Inactivation of human immunodeficiency virus type 1 reverse transcriptase by oltipraz: evidence for the formation of a stable adduct.
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)
Chavan
S J
SJ
Molecular Pharmacology and Therapeutics Program, Memorial Sloan-Kettering Cancer Center, New York, New York 10021, USA.
Bornmann
W G
WG
Flexner
C
C
Prochaska
H J
HJ
D000998:Antiviral Agents / Q000378:metabolism / Q000494:pharmacology*; D001665:Binding Sites; D002846:Chromatography, Affinity; D004256:DNA Polymerase I / Q000187:drug effects; D004795:Enzyme Stability / Q000187:drug effects; D004926:Escherichia coli / Q000235:genetics; D054303:HIV Reverse Transcriptase; D015497:HIV-1 / Q000201:enzymology* / Q000235:genetics; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D009154:Mutation; D011719:Pyrazines / Q000378:metabolism / Q000494:pharmacology*; D012194:RNA-Directed DNA Polymerase / Q000187:drug effects* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000187:drug effects / Q000302:isolation & purification; D018894:Reverse Transcriptase Inhibitors / Q000378:metabolism / Q000494:pharmacology*; D013871:Thiones; D013876:Thiophenes
D000998:Antiviral Agents; D011719:Pyrazines; D011994:Recombinant Proteins; D018894:Reverse Transcriptase Inhibitors; D013871:Thiones; D013876:Thiophenes; C026209:oltipraz; D054303:HIV Reverse Transcriptase; D012194:RNA-Directed DNA Polymerase; D004256:DNA Polymerase I; D008274:Magnesium