The benzylthio-pyrimidine U-31,355, a potent inhibitor of HIV-1 reverse transcriptase.

U-31,355, or 4-amino-2-(benzylthio)-6-chloropyrimidine is an inhibitor of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) and possesses anti-HIV activity in HIV-1-infected lymphocytes grown in tissue culture. The compound acts as a specific inhibitor of the RNA-directed DNA polymerase function of HIV-1RT and does not impair the functions of the DNA-catalyzed DNA polymerase or the Rnase H of the enzyme. Kinetic studies were carried out to elucidate the mechanism of RT inhibition by U-31,355. The data were analyzed using Briggs-Haldane kinetics, assuming that the reaction is ordered in that the template:primer binds to the enzyme first, followed by the addition of dNTP, and that the polymerase is a processive enzyme. Based on these assumptions, a velocity equation was derived that allows the calculation of all the essential forward and backward rate constants for the reactions occurring between the enzyme, its substrates, and the inhibitor. The results obtained indicate that U-31,355 acts as a mixed inhibitor with respect to the template:primer and dNTP binding sites associated with the RNA-directed DNA polymerase domain of the enzyme. The inhibitor possessed a significantly higher binding affinity for the enzyme-substrate complexes, than for the free enzyme and consequently did not directly affect the functions of the substrate binding sites. Therefore, U-31,355 appears to impair an event occurring after the formation of the enzyme-substrate complexes, which involves either inhibition of the phosphoester bond formation or translocation of the enzyme relative to its template:primer following the formation of the ester bond. Moreover, the potency of U-31,355 depends on the base composition of the template:primer in that the inhibitor showed a much higher binding affinity for the enzyme-poly (rC):(dG)10 complexes than for the poly (rA):(dT)10 complexes.

The quinoline U-78036 is a potent inhibitor of HIV-1 reverse transcriptase.

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.

Enzymatic kinetic studies with the non-nucleoside HIV reverse transcriptase inhibitor U-9843.

The polymer of ethylenesulfonic acid (U-9843) is a potent inhibitor of HIV-1 RT (reverse transcriptase) and the drug possesses excellent antiviral activity at nontoxic doses in HIV-infected lymphocytes grown in tissue culture. The drug also inhibits RTs isolated from other species such as AMV and MLV retroviruses. Enzymatic kinetic studies of the HIV-1 RT catalyzed RNA-directed DNA polymerase function, using synthetic template:primers, indicate that the drug acts generally noncompetitively with respect to the template:primer binding site but the specific inhibition patterns change somewhat depending on the drug concentration. The inhibitor acts noncompetitively with respect to the dNTP binding sites. Hence, the drug inhibits this RT polymerase function by interacting with a site distinct from the template:primer and dNTP binding sites. In addition, the inhibitor also impairs the DNA-dependent DNA polymerase activity of HIV-1 RT and the RNase H function. This indicates that the drug interacts with a target site essential for all three HIV RT functions addressed (RNA- and DNA-directed DNA polymerases, RNase H).

Anti-human immunodeficiency virus effects of dextran sulfate are strain dependent and synergistic or antagonistic when dextran sulfate is given in combination with dideoxynucleosides.

The effects of three molecular weight ranges of dextran sulfate on five different human immunodeficiency virus (HIV) isolates (from patients with acquired immunodeficiency syndrome), alone and in combination with dideoxynucleosides, were investigated in vitro. The higher the molecular weight range of dextran sulfate, the more potent the activity as assessed by a quantitative syncytium formation assay. Although all five HIV isolates had similar susceptibilities to the inhibitory effects of dideoxynucleosides, the two clinical isolates of HIV (HIV type 1 [HIV-1] TM and SP) exhibited a pattern of reduced susceptibility to dextran sulfate when compared with the two cloned isolates (HIV-1 WMF and HIV-2 ROD) and a prototype laboratory strain (HIV-1 IIIB). In combination with dideoxynucleosides, the high-molecular-weight range of dextran sulfate (500,000) resulted in an antagonistic response directed against the two clinical isolates of HIV (HIV-1 TM and SP) when the antiviral concentrations of dextran sulfate were in the ineffective range. Additive or synergistic effects were seen with the other three HIV isolates and all five HIV isolates when the low-molecular-weight range of dextran sulfate (8,000) was used. The results of these studies raise issues on the impact of drug-resistant strains on disease progression and the use of dextran sulfate in combination with nucleoside analogs for the clinical management of HIV disease.

