Inactivation of human immunodeficiency virus type 1 reverse transcriptase by oltipraz: Evidence for the formation of a stable adduct

Surendra J. Chavan, William G. Bornmann, Charles Flexner, Hans J. Prochaska

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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≅ 10 μM). 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 or280Cys) were the target(s) for oltipraz, and we synthesized [Me14C]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 transformedEscherichia colistrain DH5α (A. Hizi, M. Shaharabany, R. Tal, and S. H. Hughes (1992)J. Biol. Chem.267, 1293-1297) via a purification procedure that included (NH4)2SO4fractionation 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 MgCl2had 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 μM;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 μM[Me14C]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 4Murea. Our results indicate that: (a) oltipraz does not act like a reactive sulfhydryl reagent in the case of RT; (b) oltipraz exhibits specificity for the proteins it inactivates; and (c) the inactivation is due to the stoichiometric formation of a stable adduct. Studies are underway to determine the target for oltipraz on RT.

Original languageEnglish (US)
Pages (from-to)143-152
Number of pages10
JournalArchives of Biochemistry and Biophysics
Volume324
Issue number1
DOIs
StatePublished - Dec 1 1995

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Keywords

  • 1, 2-dithiole-3-thiones
  • Anticarcinogen
  • Dye-ligand chromatography
  • Enzyme inhibitor
  • HIV

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology

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