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-thi which is undergoing clinical evaluation as an anticarcinogen, also inhibits HIV-1 replicat ion (IC50 congruent to 10 mu M). 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 i nfected ACH-S cells (H. J, Prochaska, W. G. Bornmann, P. Baron, and B. Polsky (1995) Mel. Pharmacol. 48, 15-20). Since a nucleophilic amino acid is a likely target for oltipraz, we assessed whether the conserve d cysteine residues of HIV-1 RT ((38)Cys or (280)Cys) were the target( s) for oltipraz, and we synthesized [Me C-14]oltipraz to determine if oltipraz forms a stable adduct with RT, Thus, HTV-2 RT as well as wild type, (38)Cys-->Ser, (280)Cys-->Ser, and the Cys-->Ser double mutant o f HIV-1 RT were purified from the lysates of transformed Escherichia c oli strain DH5 alpha (A, Hizi, M, Shaharabany, R, Tal, and S, H. Hughe s (1992) J. Biol. Chem. 267, 1293-1297) via a purification procedure t hat included (NH4)(2)SO4 fractionation followed by gel filtration, dye -ligand, and ion-exchange chromatographies. Procion yellow H4R was cho sen as the dye-ligand chromatography since it was the most potent and selective inhibitor of RT among seventy reactive dyes that were screen ed. Mono Q anion-exchange chromatography with diethanolamine (pH 9) re sulted in the generation of heterodimeric RT from a predominantly homo dimeric enzyme preparation. Because the instability of dilute RT prepa rations at room temperature rendered the kinetic evaluation of inactiv ation difficult, we sought to identify conditions that prevent denatur ation of these enzymes, High concentrations (25 mM) of MgCl2 had a sta bilizing effect. Oltipraz behaved kinetically as an irreversible inhib itor of all RTs purified, and the kinetic constants for the inactivati on of these enzymes were not significantly different from wild-type HI V-1 RT (K-i = 17.0 +/- 4.1 mu M; k(3) = 0.214 +/- 0.051 h(-1)). In sta rk contrast, oltipraz neither inhibited nor inactivated the Klenow fra gment of DNA polymerase I, whose subdomain structure is similar to the p66 subunit of RT. Wild-type RT was incubated with 60 mu M [Me C-14]o ltipraz for 4 h and was then subjected to gel filtration chromatograph y. The [C-14] label comigrated with RT with a stoichiometry of binding of 0.88 +/- 0.05 oltipraz per inactivated RT subunit (N = 3 experimen ts), and the [C-14] label remained bound after treatment with 4 M urea . Our results indicate that: (a) oltipraz does not act like a reactive sulfhydryl reagent in the case of RT; (b) oltipraz exhibits specifici ty 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. (C) 1995 Academic Press, Inc.