       Document 0251
 DOCN  M9630251
 TI    Inactivation of human immunodeficiency virus type 1 reverse
       transcriptase by oltipraz: evidence for the formation of a stable
       adduct.
 DT    9603
 AU    Chavan SJ; Bornmann WG; Flexner C; Prochaska HJ; Molecular Pharmacology
       and Therapeutics Program, Memorial; Sloan-Kettering Cancer Center, New
       York, New York 10021, USA.
 SO    Arch Biochem Biophys. 1995 Dec 1;324(1):143-52. Unique Identifier :
       AIDSLINE MED/96095703
 AB    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)
 DE    Antiviral Agents/METABOLISM/*PHARMACOLOGY  Binding Sites
       Chromatography, Affinity  Comparative Study  DNA Polymerase I/DRUG
       EFFECTS  Enzyme Stability/DRUG EFFECTS  Escherichia coli/GENETICS
       HIV-1/*ENZYMOLOGY/GENETICS  Kinetics  Magnesium/PHARMACOLOGY  Mutation
       Pyrazines/METABOLISM/*PHARMACOLOGY  Recombinant
       Proteins/BIOSYNTHESIS/DRUG EFFECTS/ISOLATION & PURIF  Reverse
       Transcriptase Inhibitors/METABOLISM/*PHARMACOLOGY  RNA-Directed DNA
       Polymerase/*DRUG EFFECTS/GENETICS/ISOLATION &  PURIF/METABOLISM
       Support, U.S. Gov't, P.H.S.  JOURNAL ARTICLE

       SOURCE: National Library of Medicine.  NOTICE: This material may be
       protected by Copyright Law (Title 17, U.S.Code).

