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 cisplatin hydrolytes with methionine, cysteine, and plasma ultrafiltrate studied by a combination of HPLC and NMR techniques

The reactions of cis-[PtCl(NH3)2(H2O)]+ with L-methionine have been studied by 1D 195Pt and 15N NMR, and by 2D[1H, 15N] NMR. When the platinum complex is in excess, the initial product, cis-[PtCl(NH3)2(Hmet-S)]+ undergoes slow ring closure to [Pt(NH3)2(Hmet-N,S)]2+. Slow ammine loss then occurs to give the isomer of [PtCl(NH3)(Hmet-N,S)]+ with chloride trans to sulfur. When methionine is in excess, a reaction sequence is proposed in which trans-[PtCl(NH3)(Hmet-S)2]+ isomerises to the cis-isomer, with subsequent ring closure reactions leading to cis-[Pt(Hmet-N,S)2]2+. Near pH 7, methionine is unreactive toward cis-[PtCl(OH)(NH3)2]. By contrast, L-cysteine reacts readily with cis-[PtCl(OH)(NH3)2] at pH 7, but there were many reaction products, including bridged species. Cis-[PtCl(OH)(NH3)2] reacts with reduced thiols in ultrafiltered plasma but these are oxidized if the plasma is not fresh or appropriately stored. With very low concentrations of the platinum complexes (35.5 microM), HPLC experiments (UV detection at 305 nm) indicate that the thiolate (probably cysteine) reactions become simpler as bridging becomes less important.     

Guanine-06 methylation reduces the reactivity of d(GpG) towards platinum complexes.

6-methylated guanine dinucleotides were used to study the influence of hydrogen bonding on the specific binding of the antitumor drug cDDP, cis-PtCl2(NH3)2, to DNA. In this interaction, the guanine-06 site appears to be important in explaining the preference for a pGpG-N7(1),N7(2) chelate, which results from H-bridge formation with the ammine ligand of cDDP. Guanine-06 methylated dinucleotides and the nonmodified dinucleotides were reacted with [Pt(dien)Cl]+, cis-PtCl2(NH3)2, and cis-[Pt(NH3)2(H2O)2]2+ and the reaction products were characterized by 1H NMR using pH titrations. Methylation at guanine-06 clearly reduces the preference for the guanine. In competition experiments monitored by NMR and experiments using UV spectrophotometry a decreasing reactivity towards [Pt(dien)(H2O)]2+ and cis-[Pt(NH3)2(H2O)2]2+ was found, in the order of d(GpG) greater than d(GomepG) greater than d(GpGome) greater than d(GomepGome). The difference in reactivity between 5' guanine methylation and 3' guanine methylation is ascribed to differences in the H-bond formation with the backbone phosphate. The resulting reduced stacking of the bases in both modified dinucleotides, compared to the bases in d(GpG), results in a preference for the 3' guanine over 5'.     

Sterically hindered cisplatin derivatives with multiple carboxylate auxiliary arms: synthesis and reactions with guanosine-5'-monophosphate and plasmid DNA.

Two novel sterically hindered cisplatin derivatives with the ligand L=NH(2)C(CH(2)CH(2)COOH)(3) were prepared: cis-PtCl(2)L(2) and cis-PtCl(2)L(NH(3)). The starting compound for the syntheses was NH(2)C(CH(2)CH(2)COOtBu)(3), also known as a building block for dendrimers. cis-PtCl(2)L(2) was prepared from K(2)PtCl(4) in an unusual two-phase reaction in water-chloroform, followed by deprotection of the tert-butyl protective groups with formic acid to yield a water-soluble complex. The mixed-ligand compound cis-PtCl(2)L(NH(3)) was prepared from [PPh(4)][PtCl(3)(NH(3))] in methanol, with subsequent deprotection in formic acid. DNA-binding properties of the two compounds were investigated using the model base guanosine-5'-monophosphate (5'-GMP) and pBR322 plasmid DNA. While cisplatin [cis-PtCl(2)(NH(3))(2)] induced an unwinding of 12 degrees in pBR322 plasmid DNA, cis-PtCl(2)L(NH(3)) induced only 3 degrees unwinding, which is indicative of a monofunctional binding mode. Remarkably, cis-PtCl(2)L(2) did not induce any distortion in plasmid DNA, which strongly suggests that the compound does not bind to DNA. Test reactions with 5'-GMP, monitored by 1H and 195Pt NMR, confirmed that cis-PtCl(2)L(2) is unable to bind to DNA, whereas cis-PtCl(2)L(NH(3)) binds only one nucleotide. Apparently, binding of platinum to nucleotides at the coordination site cis with respect to the ligand L is prevented by steric crowding. Thus, cis-PtCl(2)L(NH(3)) must bind DNA monofunctionally at the trans position. Besides, both compounds have a chloride replaced by one of the carboxylate arms, forming a a seven-membered chelate ring. In theory, cis-PtCl(2)L(2) could also form a second chelate ring, but this was not observed.     

