Cytotoxicity and DNA binding studies of several platinum (II) and palladium (II) complexes of the 2,2'-bipyridine and an anion of 2-pyridinecarboxylic/2-pyrazinecarboxylic acid.

Four water soluble complexes of the type [M(bpy)(a-x)]NO3, where M is Pd(II) or Pt(II), bpy is 2,2-bipyridine, and a-x is anion of 2-pyridinecarboxylic acid or 2-pyrazinecarboxylic acid, have been found to bind calf thymus DNA, possibly through hydrogen binding. [M(bpy)(2-py)]NO3 complexes (2-py is an anion of 2-pyridinecarboxylic acid) show I.D.50 values smaller than cisplatin whereas [M(bpy)(2-pyz)]NO3 complexes (2-pyz is an anion of 2-pyrazinecarboxylic acid) show I.D.50 values larger than cisplatin against P388 cancer cells.

Some potential antitumor 2,2'-dipyridylamine Pt(II)/Pd(II) complexes with amino acids: their synthesis, spectroscopy, DNA binding, and cytotoxic studies.

Four new palladium(II) and platinum(II) complexes of formula [M(dipy)(AA)]+ (where dipy is 2,2'-dipyridylamine, AA is an anion of glycine or L-alanine, and M is Pd(II) or Pt(II)) have been synthesized and characterized with amino acids binding as bidentate ligands. These complexes are 1:1 electrolyte in conductivity water. Of the above four complexes, the two L-alanine complexes show ID50 values against P388 lymphocytic leukemia cells lower than cis-diamminedichloroplatinum(II), whereas the two glycine complexes show ID50 values higher than cisplatin. The interaction of calf thymus DNA with the above complexes shows significant spectral changes in the presence of [Pt(dipy)(gly)]Cl, [Pd(dipy)(ala)]Cl, and [Pt(dipy)(ala)]Cl and the mode of binding between these complexes and DNA seems to be noncovalent.

Synthesis, characterization, cytotoxic, and DNA binding studies of some platinum (II) complexes of 1,2-diamine and alpha-diimine with 2-pyridinecarboxylate anion.

Seven new water-soluble cationic complexes of general formula [Pt(2-pyc)(N-N)]+ (where N-N is 2NH3, ethylenediamine (en), 1,2-diaminopropane (1,2-dap), 1,3-diaminopropane (1,3-dap), (+/-) trans-1,2-diaminocyclohaxane (dach), 2,2'-dipyridylamine (dpa) or 1,10-phenanthroline (phen), and 2-pyridinecarboxylate anion) have been prepared. These complexes have been characterized by conductance measurements, and by ultraviolet-visible, infrared (IR), and 1H nuclear magnetic resonance (NMR) spectroscopy. The COSY (correlated spectroscopy) spectra of [Pt(2-pyc)(dpa)]+ and [Pt(2-pys)(dpa)]+ further support the structures of the above complexes with three nitrogen and one oxygen donor atoms in the first coordination sphere of platinum(II) with 1,2-diamine or alpha-diimine and 2-pyridinecarboxylate anion behaving as bidentate ligands. One of the compounds, [Pt(2-pyc)(dpa)]Cl, also shows a birefringence property in water. These compounds inhibit the growth of P388 lymphocytic leukemia cells. [Pt(2-pyc)(dpa)]+ shows I.D.50 value comparable to cisplatin. However, six other complexes show higher I.D.50 values than cisplatin. In addition, the inhibition studies also suggest that their target is DNA. Therefore, the interactions of four of the above complexes with calf thymus DNA have been studied by ultraviolet and fluorescence spectral methods. These studies suggest that [Pt(2-pyc)(NH3)2]+ and [Pt(2-pyc)(1,2-dap)+ bind to DNA by noncovalent interactions. On the other hand, [Pt(2-pyc)(dpa)]+ and [Pt(2-pyc)(phen)]+ bind to DNA by covalent monofunctional binding. The latter two complexes have also been interacted with PUC19 DNA. The gel electrophoresis studies of these interactions suggest that these complexes bind to DNA, and this binding leads to a conformational change in DNA.

Fluorometric measurement of reverse transcriptase activity with 4',6-diamidino-2-phenylindole.

We describe a rapid fluorometric assay for reverse transcriptase (RT) activity. After RT is incubated in the presence of poly(A).oligo(dT) and dTTP for up to 1 h, the reaction is stopped with EDTA and aliquots are added to cuvettes containing 4',6-diamidino-2-phenylindole (DAPI). DAPI fluorescence, which is increased upon binding the RNA.DNA heteroduplex, is measured after 30 min and is linearly dependent on the enzymatic reaction time and the amount of active RT added to the enzyme assay. The increased fluorescence correlates well with the incorporation of [alpha-32P]dTTP into DNA (r2 = 0.986). However, similar assays with the Klenow fragment using poly(dA).oligo(dT) did not result in increased fluorescence under conditions wherein incorporation of [alpha-32P]dTTP into DNA was documented. Thus, the poly(A).poly(dT) [RNA.DNA] heteroduplex must differ from the poly(dA).poly(dT) [DNA.DNA] duplex in a manner that allows for a perturbation of DAPI fluorescence. The relative specific activities of RT in crude preparations measured with the fluorometric assay were comparable to conventional isotopic enzyme assays as were determinations for the type of inhibition and the kinetic constants of purified RT with inhibitors such as zidovudine 5'-triphosphate, nevirapine, and oltipraz.(ABSTRACT TRUNCATED AT 250 WORDS)

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)

Inhibition of human immunodeficiency virus type 1 long terminal repeat-driven transcription by an in vivo metabolite of oltipraz: implications for antiretroviral therapy.

