A circular dichroism study of DNA.platinum complexes. Differentiation between monofunctional, cis-bidentate and trans-bidentate platinum fixation on a series of DNAs

The circular dichroism (CD) spectra of a series of DNA . platinum complexes are presented. The following platinum compounds, [Pt(dien)Cl]Cl, cis-Pt(NH3)2Cl2, cis-Pt(en)Cl2, trans-Pt-(NH3)2Cl2, K[Pt(NH3)Cl3] and K2[PtCl4] were complexed with the DNA extracted from bacteria Micrococcus lysodeikticus (72% dG + dC), Escherichia coli (50% dG + dC), Clostridium perfringens (32% dG + dC) and salmon sperm (41% dG + dC). Strong differences were found between the different DNA . Pt complexes. Three types of spectra clearly demonstrate the different platinum binding modes on DNA. In the first type, the platinum compound, i.e. [Pt(dien)Cl]Cl, is fixed to DNA with only one bond (monofunctional complex formation) and no significant change of the CD positive band of DNA is found. The main feature of the second type is a continuous intensity decrease of the positive band as observed for trans-Pt(NH3)2Cl2 (trans-bidentate complex formation). The third type concerns the cis-bidentate platinum fixation obtained with cis-Pt(NH3)2Cl2, cis-Pt(en)Cl2, K[Pt(NH3)Cl3] and K2[PtCl4]. The CD spectra are in this case characterized by an increase in the positive Cotton effect which is dG + dC-dependent up to an rb value around 0.10 (where rb = number of platinum atoms bound per nucleotide), followed by a decrease until DNA saturation with platinum is reached. A linear decrease in the amplitude of the negative band is detected in all the complexes except in the case of the monofunctional DNA . Pt complexes. For the cis-bidentate and trans-bidentate platinum fixation, a continuous bathochromic shift occurs.     

Antitrypanosomal action of cis-diamminedichloroplatinum (II) analogs

The present report deals with the alterations produced by cis-diamminedichloroplatinum (II) (DDP), and 2 of its analogs: cis-Pt(II)(tranylcypromine)2Cl2 and cis-Pt(II)(benzothiazole)2Cl2 in cultured epimastigote forms of Trypanosoma cruzi. Studies have been performed at the ultrastructural level and the inhibitory effect of these complexes on macromolecule synthesis, evaluated by 3H-thymidine, 3H-uridine, and 3H-leucine incorporation, has been investigated. DDP at concentrations of 50 and 100 micrograms/ml does not inhibit significantly the incorporation of radioactive precursors, but a clear decrease was observed with the 2 analogs. Eight hours of treatment at a concentration of 10 micrograms/ml rendered in all 3 cases an increase in autophagic vacuoles and lipids as well as an abnormal condensation of the nucleus chromatin.     

X-ray photoelectron spectroscopy of DNA-Pt complexes. Evidence of O6 (Gua)-N7 (Gua) chelation of DNA with cis- dichlorodiamine platinum(II).

The binding energies of nitrogen, oxygen, phosphorus, chlorine and Pt in several DNA - Pt (II) complexes are reported and discussed. The nitrogen band of DNA is slightly shifted upon complexation with Pt. Oxygen binding energies in the complexes studied clearly show that cis-Pt(NH3)2Cl2 forms a specific chelate N7(Gua) - O6 (Gua) with DNA as opposed to trans-Pt(NH3)2Cl2 and the other Pt compounds which react only with the N7(Gua) site of DNA.     

DNA-platinum interactions in vitro with trans- and cis-Pt(NH3)2Cl2.

In the present study the nature and the hydrolysis of DNA-Pt complexes with the platinum compounds, [Pt(dien)Cl]Cl, trans- and cis-Pt(NH3)2Cl2, using potentiometric chloride determinations, have been investigated. The trans-Pt(NH3)2Cl2 and the [Pt(dien)Cl]Cl react with the GC planes at the N7(G) sites, while the cis-Pt(NH3)2Cl2 compound reacts with the GC planes and forms a chelate by using the N7(G) and O6(G) sites. The complex is a specific 1:1 Pt:DNA adduct. The platinum atom in cis-Pt(NH3)2Cl2 liberates both chlorine atoms on chelation. A mechanism for the in vivo antitumor activity of the cis-Pt(NH3)2Cl2 is proposed and the structure activity relationship is discussed.     

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.     

Reactions of the UVRABC excision nuclease with DNA damaged by diamminedichloroplatinum(II).