Cellular pharmacology and anti-HIV activity of 2',3'-dideoxyguanosine.

The antiviral activity, uptake, and metabolism of 2',3'-dideoxyguanosine was investigated in human immunodeficiency virus- (HIV) infected and noninfected human cells. 2',3'-Dideoxyguanosine had anti-HIV activity (effective dose 50%: 0.1-1.0 microM) in H-9 and MT-2 cells. The addition of excess (greater than or equal to 30 microM) guanosine, deoxyguanosine, or 8-aminoguanosine had no effect on the anti-HIV activity of 2',3'-dideoxyguanosine. In [8-3H]2',3'-dideoxyguanosine-exposed cells, the intracellular radioactivity was twofold higher than the extracellular. When guanosine, deoxyguanosine, or 8-aminoguanosine was preincubated or added simultaneously to 2',3'-dideoxyguanosine, uptake of 2',3'-dideoxyguanosine was reduced by 28 to 34%, whereas addition of p-nitrobenzylmercaptopurine riboside (20 microM) had no effect. In metabolism studies using H-9 cells, dideoxyguanosine triphosphate could not be detected despite a 24-h incubation of 2',3'-dideoxyguanosine at effective anti-HIV concentrations. The addition of excess (greater than or equal to 30 microM) guanosine, deoxyguanosine, and 8-aminoguanosine, while inhibiting the catabolism of 2',3'-dideoxyguanosine, did not enhance the anabolic conversion of 2',3'-dideoxyguanosine to dideoxyguanosine triphosphate. Our failure to detect the formation of dideoxyguanosine triphosphate and the lack of reversal of antiviral effects by natural purine nucleosides raises questions on the role of this metabolite in the anti-HIV activity of 2',3'-dideoxyguanosine.

Uptake of 2',3'-dideoxyadenosine in human immunodeficiency virus-infected and noninfected human cells.

The uptake of 2',3'-dideoxyadenosine was examined in a human immunodeficiency virus (HIV) infected and uninfected T cell line (H9 cells), a B cell line (Namalwa), and in normal peripheral blood mononuclear cells. After a 10-minute incubation at ambient temperature, the intracellular 2',3'-dideoxyadenosine-derived radioactivity was 8- to 16-fold higher than the extracellular radioactivity. In metabolically inactive cells (0 degrees C), the intracellular and extracellular 2',3'-dideoxyadenosine-derived radioactivities were nearly equal. In infected and noninfected H9 cells, a large excess of p-nitrobenzylmercaptopurine riboside or pyrimidine nucleosides weakly inhibited the uptake of 2',3'-dideoxyadenosine (7-30%), whereas deoxycoformycin was a stronger inhibitor (50-80%). Purine nucleosides minimally enhanced the uptake (10-20%). The cellular uptake was not associated with the accumulation of dideoxyadenosine triphosphate. In normal peripheral blood mononuclear cells, the uptake of 2',3'-dideoxyadenosine was inhibited by all agents except 2'-deoxyadenosine (15% enhancement). In contrast to H9 cells, the formation and accumulation of dideoxyadenosine triphosphate paralleled the uptake of dideoxyadenosine. The results of these studies suggest that the major route of transport of 2',3'-dideoxyadenosine into cells is by simple diffusion and that different metabolic patterns exist among cell lines and normal peripheral blood mononuclear cells. An understanding of these cellular differences could aid in the development of therapeutic strategies directed against HIV.