Metabolite III (MIII, 7-methyl-6,8-bis(methylthio)pyrrolo[1,2-alpha]pyrazine), a major in vivo metabolite of oltipraz (OLT, 5-pyrazinyl-4-methyl-1,2-dithiole-3-thione), appears to disrupt human immunodeficiency virus type 1 (HIV-1) replication at a point distal to integration of the viral genome into host DNA. We report that MIII (but not OLT) is a nontoxic inhibitor of long terminal repeat (LTR)-driven expression of beta-galactosidase in phorbol-12-myristate-13-acetate (PMA)-stimulated and unstimulated 293.27.2 cells (ED50 = 14 +/- 1 and 41 +/- 4 microM, respectively). Electrophoretic mobility-shift assays (EMSA) reveal that MIII does not significantly reduce the PMA-induced DNA binding activities of NF-kappa B or AP-1. Although the mechanism by which MIII inhibits LTR-driven transcription remains unclear, the antiviral synergism of OLT and MIII in vitro are likely due their independent activities. Whether this translates into antiviral synergy in vivo is being examined by comparing OLT and MIII pharmacokinetics to the pharmacodynamic effects of orally-administered OLT in patients with p24 antigenemia.

Oltipraz, a novel inhibitor of human immunodeficiency virus type 1 (HIV-1) replication.

Glutathione (GSH) levels are markedly depleted in patients infected with human immunodeficiency virus type 1 (HIV-1) and supplementation of media with high concentrations (5-20 mM) of low-molecular weight thiols prevents HIV-1 replication in cultured cells. We were intrigued whether chemopreventive enzyme inducers might represent a more pharmacologically feasible method to inhibit HIV-1 replication since these compounds elevate intracellular concentrations of GSH at nontoxic doses in vivo. After establishing that all inducers surveyed were able to elevate GSH levels in human T-cell and monocytoid cell lines, we were surprised to find that oltipraz (5-pyrazinyl-4-methyl-1,2-dithiole-3-thione) was uniquely able to inhibit HIV-1 replication (IC50 = 5-15 microM). Oltipraz and other antiviral 1,2-dithiole-3-thiones (DTTs) appear to inhibit acute HIV-1 replication by inactivating reverse transcriptase (RT). However, among DTTs that inhibit HIV-1 replication in acutely infected cells, only oltipraz was able to inhibit HIV-1 replication in a chronic infection model. Thus, in addition to inactivating RT, oltipraz appears to have an additional antiviral mechanism distal to viral integration. Our laboratories are attempting to determine the mechanism by which oltipraz inhibits HIV-1 replication in chronically infected cells; we are also attempting to determine the bioorganic mechanism for the inactivation of RT. Since the covalent modification of schistosomal proteins and transcription factor(s) are thought to be responsible for the antiparasitic and chemopreventive activities of DTTs, respectively, our studies should be relevant to understanding the diverse medicinal properties of DTTs. Oltipraz, an antischistosomal drug undergoing clinical evaluation as an anticarcinogen, inhibits HIV-1 replication at concentrations achievable in human serum. It is intriguing to consider oltipraz as a therapeutic agent not only for its antiretroviral activity, but also for the prevention of HIV-1 associated neoplasms.

Reduction in T cell apoptosis in patients with HIV disease following antiretroviral therapy.

Patients with HIV infection manifest increased T lymphocyte apoptosis. This study investigated the influence of antiretroviral therapy (ART) upon lymphocyte apoptosis in 23 HIV-infected adults naive to protease inhibitors. Patients were enrolled in a treatment trial consisting of Nelfinavir (NFV), d4T, or NFV + d4T for 24 weeks, followed by triple therapy (NFV + reverse transcriptase inhibitors) for an additional 24 weeks. Spontaneous T cell apoptosis in cultured PBMC decreased by 23.67 +/- 18.2% (P < 0.006) at 48 weeks and plasma HIV RNA decreased by 1.79 +/- 0.59 log(10) RNA copies/ml (P < 0.001). The absolute decrease and slope of T cell apoptosis correlated with plasma virus load and with activated CD8 T cells and was inversely correlated with CD4 T cells. We conclude that reduction in chronic antigenic stimulation and the absence of cellular signals elicited by viral products contribute to the rescue of T lymphocytes from apoptosis, which facilitates immunologic recovery in ART-treated patients.