DNA and glutathione interactions in cell-free media of asymmetric platinum(II) complexes cis- and trans-[PtCl2(isopropylamine)(1-methylimidazole)]: relations to their different antitumor effects

The global modification of mammalian and plasmid DNAs by the novel platinum compounds cis-[PtCl₂(isopropylamine)(1-methylimidazole)] and trans-[PtCl₂(isopropylamine)(1-methylimidazole)] and the reactivity of these compounds with reduced glutathione (GSH) were investigated in cell-free media using various biochemical and biophysical methods. Earlier cytotoxicity studies had revealed that the replacement of the NH₃ groups in cisplatin by the azole and isopropylamine ligands lowers the activity of cisplatin in both sensitive and resistant cell lines. The results of the present work show that this replacement does not considerably affect the DNA modifications by this drug, recognition of these modifications by HMGB1 protein, their repair, and reactivity of the platinum complex with GSH. These results were interpreted to mean that the reduced activity of this analog of cisplatin in tumor cell lines is due to factors that do not operate at the level of the target DNA. In contrast, earlier studies had shown that the replacement of the NH₃ groups in the clinically ineffective trans isomer (transplatin) by the azole and isopropylamine ligands results in a radical enhancement of its activity in tumor cell lines. Importantly, this replacement also markedly alters the DNA binding mode of transplatin, which is distinctly different from that of cisplatin, but does not affect reactivity with GSH. Hence, the results of the present work are consistent with the view and support the hypothesis systematically tested by us and others that platinum drugs that bind to DNA in a fundamentally different manner from that of conventional cisplatin may have altered pharmacological properties.     

Spectral and redox studies on mixed ligand complexes of cobalt(III) phenanthroline/bipyridyl and benzoylhydrazones, their DNA binding and antimicrobial activity

Cobalt(III) complexes of the type [Co(N-N)2L](ClO4)2.H2O [where L=anionic form of para-substituted benzaldehyde-benzoylhydrazone (BHBX-); X=H, Me, OMe, OH, Cl or NO2; N-N=2,2'-bipyridine (bpy) or 1,10-phenanthroline (phen)] have been synthesized and characterized through UV-Vis, IR, NMR and electrochemical studies. The IR spectral frequencies support the mode of coordination of BHBX to the metal through the imino nitrogen and enolic oxygen atoms. The electronic absorption spectra exhibit metal to ligand charge transfer (MLCT) transition around 450 nm together with intraligand (IL) bands that are comparable to that of [Co(phen/bpy)3]3+. In acetonitrile solution these complexes show two well defined redox couples corresponding to Co(III/II) and Co(II/I) processes. Binding of these complexes with herring sperm DNA have been investigated by spectroscopic and voltammetric methods. The lower binding constant values of these complexes with respect to the [Co(phen/bpy)3]3+ are ascribed to the polar interaction of the substituted benzoylhydrazone moiety with the sugar-phosphate backbone of the DNA. The UV spectrum shows reasonable hypochromism with slight (2-4 nm) red shift, while the cyclic voltammogram shows decrease in current intensity along with a very small shift in the formal potential of the Co(III/II) redox couple. These experimental results indicate that phen mixed ligand complexes bind to DNA through an intercalative mode more effectively than their bpy counterparts. These complexes are also found to have good antimicrobial activity.     

Comparison of chemical reactivity, cytotoxicity, interstrand cross-linking and DNA sequence specificity of bis(platinum) complexes containing monodentate or bidentate coordination spheres with their monomeric analogues

The properties of a new bis(platinum) complex containing two monodentate coordination spheres, [(trans-PtCl(NH3)2)2H2N(CH2)4NH2]Cl2 (1,1/t,t), are reported. Comparison is made with respect to chemical reactivity, in vitro biological activity in murine and tumor cells, DNA conformational changes, cross-linking efficiency, and sequence specificity between this complex and the previously reported complex containing two bidentate platinum atoms, [(Pt(mal)(NH3))2H2N(CH2)4NH2] (2,2/c,c), as well as with their respective monomeric analogues, [PtCl(dien)]Cl and cis-[PtCl2(NH3)2](cis-DDP). While both bis(platinum) complexes are active against cis-DDP-resistant cells, the monodentate bis(platinum) complex (1,1/t,t) has a lower resistance factor than the complex with bidentate coordination spheres (2,2/c,c). More importantly, this property is repeated in a human ovarian carcinoma cell line. DNA-binding studies show that DNA interstrand cross-linking is more efficient for the 1,1/t,t complex. DNA sequencing studies employing the exonuclease activity of T4-polymerase demonstrate that there are a variety of binding sites; some are common to all complexes and some common to both bis(platinum) complexes, while the monodentate 1,1/t,t species also reacts at unique sites, not attacked by any of the other complexes studied. The circular dichroism of CT DNA modified by the 1,1/t,t complex is also unique and is not seen for any of the other agents.     