Mechanism of the reversal reaction of platinated DNA with thiourea studied by platinated 5'-GMP

The reversal reactions of cis-[Pt(NH3)2(5'-GMP)2] 2-(1) and trans-[Pt(NH3)2(5'-GMP)2] 2-(2) with thiourea were examined by reversed phase HPLC and monothioureido intermediate cis-[Pt(NH3)2(5'-GMP) (tu)] (4) was detected. This result suggested that Pt-[5'-GMP-N(7)] bond was more labile than Pt-NH3 bond and the release of ammonia from cis-Pt(II)-DNA base complexes is a result of trans-labilizing effect of sulfur containing molecule displaced with DNA base.     

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.     

Investigation of sulfur containing amino acids at the lipoxygenase active site using a platinum complex.

Inactivation of native soybean lipoxygenase-1 was observed upon preincubation with (NEt4)[PtCl3(P(Bun)3)]. Removal of the platinum complex(es) from the inactivated enzyme by treatment with sodium diethyldithiocarbamate (Naddtc) which reverses methionine but not cysteine binding, restores most of the activity. Linoleic acid, an enzyme substrate, protects it from inactivation. The quenching of the fluorescence of the putative active site tryptophans which accompanies inactivation disappears after Naddtc reactivation. The (NEt4)[PtCl3(P(Bun)3)]-inactivated enzyme iron(II) cannot be oxidized at variance with that of the native or Naddtc reactivated enzyme, as checked by EPR spectroscopy. These results show that at least one methionine is close to the iron binding site in soybean lipoxygenase-1.     

Cisplatin interaction with cysteine and methionine, a theoretical DFT study

Interactions of hydrated cisplatin complexes with sulphur-containing amino acids cysteine and methionine were explored. The square-planar cis-[Pt(NH3)2(H2O)X]+ complexes (where X=Cl- and OH-) were chosen as mono- and dihydrated reactants. Calculations using density functional theory (DFT) techniques with B3LYP functional were performed. The isolated molecules and the supermolecular approaches were employed for the determination of the reaction energies. Bond dissociation energies (BDE) were estimated in the model of isolated molecules and supermolecules were used for the determination of the association energies between the two interacting parts. Formation of monodentate complexes by replacing the aqua-ligand with the S, N, and O-sites of both amino acids represents an exothermic process. The highest BDE was found in cysteine structures for the Pt-S coordination. The bonding energy is about 114 kcal/mol, which is comparable with cisplatin-guanine adducts. Analogous BDE for methionine complexes is smaller by about 40 kcal/mol. This correlates well with the known fact that cysteine forms irreversible cisplatin adducts while similar adducts in the methionine case are reversible. The formation of chelate structures is an exothermic reaction only for the hydroxo-form of reactants in the supermolecular approach where additional association interactions between the released water and chelate molecules sufficiently stabilize the final product.     

Coordination chemistry in biological media: reactions of antitumor Pt(II) and Au(III) complexes with cell culture media

Reactions of cis-PtCl2(NH3)2 (1), Pt(1,2-diaminoethane)Cl2 (2), PtCl4(2-) (3), and AuCl4- (4) with intact cell culture media have been studied by spin-echo 500 MHz proton NMR spectroscopy. This has allowed us to observe reactions of components of the media at submillimolar concentrations. Upon the addition of 400 microM 1, 2, or 3 to the media, the S-methyl peak of methionine decreases in intensity and in each case a new peak appears which we have tentatively assigned to the S-CH3 of Pt(N,S-L-Met)2. In the spectra of the media with 2, an additional peak appears, assignable to the S-CH3 of Pt(1,2-diaminoethane)(N,S-L-Met). Upon the addition of Au(III) to the media, the S-CH3 peak of methionine also decreases in intensity and new peaks appear in the 2.6 to 2.8 ppm region, including a peak identified as the S(O)-CH3 peak of methionine sulfoxide. The other peaks are assignable to Au(I)-S(Met) species. Practical methods of following the reactions of metal complexes in cell culture media are becoming of wider significance with the increasing use of cell cultures for drug screening instead of animal tests.     