Mutants of Escherichia coli, which are blocked in excision repair (uvrA6, uvrB5, or uvrC34) are exceptionally sensitive to the antitumor drug cis-Pt(II)(NH3)2Cl2 (cis-DDP) but not the trans isomer. Plasmid DNA, damaged by either the cis or trans compound and treated with the UVRABC excision nuclease was cut as shown by conversion of supercoiled DNA to relaxed forms. All three protein products of the uvrA, uvrB, and uvrC genes were required for incision. End-labeled fragments damaged with cis-DDP and reacted with the UVRABC nuclease were cut at the 8th phosphodiester bond 5' and at the 4th phosphodiester bond 3' to adjacent GG's. DNA treated with trans-DDP was not cut appreciably at adjacent GG's by the repair enzyme as subsequent analysis of reaction products after enzyme digestion gave a pattern similar to those obtained with control untreated fragments. The results indicate that the UVRABC nuclease may promote cell survival by the removal of adjacent GG's which are crosslinked by cis-Pt(II)(NH3)2Cl2.     

Effect of biologically active compounds of divalent platinum on the properties of the liquid crystal "microphase" of DNA

The optical properties of the "microphases" modeling the state of the DNA molecule in the cell and formed of both the low molecular DNA and the DNA complexes with cis- and trans-isomers of dichlorodiamine platininum (II) were studied. It was shown that the intensive band characteristic of the circular dichroism spectrum of the initial DNA "microphase" was decreasing with binding of DNA to cis-Pt (II) or trans-Pt (II). The effect of cis-Pt (II) on the "microphase" optical properties was more significant than that of trans-Pt (II). The effect correlated with the biological activity of the cis- and trans-compounds of platinum. Possible causes of the decrease in the optical activity of the DNA "microphase" are discussed.     

Dissociation of double-stranded poly(I) . poly(C) by cis-diammine-dichloro-Pt(II).

The covalent binding of cis-Pt(NH3)2Cl2 on the double stranded poly(I) . poly(C) induced an irreversible dissociation of the two strands. This dissociation was evidenced mainly by poly(I)-Agarose affinity chromatography which allowed to recover free strands of cis-Pt(NH3)2Cl2-poly(I) from a cis-Pt(NH3)2Cl2-poly(I) . poly(C) complex, by density equilibrium centrifugation where free poly(C) could be isolated, and by acid titrations of the metal-poly(I) . poly(C) complexes. The separation of the two strands of the polyribonucleotide upon cis-Pt(NH3)2Cl2 fixation was shown not to exceed 90--95%. A dissociation curve of the polynucleotide double helix as a function of the amount of bound cis-Pt(NH3)2Cl2 was determined and was shown to be of a characteristic cooperative effect. The fixation of the paltinum compound to poly(I) . poly(C) seemed also to be cooperative.     

Interaction of platinum compounds with short oligodeoxynucleotides containing guanine and cytosine

The products resulting from reaction of cis-Pt(NH3)2Cl2 with d(CpCpGpG), d(GpCpG), d(pCpGpCpG), d(pGpCpGpC) and d(CpGpCpG) and from reaction of [Pt(dien)Cl]Cl with d(CpCpGpG) and d(GpCpG) have been characterized with the aid of proton NMR spectroscopy, circular dichroic spectroscopy and Pt analysis. The binding sites of the Pt compounds were determined by pH-dependent NMR spectroscopy. Binding of the two Pt compounds invariably occurs at the guanine N7 atoms. In all compounds containing [cis-Pt(NH3)2]2+ chelates are formed by coordination of platinum to two guanines of the same oligonucleotide. The resulting intrastrand-cross-linked oligonucleotides contain either d(GpG) . cisPt units, or d(GpCpG) . cisPt units. In the latter case the middle cytosine is not coordinated to platinum. As a result the conformational changes originating from these two chelates are different from each other. In the case of [Pt(dien)Cl]Cl as a starting product, two types of oligonucleotide adducts are formed, i.e. those with one Pt atom/molecule and those with two Pt atoms/molecule. The NMR spectra of the adducts containing only one Pt(dien)2+ show that only one adduct is formed, although two guanine bases are present. This indicates a preference for one of the N7 atoms in the molecule.     

Genotoxicity of cis-PtII complex of 3-aminoflavone in comparison with cis-DDP in A549 cells evaluated by comet assay

Cis-diamminedichloroplatinum(II) (cis-DDP) is one of the most successful antineoplastic drugs. However, besides effectiveness it gives many side effects. Therefore, current studies are concentrated on searching for new analogs equally effective in chemotherapy but less toxic. Comparison of genotoxic properties of cis-Pt(II) complex of 3-aminoflavone and cis-DDP in a comet assay with and without H2O2 application was performed in A549 cell line. The higher tail moment values were noticed for the former compound contrary to the latter one in both variants. It suggests mainly DNA breaks (besides cross-links) appearance after cis-Pt(II) complex of 3-aminoflavone application and might indicate DNA degradation in comparison with cis-DDP.     