Toxicity of platinum(II) amino acid (N,O) complexes parallels their binding to DNA as measured in a new solid phase assay involving a fluorescent HMG1 protein construct readout

 The compound [Pt(lysine)Cl₂] (Kplatin) was previously identified in a study of platinum amino acid complexes as a potential antitumor drug candidate. The DNA binding properties, high mobility group (HMG)-domain protein affinity for the platinated DNA, and cytotoxicity against HeLa cells of Kplatin and three related (N,O) chelated platinum(II) amino acid complexes, [Pt(arginine)Cl₂] (Rplatin), K[Pt(N_e-acetyllysine)Cl₂] (NacKplatin), and K[Pt(norleucine)Cl₂] (Norplatin), are reported. The four complexes have identical PtCl₂(N,O) coordination environments. A new solid phase screening methodology was devised in which platinated DNA probes are covalently attached to a nylon support and tested for their ability to bind a fluorescently labeled HMG-domain protein. The fluorescent HMG-domain protein was generated by expressing a fusion of the green fluorescent protein (GFP) with recombinant rat HMG1. Binding revealed by the solid phase method correlated well with the results of gel mobility shift and HeLa cytotoxicity assays. These results suggest that the net charge on the complex, rather than the nature of the side chain, is the most important factor underlying the DNA binding properties and toxicity of amino acid (N,O) chelated platinum complexes. This property explains why Kplatin was previously selected from the pool of platinum amino acid complexes based on the ability of its DNA adducts to bind HMG1. Received: 3 February 1999 / Accepted: 7 April 1999     

The new antitumor compound, cis-[Pt(NH3)2(4-methylpyridine)Cl]Cl, does not form N7,N7-d(GpG) chelates with DNA. An unexpected preference for platinum binding at the 5'G in d(GpG)

The reaction of the antitumor active agent cis-[Pt(NH3)2(4-mepy)Cl]Cl (4-mepy stands for 4-methylpyridine) with d(GpG) has been investigated by 1H magnetic resonance spectroscopy. Initially, two mononuclear complexes cis-Pt(NH3)2(4-mepy)[d(GpG)-N7(1)] 1 and cis-Pt(NH3)2(4-mepy)[d(GpG)-N7(2)] 2 are formed in an unexpected ratio 65:35, as determined by 1H NMR and enzymatic digestion techniques. Both products react further with a second equivalent of cis-[Pt(NH3)2(4-mepy)Cl]Cl forming the dinuclear platinum complex [cis-Pt(NH3)2(4-mepy)]2[mu-d(GpG)- N7(1),N7(2)] 3. With [Pt(dien)Cl]Cl and [Pt(NH3)3Cl]Cl similar complexes are formed. No evidence was found for the formation of chelates cis-Pt(NH3)(4-mepy) [d(GpG)-N7(1),N7(2)], which would be formed upon ammonia release from the mononuclear complexes 1 and 2. Even addition of strong nucleophiles, like sodium diethyldithiocarbamate, thiourea, cysteine, or methionine, before or after reaction, do not induce the formation of a chelate. Under all conditions the N-donor ligands remain coordinated to Pt in 1,2 and 3. In addition, the results of bacterial survival and mutagenesis experiments with E. coli strains show that the in vivo formation of bifunctional adducts in DNA, comparable to those induced by cis-Pt(NH3)2Cl2, by treatment of cells with cis-[Pt(NH3)2(4-mepy)Cl]Cl is unlikely. Also, a mechanism of binding and intercalation is not supported by experimental data. All experiments suggest that the mechanism of action of this new class of antitumor agents must be different from that of cis-Pt(NH3)2Cl2.