoxic doses in HIV-1-infected lymphocytes grown in tissue culture. Enzymatic kinetic studies of the HIV-1 reverse transcriptase (RT)-catalyzed RNA-directed DNA polymerase function were carried out in order to determine whether the inhibitor interacts with the template-primer or deoxyribonucleotide triphosphate (dNTP) binding sites of the polymerase. The data were analyzed using steady-state or Briggs-Haldane kinetics assuming that the template-primer binds to the enzyme first followed by the dNTP and that the polymerase functions processively. The calculated rate constants are in agreement with this model. The results show that the inhibitor acts as a mixed to noncompetitive inhibitor with respect to both the template-primer and the dNTP binding sites of the enzyme. Hence, U-78036 inhibits the RNA-directed DNA polymerase activity of RT by interacting with a site distinct from the template-primer and dNTP binding sites. Moreover, the potency of U-78036 is dependent on the base composition of the template-primer. The equilibrium constants for various enzyme-substrate-inhibitor complexes were at least seven times lower for the poly(rC).(dG)10-catalyzed system than the one catalyzed by poly(rA).(dT)10. In addition, the inhibitor does not impair the DNA-dependent DNA polymerase activity and the RNase H function of HIV-1 RT nor does it inhibit the RNA-directed DNA polymerase activity of the HIV-2, avian myoblastoma virus, and murine leukemia virus RT enzymes.
Highlights
The data were analyzed using steady-state or Briggs-Haldane kinetics assuming that thetemplate-primer bindsto thenzyme first followed by the dNTP and that the polymerase functions processively
The results show that the inhibitor acts as a mixed to noncompetitive inhibitor withrespect to both the template-primer and the dNTP binding siotfesthe enzyme
Antiviral Actiuity-The quinoline U-78036 is a potent inhibitor of HIV-1 RT and hsahsown excellent antiviralactivity at nontoxic doses in HIV-1-infected lymphocytes grown in tissue culture
Summary
Preparations were purchased from Life Technologies, Inc. These enzymes were assayed in the same standard reaction mixture as described above for the HIV RT. An ordered mechanism was assumed, whereby the template-primer complex binds first to the enzyme, followed by the addition of dNTP [29, 30]. U-78036,an Inhibitor of HIV-1 ReTvrearnsescriptase tions, the formation of the phosphoester bond can be consid- The zero-one matrix is transformed into a B matrix in ered as irreversible, since the reverse reaction occurs at an which the diagonal element in the ithrow is equal to thesum extremely slow rateandthe dissociation of the enzyme- of the elements in the ithrow of matrix A , while all the other product complex into its components isalso negligible during elements just equal the negatives of the corresponding elethe initial reaction phase.
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