Inactivation of human immunodeficiency virus type 1 reverse transcriptase by oltipraz: Evidence for the formation of a stable adduct Journal Article
| Authors: | Chavan, S. J.; Bornmann, W. G.; Flexner, C.; Prochaska, H. J. |
| Article Title: | Inactivation of human immunodeficiency virus type 1 reverse transcriptase by oltipraz: Evidence for the formation of a stable adduct |
| 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. © 1995 Academic Press, Inc. |
| Keywords: | mutation; nonhuman; comparative study; human immunodeficiency virus infection; t lymphocyte; enzyme inhibition; pyrazines; drug effect; enzyme inhibitor; kinetics; escherichia coli; enzyme inactivation; recombinant proteins; binding sites; human immunodeficiency virus; virus replication; hiv; human immunodeficiency virus 1; hiv-1; antiviral agents; magnesium; dna polymerase i; enzyme stability; chromatography, affinity; reverse transcriptase inhibitors; rna directed dna polymerase; priority journal; article; support, u.s. gov't, p.h.s.; human immunodeficiency virus 2; rna-directed dna polymerase; oltipraz; 1, 2-dithiole-3-thiones; anticarcinogen; dye-ligand chromatography; hiv-1 reverse transcriptase |
| Journal Title: | Archives of Biochemistry and Biophysics |
| Volume: | 324 |
| Issue: | 1 |
| ISSN: | 0003-9861 |
| Publisher: | Elsevier Inc. |
| Date Published: | 1995-12-01 |
| Start Page: | 143 |
| End Page: | 152 |
| Language: | English |
| DOI: | 10.1006/abbi.1995.9916 |
| PUBMED: | 7503549 |
| PROVIDER: | scopus |
| DOI/URL: | |
| Notes: | Article -- Export Date: 28 August 2018 -- Source: Scopus |
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