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Inactivation of human immunodeficiency virus type 1 reverse transcriptase by oltipraz: evidence for the formation of
S J Chavan1, W G Bornmann, C Flexner
1Molecular Pharmacology and Therapeutics Program, Memorial Sloan-Kettering Cancer Center, New York, New York 10021, USA.
Abstract:
Oltipraz (5-pyrazinyl-4-methyl-1,2-dithiole-3-thione), which is undergoing clinical evaluation as an anticarcinogen, also inhibits HIV-1 replication (IC50 approximately equal to 10 microM). The inactivation of RT appears to be a relevant antiviral mechanism since oltipraz blocks viral replication in acutely infected T-cell lines, but is ineffective in chronically infected ACH-2 cells (H. J. Prochaska, W. G. Bornmann, P. Baron, and B. Polsky (1995) Mol. Pharmacol. 48, 15-20). Since a nucleophilic amino acid is a likely target for oltipraz, we assessed whether the conserved cysteine residues of HIV-1 RT (38Cys or 280Cys) were the target(s) for oltipraz, and we synthesized [Me 14C]oltipraz to determine if oltipraz forms a stable adduct with RT. Thus, HIV-2 RT as well as wild-type, 38Cys-->Ser, 280Cys-->Ser, and the Cys-->Ser double mutant of HIV-1 RT were purified from the lysates of transformed Escherichia coli strain DH5 alpha (A. Hizi, M. Shaharabany, R. Tal, and S. H. Hughes (1992) J. Biol. Chem. 267, 1293-1297) via a purification procedure that included (NH4)2SO4 fractionation followed by gel filtration, dye-ligand, and ion-exchange chromatographies. Procion yellow H4R was chosen as the dye-ligand chromatography since it was the most potent and selective inhibitor of RT among seventy reactive dyes that were screened. Mono Q anion-exchange chromatography with diethanolamine (pH 9) resulted in the generation of heterodimeric RT from a predominantly homodimeric enzyme preparation. Because the instability of dilute RT preparations at room temperature rendered the kinetic evaluation of inactivation difficult, we sought to identify conditions that prevent denaturation of these enzymes. High concentrations (25 mM) of MgCl2 had a stabilizing effect. Oltipraz behaved kinetically as an irreversible inhibitor of all RTs purified, and the kinetic constants for the inactivation of these enzymes were not significantly different from wild-type HIV-1 RT (Ki = 17.0 +/- 4.1 microM; k3 = 0.214 +/- 0.051 h-1). In stark contrast, oltipraz neither inhibited nor inactivated the Klenow fragment of DNA polymerase I, whose subdomain structure is similar to the p66 subunit of RT. Wild-type RT was incubated with 60 microM [Me 14C]oltipraz for 4 h and was then subjected to gel filtration chromatography. The [14C] label comigrated with RT with a stoichiometry of binding of 0.88 +/- 0.05 oltipraz per inactivated RT subunit (N = 3 experiments), and the [14C] label remained bound after treatment with 4 M urea.(ABSTRACT TRUNCATED AT 400 WORDS)
Insights
Oltipraz inhibits HIV-1 replication by irreversibly inactivating reverse transcriptase (RT). This anticarcinogen targets conserved cysteine residues in RT, forming a stable adduct and blocking viral activity.
Area of Science:
- Biochemistry
- Virology
- Drug Discovery
Background:
- Oltipraz, an anticarcinogen, demonstrates inhibitory activity against HIV-1 replication.
- HIV-1 reverse transcriptase (RT) is a key target for antiviral therapies.
- The mechanism of oltipraz's antiviral effect, particularly its interaction with RT, requires elucidation.
Purpose of the Study:
- To investigate whether conserved cysteine residues (38Cys or 280Cys) in HIV-1 RT are the targets of oltipraz.
- To determine if oltipraz forms a stable adduct with HIV-1 RT.
- To characterize the kinetics of oltipraz-mediated inactivation of HIV-1 RT.
Main Methods:
- Purification of wild-type and mutant HIV-1 RT (including 38Cys-->Ser and 280Cys-->Ser variants) and HIV-2 RT from Escherichia coli.
- Development of a purification strategy involving ammonium sulfate fractionation, gel filtration, dye-ligand chromatography (Procion yellow H4R), and ion-exchange chromatography (Mono Q).
- Kinetic analysis of RT inactivation by oltipraz and assessment of [Me 14C]oltipraz binding to RT using gel filtration chromatography.
Main Results:
- Oltipraz acted as an irreversible inhibitor of purified wild-type and mutant HIV-1 RT and HIV-2 RT, with similar kinetic constants.
- Oltipraz did not inhibit or inactivate the Klenow fragment of DNA polymerase I, suggesting specificity for RT.
- [Me 14C]oltipraz formed a stable adduct with wild-type RT, with a stoichiometry of approximately 0.88 oltipraz molecules per RT subunit, even after urea treatment.
Conclusions:
- Conserved cysteine residues in HIV-1 RT are likely targets for oltipraz-mediated inactivation.
- Oltipraz forms a stable covalent bond with HIV-1 RT, contributing to its antiviral mechanism.
- Oltipraz exhibits specificity for HIV RT, differentiating it from other DNA polymerases.