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.     

Interaction of cisplatin with methionine- and histidine-containing peptides: competition between backbone binding,macrochelation and peptide cleavage

The pH- and time-dependent reaction of cis-[PtCl2(NH3)2] with the methionine- and histidine-containing peptides H-Gly-Met-OH, H-Gly-Gly-Met-OH, Ac-His-Gly-Met-OH, and Ac-His-(Ala)3-Met-OH at 313 K has been investigated by ion-pairing reverse phase HPLC and NMR spectroscopy. For equimolar solutions (c=0.8 mM, pH approximately equals 3 or 8.8), initial formation of the kinetically favored S-bound complex is followed by relatively rapid metallation of the neighboring methionine amide nitrogen NM to afford a kappa2NM,S six-membered chelate. The strong trans effect of the methionine S then favors facile NH3 substitution, leading to generation of tridentate complexes such as [Pt(H-Gly-MetH(-1)-OH)-kappa3NG,NM,S)(NH3)]+ or [Pt(H-Ac-His-GlyH(-1)-MetH(-1)-OH-kappa3NG,NM,S)(NH3)]. In the case of H-Gly-Gly-Met-OH, this reaction is accompanied by loss of a second NH3 ligand in alkaline solution to generate the tetradentate kappa4NG1,NG2,NM,S species. In contrast, cleavage of the backbone C(O)-N bond to the second metallated amide nitrogen after t>100 h is common to the tridentate complexes of the tri- and pentapeptides at pH<5. Although an imidazole-coordinated kappa2N3H,S macrochelate is formed throughout the whole range 2.5 < or = pH < or = 10 for Ac-His-Gly-Met-OH, it slowly decays (t=10-1000 h) to the thermodynamically more stable tridentate kappa3NG,NM,S complex. All major final products were separated and fully characterized by NMR and MS.     

Reaction of cis- and trans-[PtCl2(NH3)2] with reduced glutathione inside human red blood cells, studied by 1H and 15N-[1H] DEPT NMR

Reactions of cis- and trans-[PtCl2(NH3)2] with glutathione (GSH) inside intact red blood cells have been studied by 1H spin-echo nuclear magnetic resonance (NMR). Upon addition of trans-[PtCl2(NH3)2] to a suspension of red cells, there was a gradual decrease in the intensity of the resonances for free GSH, and new peaks were observed that were assignable to coordinated GSH protons in trans-[Pt(SG)Cl(NH3)2], trans-[Pt(SG)2(NH3)2], and possibly the S-bridged complex trans-[[NH3)2PtCl)2SG]+. Formation of trans-[Pt(SG)2(NH3)2] inside the cell was confirmed from the 1H NMR spectrum of hemolyzed cells, which were ultrafiltered to remove large protein molecules; the ABM multiplet of the coordinated GSH cys-beta CH2 protons was resolved using selective-decoupling experiments. Seventy percent of the total intracellular GSH was retained by the ultrafiltration membrane, suggesting that the mixed complex trans-[Pt(SG)(S-hemoglobin)(NH3)2] also is a major metabolite of trans-[PtCl2(NH3)2] inside red cells. The reaction of cis-[PtCl2(NH3)2] with intracellular GSH was slower; only 35% of the GSH had been complexed after a 4-hr incubation compared to 70% for the trans isomer. There was a gradual decrease in the intensity of the GSH 1H spin-echo NMR resonances, but no new peaks were resolved. This was interpreted as formation of high-molecular weight Pt:GSH and mixed GS-Pt-S(hemoglobin) polymers. By using a 15N-[1H] DEPT pulse sequence, we were able to study the reaction of cis-[PtCl2(15NH3)2] with red cells at concentrations as low as 1 mM. 15NH3 ligands were released, and no resonances assignable to Pt-15NH3 species were observed after a 12-hr incubation.     