Effect of pKM101 on cell killing and specificity of mutation induction by cis-diaminedichloroplatinum(II) in Escherichia coli K-12.

Cell killing and mutation induction in the lacI gene of Escherichia coli by cis-Pt(NH3)2Cl2 were studied in cells with different repair capacities, with and without pKM101. The presence of the plasmid pKM101 made repair-proficient cells more susceptible to killing by cis-Pt(NH3)2Cl2 and strongly enhanced mutation induction by that compound. Both effects were shown to be dependent upon excision repair. Characterization of the induced mutations in the lacI gene after cis-Pt(NH3)2Cl2 treatment of E. coli cells, by the LacI system, revealed that the mutagenic specificity of the Pt compound was strongly influenced by the presence of the pKM101 plasmid. With pKM101, 23% of the induced amber and ochre mutations resulted from substitutions at AT base pairs, whereas these mutations were hardly induced in cells without pKM101. These results suggest that pKM101-induced repair differs from normal SOS repair.     

Effects in bacterial systems of Pt(II) complexes with antitumour activity.

A series of alicyclic amine complexes of the cis-Pt(am)2Cl2 type, showed specific antitumour activity against various animal tumours. We have tested these compounds in a number of bacterial systems indicative of their interaction with bacterial DNA.     

Interaction of cis-Pt(ino)2Cl2 with amino acids

The reactions of cis-Pt(ino)2Cl2 with the amino acids Gly, L-Ala, L-Val, L-Ileu, L-Phe and L-Pro were studied in methanolic solutions. The (1:1) adducts of the formulas cis-[Pt(ino)2(am-ac)]Cl were isolated from these reactions in the solid state, which in turn produce the cis-[Pt(ino)2(am-acH)Cl]Cl complexes, by treating the former with equivalent amounts of HCl, in aqueous solutions. The complexes were characterized with elemental analysis, conductivity measurements, IR, 1H NMR, and 13C NMR spectra. The results show that the purine ring of inosine interacts with the aliphatic side chain of the amino acids. The platination increases the percentage of the C3'-endo-anti conformation of the sugar part of inosine.     

A study of interactions of platinum (II) compounds with DNA by means of CD spectra of solutions and liquid crystalline microphases of DNA

The optical properties of the DNA complexes with divalent platinum compounds of the cis-diamine type differing both in the nature of anionic and neutral ligands and in the spatial arrangement about the platinum atom were studied. The platinum compounds cis-[Pt(NH3)2Cl2], [Pt(en)Cl2], [Pt(tetrameen)Cl2], cis-[Pt(NH3)2NO2Cl], and cis-[PtNH3(Bz)Cl2] at small values of r (r is the molar ratio of a platinum compound to DNA nucleotides in the reaction mixture) were found to induce an increase in the amplitude of the positive band in the circular dichroic (CD) spectrum of linear DNA. All the compounds listed except cis-[Pt(NH3)2NO2Cl] caused a sharp decrease of the amplitude of the negative band in the CD spectrum of a liquid crystalline microphase of DNA formed in solution in the presence of poly(ethylene glycol). All these platinum compounds (except [Pt(tetrameen)Cl2]) exhibit biological (antimitotic, antitumour, etc.) activity. The platinum compounds trans-[Pt(NH3)Cl2], trans-[Pt(NH3)2NO2Cl], cis-[PtNH3PyCl2], cis-[Pt(NH3)2(NO2)2], and [Pt(NH3)3Cl]Cl exhibiting a low (if any) biological activity, either induced a decrease of the amplitude of the positive band in the CD spectrum of linear DNA, or did not affect the CD spectrum at all. The effect of these platinum compounds on the CD spectrum of the liquid crystalline microphase of DNA was either weak or absent. It is assumed that the specific biological action of platinum compounds of the cis-diamine type is determined by the polydentate binding to DNA: in addition to the cis-bidentate covalent binding of platinum to DNA nitrogen bases, a hydrogen bond formation between the DNA and cis-amino ligands occurs by means of protons at nitrogen atoms.     

Optical properties of DNA complexes with antitumor compounds of bivalent platinum

The optical properties of the DNA complexes with the compounds of bivalent platinum were studied. The compounds differed by the nature of the anionic and neutral ligands and their spatial arrangement about the platinum atom. It was shown that the same as cis-[Pt (NH3)2Cl2] the platinum compounds with the biological activity, i.e. [Pt (en) Cl2], cis-[PtNH3 (Bz) Cl2] and cis-[Pt (NH3)2NO2Cl] induced at low values of r (a ratio of the number of the platinum moles added to the number of the DNA nucleotide moles in the solution) an increase in the amplitude of the positive band in the spectrum of the circular dichroism (CD) of the linear DNA and a marked decrease in the amplitude of the negative band in the spectrum of the CD of the liquid crystalline microphase of DNA formed in the presence of polyethyleneglycol. By the character of the action on the CD spectrum of the linear and condensed DNA [Pt (tetrameen)Cl2] which had no selective antimitotic effect might be referred to the above platinum compounds. Trans-[Pt (NH3)2NO2Cl], [PtNH3PyCl2], cis-[Pt (NH3)2(NO2)2] and [Pt (NH3)3Cl]Cl having no biological activity either induced only a decrease in the amplitude of the positive band in the CD spectrum of the linear DNA or had no effect on the CD spectrum. The effect of these compounds on the CD spectrum of the liquid crystalline microphase of DNA was slightly pronounced or not observed.     