Bending studies of DNA site-specifically modified by cisplatin, trans-diamminedichloroplatinum(II) and cis-[Pt(NH3)2(N3-cytosine)Cl]+

Duplex oligonucleotides containing a single intrastrand [Pt(NH3)2]2+ cross-link or monofunctional adduct and either 15 or 22 bp in length were synthesized and chemically characterized. The platinum-modified and unmodified control DNAs were polymerized in the presence of DNA ligase and the products studied on 8% native polyacrylamide gels. The extent of DNA bending caused by the various platinum-DNA adducts was revealed by their gel mobility shifts relative to unplatinated controls. The bifunctional adducts cis-[Pt(NH3)2[d(GpG)]]+, cis-[Pt(NH3)2[d(ApG)]]+, and cis-[Pt(NH3)2[d(G*pTpG*)]], where the asterisks denote the sites of platinum binding, all bend the double helix, whereas the adduct trans-[Pt(NH3)2[d(G*pTpG*)]] imparts a degree of flexibility to the duplex. When modified by the monofunctional adduct cis-[Pt(NH3)2(N3-cytosine)(dG)]Cl the helix remains rod-like. These results reveal important structural differences in DNAs modified by the antitumor drug cisplatin and its analogs that could be important in the biological processing of the various adducts in vivo.     

Similar binding of the carcinostatic drugs cis-[Pt(NH3)2Cl2] and [Ru(NH3)5Cl] Cl2 to tRNAphe and a comparison with the binding of the inactive trans-[Pt(NH3)2Cl2] complex - reluctance in binding to Watson-Crick base pairs within double helix.

A comparative study of the binding of square planar cis- and trans-[Pt(NH3)2Cl2] complexes and the octahedral [Ru(NH3)5(H2O)]3+ complex to tRNAphe from yeast was carried out by X-ray crystallography. Both of the carcinostatic compounds, cis-[Pt(NH3)2Cl2] and [Ru(NH3)5(H2O)]3+ show similarities in their mode of binding to tRNA. These complexes bind specifically to the N(7) positions of guanines G15 and G18 in the dihydrouridine loop. [Ru(NH3)5(H2O)]3+ has an additional binding site at N(7) of residue G1 after extensive soaking times (58 days). A noncovalent binding site for ruthenium is also observed in the deep groove of the acceptor stem helix with shorter (25 days) soaking time. The major binding site for the inactive trans-[Pt(NH3)Cl2] complex is at the N(1) position of residue A73, with minor trans-Pt binding sites at the N(7) positions of residues Gm34, G18 and G43. The similarities in the binding modes of cis-[Pt(NH3)2Cl2] and [Ru(NH3)5(H2O)]3+ are expected to be related to their carcinostatic properties.     

Effects of differentiation-inducing agents on synthesis, maturation and secretion of cathepsin D in U937 and HL-60 cells

Treatment of human monocyte U937 and promyelocyte HL-60 cultures with agents known to induce differentiation (12-O-tetra-decanoylphorbol 13-acetate, calcitriol and dimethylsulfoxide) accelerates the maturation of cathepsin D and enhances the incorporation of [35S]methionine into cathepsin D. The most pronounced effects are obtained with calcitriol, which at a concentration of 10(-7) M increases the incorporation of [35S]methionine into cathepsin D from 0.08% to 0.4% of the detergent-soluble radioactivity. In addition, this treatment enhances the secretion of cathepsin D from about 8% to greater than or equal to 16% of the newly synthesized enzyme. In the presence of 10mM NH4Cl approximately half of the produced cathepsin D is secreted in both control and calcitriol-treated cells. It appears that in U937 cells two mechanisms are involved in sorting of cathepsin D. One of these is sensitive to NH4Cl and its efficiency is selectively decreased in cells pretreated with calcitriol.     

The preparation, characterisation, and DNA adduct profile of 2-amino-2-methyl-3-butanoneoximedichloroplatinum(II), a platinum(II) complex designed to bind to GpA sequences of DNA

The complex, 2-amino-2-methyl-3-butanoneoximedichloroplatinum(II), [Pt(ambo)Cl2], was chosen because of its potential to bind to GpA sequences of duplex DNA. Crystals of [Pt(ambo)Cl2] are monoclinic, space group, P2(1)/n, a = 6.799(4), b = 17.642(5), c = 8.193(2) A, beta = 102.10(3) degrees, Z = 4, R = 0.033 (1864 F). The binding of [Pt(ambo)Cl2] to salmon-sperm DNA was studied using enzymatic digestion and HPLC analysis. [Pt(ambo)Cl2] was found to form fewer GpG and ApG intrastrand adducts and more monofunctional adducts than [Pt(en)Cl2]. Binding to GpA sequences could not be established, but [Pt(ambo)Cl2] forms substantially more adducts with adenine than does [Pt(en)Cl2].     

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.     