Distribution of a platinum anti-tumour drug in HeLa cells by analytical electron microscopy

The distribution of Pt in HeLa cell sections after cell treatment with cis-Pt(NH3)2Cl2 dissolved in dimethylsulphoxide was probed by analytical electron microscopy. Primary targets were the nucleolus and the inner side of the nuclear double membrane. Even after solvolysis in dimethylsulphoxide the drug reached similar sites. It is suggested that cell death may be due to Pt inhibition at the initiation sites of DNA synthesis.     

Pentamidine is an antiparasitic and apoptotic drug that selectively modifies ubiquitin

We have determined the cytotoxic properties of pentamidine isethionate (2) towards the promastigotes of the protozoan parasite Leishmania infantum. The leishmanicidal activity of 2 was 60 times higher after 72 h of incubation than that of cisplatin (4). The pentamidine salt 2 induced a higher amount of programmed cell death (PCD) than cisplatin, which is associated with inhibition of DNA synthesis and cell-cycle arrest in the G2/M phase. Circular dichroism (CD) data indicate that binding of 2 to calf-thymus DNA (CT-DNA) induces conformational changes in the DNA double helix, consistent with a B-->A transition. Moreover, the interaction of 2 with ubiquitin led to a 6% increase in the beta-sheet content of the protein as observed by CD spectroscopy. Fluorescence-spectroscopy studies agreed with the CD data, showing that the pentamidine portion of 2 induces a significant decrease in the fluorescence of the Ub residues Phe4 and Phe45 located on the beta-cluster of the molecule, but not of Tyr59 on the alpha-cluster. These data indicate that pentamidine specifically modifies the beta-cluster, i.e., the 'basic face' of ubiquitin. Our results suggest that the biochemical mechanism of action of pentamidine may be a consequence of its dual binding to DNA and proteins.     

Structure-antitumor activity relationship for new analogs of thecis-dichloro(l,2-diamino cyclohexane) platinum(II) complex

In 1977, Gale and associates reported the synthesis and antitumor activity of a series of Pt(II) complexes containing 1,2-diaminocyclohexane as the ligand. Certain of these complexes showed superior activity and greater water solubility compared to cis-Pt(NH₃)₂Cl₂ complexes (“Neoplatin”). In this paper we report the synthesis and antitumor activity of some 40 new water soluble platinum(II) and platinum(IV) complexes. The following classes of the cis-Pt(L)Cl₂ complexes were obtained, where L = 1,2-diaminocyclohexane: (a) cis-Pt(L)(X), where X is a derivative of homophthalic acid or a derivative of 1,3-benzendicarboxylic acid, (b) cis-Pt(L)(X)(OH)₂ and cis-Pt(L)(X)(Cl)₂ complexes, where L and X are the above-mentioned ligands, (c) cis-Pt(L)(X)₂ complexes where X is the monoanion of an organic xanthate or dithiocarbamate and L = 1,2-diaminocyclohexane, (d) their corresponding Pt(IV) analogues, Pt(L)(X)₂(OH)₂ and Pt(L)(X)₂(Cl)₂. All complexes were assayed against P388 tumors and/or KB cell-bearing mice. The observed antitumor activities were correlated with the structures and spectra of the complexes as well as with the results of EHMO calculations performed on the leaving ligand molecules. A number of the most active complexes were also tested for activity against ADJ/PC6 Yoshida and S-180 tumors in vivo.     

Differential response to mitomycin-C- and cis-diamminedichloroplatinum(II)-induced damage in normal human fibroblasts during confluent holding

Confluent cultures of normal human fibroblasts were treated with the chemotherapeutic agents, mitomycin C (MMC) and cis-diamminedichloroplatinum(II) (cis-Pt(II]. This treatment induced a decrease in clonogenic cell survival. Recovery of the cytotoxic effect was observed in the case of cis-Pt(II)-treated cultures maintained at confluence for 1-5 days. No such recovery was observed after treatment with MMC. These data suggest that contrary to potential lethal damage induced by cis-Pt(II) which is repaired in confluent cells, DNA damage induced by MMC is not repaired in confluent cells.