The influence of methionine-containing peptides on the reaction of carboplatin with 5'-guanosine monophosphate: a comparison with cisplatin

The pH- and time-dependent reaction of the anticancer drug carboplatin, [Pt(cbdca-kappa(2)O,O')(NH(3))(2)] (cbdca=cyclobutane-1,1-dicarboxylate), with the tripeptides H-glyglymet-OH (glycylglycyl-L-methionine) and Ac-glyglymet-OH at 313 K was investigated by high-performance liquid chromatography, NMR and mass spectrometry. The relative stability of the initial ring-opened kappaS complex [Pt(cbdca-kappaO)(Ac-glyglymet-OH-kappaS)(NH(3))(2)] leads to increased formation of the kinetically favoured kappaS:kappaS' bis-adduct [Pt(Ac-glyglymet-OH-kappaS)(2)(NH(3))(2)](2+) in comparison with cisplatin. As a result a second 1:2 reaction pathway kappaS-->kappaS:kappaS'-->kappa(2)N(M), S:kappaS'-->kappa(3)N(G2),N(M), S:kappaS', where N(M) and N(G2) represent, respectively, metallated methionine and glycine nitrogen atoms, competes with the 1:1 route kappaS-->kappa(2)N(M), S-->kappa(3)N(G2),N(M), S also observed for cisplatin. Cleavage of N-acetylglycine at the backbone C(O)-N bond to the second gly residue (G2) is observed after 100 h for the respective tridentate complexes [Pt(Ac-glyglyH(-1)metH(-1)-OH-kappa(3)N(G2),N(M), S) (Ac-glyglymet-OH-kappaS)] and [Pt(Ac-glyglyH(-1)metH(-1)-OH-kappa(3)N(G2),N(M), S)(NH(3))] at pH <5.2. The longevity of the initial kappaS complex leads to about an eight-fold increase in the rate of formation of the kappaN7:kappaN7' bis-adduct [Pt(5'-GMP-kappaN7)(2)(NH(3))(2)](2-) for the reaction of carboplatin with 5'-GMP(2-) at pH 7 in the presence of Ac-glyglymet-OH. A mixed-ligand kappaS:kappaN7 species [Pt(5'-GMP-kappaN7)(Ac-glyglymet-OH-kappaS)(NH(3))(2)] provides the major precursor for this 1:2 nucleotide complex and kappaN7 coordination of 5'-GMP(2-) is also observed in the kappa(2)N(M),S:kappaN7 complex [Pt(5'-GMP-kappaN7)(Ac-glyglymetH(-1)-OH-kappa(2)N(M),S)(NH(3))(2)](-) formed by substitution of the ammine ligand trans to the methionine sulphur. As the intermediate kappaS:kappaN7 species is formed rapidly within the first 10 h of reaction, these results suggest that the transfer reaction pathway kappaS-->kappaS:kappaN7-->kappaN7:kappaN7' involving kappaS platinated peptides could play an important role in accelerating the rate of DNA binding for carboplatin.     

Cellular pharmacology of polynuclear platinum anti-cancer agents

Study of the cellular pharmacology of the dinuclear platinum complexes, BBR3005 ([?trans-PtCl(NH3)2?2H2N(CH2)6NH2]2+), BBR3171 ([?cis-PtCl(NH3)2?2H2N(CH2)6NH2]2+) and the trinuclear platinum complex, BBR3464 ([?trans-PtCl(NH3)2?2 mu-?trans-Pt(NH3)2(H2N(CH2)6NH2)2?]4+) was undertaken in wild type and cisplatin-resistant L1210 murine leukemia cell lines. All complexes are potent cytotoxic agents against the wild type cell line. Only BBR3464 shows enhanced activity against the cisplatin-resistant cell line following a brief exposure. This enhanced activity is attributable, in part, to preserved accumulation, which contrasts with diminished accumulation of cisplatin and both dinuclear platinum complexes. The cisplatin-resistant cell line is relatively tolerant of DNA adducts induced by both cisplatin and BBR3464, but BBR3464 is much less affected. All complexes induce DNA interstrand cross-links. Di/trinuclear complex-induced interstrand cross-linking peaks early, suggesting rapid genomic access and interaction. Subsequent decay suggests susceptibility to DNA repair mechanisms. Peak and area-under-the-curve values for interstrand cross-linking among the complexes correlate poorly with cytotoxic effects, especially in the cisplatin-resistant cell line. This suggests that all interstrand cross-linking adducts are not equal in their cytotoxic effect, or other, non-interstrand cross-linking adducts are significant. BBR3464 has been selected for clinical development largely on the basis of results from in vivo activity and toxicity studies. These results show BBR3464 to have unique properties in the context of acquired cisplatin-resistance that enhance its candidacy as a potential anticancer agent.