New chemistry of an old molecule: cis-[Pt(NH3)2Cl2].

The discovery that cis-diamminedichloroplatinum(II) (cis-DDP) has clinically useful antitumor properties and can form platinum blues spawned an extensive investigation of its chemistry in water. Several new molecules have been synthesized, some rather old ones have been characterized for the first time, and clues have begun to emerge about the chemical interaction of cis-DDP with its likely biological target, DNA.

Specific binding of chromosomal protein HMG1 to DNA damaged by the anticancer drug cisplatin.

The mechanism of action of the anticancer compound cis-diamminedichloroplatinum(II) (cisplatin) involves covalent binding to DNA. In an effort to understand the tumor-specific cytotoxicity of such DNA damage, the interactions of these lesions with cellular proteins have been studied. One such protein has been identified as the high-mobility group protein HMG1. Recombinant rat HMG1 binds specifically (dissociation constant 3.7 +/- 2.0 x 10(-7) molar) to DNA containing cisplatin d(GpG) or d(ApG) intrastrand cross-links, which unwind and bend DNA in a specific manner, but not to DNA modified by therapeutically inactive platinum analogs. These results suggest how HMG1 might bind to altered DNA structures and may be helpful in designing new antitumor drugs.

Visualization of polymercurimethane-labeled for bacteriophage in the scanning transmission electron microscope.

Each of the 2700 coat proteins of fd bacteriophage was labeled with tetrakis(acetoxymercuri)methane (TAMM) or aquoglycylmethionineplatinum(II). The TAMM-labeled specimens reveal striking bright spots in the scanning transmission electron microscope which arise from clustering. Measurements of mass show increases consistent with the addition of four mercury atoms or one platinum atom, respectively, to each coat protein.

A mixed-valent polyiron oxo complex that models the biomineralization of the ferritin core.

A novel polyiron oxo complex, [FeIII4FeII8(O)2(OCH3)18(O2CCH3)6(CH3OH) 4.67] (1), has been prepared from ferrous acetate and lithium methoxide in methanol by slow addition of dioxygen. The three-dimensional close-packed layered structure found in 1 closely mimics that proposed for the inorganic core in the iron storage protein ferritin. The Mössbauer spectra of 1 reveal superparamagnetic relaxation at temperatures below 15 K, a property characteristic of the ferritin core. The small size and mixed-valent nature of 1 suggest that it is a reasonable model for intermediates formed in the biomineralization of iron during ferritin core formation. A related compound, with the same iron-oxygen framework found in 1 but containing only two ferric ions, has also been structurally characterized. Because the clusters exhibit properties of both discrete molecules and extended solids, they are representative of a new class of nanometer-sized compounds that bridge the molecular solid-state boundary.

Ixr1, a yeast protein that binds to platinated DNA and confers sensitivity to cisplatin.

Structure-specific recognition proteins (SSRPs) bind to DNA containing intrastrand cross-links formed by the anticancer drug cisplatin. A yeast gene encoding an SSRP, designated IXR1, was cloned and sequenced. The Ixr1 protein, a member of the high mobility group-box protein family, bound specifically to DNA modified with cisplatin but not inactive platinum compounds. A yeast strain with an inactivated IXR1 gene was half as sensitive to cisplatin and accumulated one-third as many platinum-DNA lesions after treatment with cisplatin as the parental strain. These findings suggest that SSRPs play a role in mediating the cytotoxicity of cisplatin.

Binding of cis- and trans-dichlorodiammineplatinum(II) to DNA: evidence for unwinding and shortening of the double helix.

The antitumor drug cis-dichlorodiammineplatinum(II) (cis-DDP) and the inactive trans isomer bind and produce cooperative changes in closed and nicked circular duplex DNA's. Covalent binding of both platinum complexes to the closed circular DNA alters the degree of supercoiling, presumably by disrupting and unwinding the double helix. Electron micrographs show the platinated DNA's to be shortened by up to 50 percent of their original length. At similar ratios of bound platinum per nucleotide, the electrophoretic mobilities of the DNA's in gels containing the dye ethidium bromide are the same for both isomers. The only detectable difference in the binding of the two platinum isomers is an increase in the electrophoretic mobility in nondye gels of closed circular DNA having small amounts of bound cis-DDP that is not apparent for the trans complex.

Stereochemical requirements for intercalation of platinum complexes into double-stranded DNA's.

The complexes 1,10-phenanthrolineethylenediamineplatinum(II) and 2,2'-bipyridineethylenediamineplatinum(II) have a planar, aromatic ligand system that facilitates intercalation, as shown by their ability to unwind closed circular duplex DNA. Nonbonded steric interactions can rotate the pryidine ligands out of the coordination plane in bis(pyridine)ethylenediamineplatinum(II), thus preventing intercalation. Fiber x-ray diffraction patterns of the two metallointeracalators indicate that the binding is governed by the neighbor exclusion principle.

X-ray structure of the major adduct of the anticancer drug cisplatin with DNA: cis-[Pt(NH3)2(d(pGpG))].

Crystals of the adduct of the anticancer drug cis-diamminedichloroplatinum(II), cis-DDP, with d(pGpG), its putative target on DNA in the cancer cell, have been obtained and used in an x-ray crystallographic study to elucidate the molecular structure to atomic resolution. Each of the four crystallographically independent cis-[Pt(NH3)2(d(pGpG))] molecules is comprised of a square-planar platinum atom bonded to two ammonia ligands and two N(7) atoms of guanosine nucleosides from the same chain. Base stacking of the two adjacent guanine rings is completely disrupted by coordination to the cis-(Pt(NH3)2)2+ unit. Comparison of the backbone and deoxyribose ring torsion angles with those found by previous (nuclear magnetic resonance spectroscopy) studies of this adduct in solution demonstrates that the solid state geometry is substantially the same as that in solution. The relevance of these results to the molecular mechanism of action of cis-DDP is discussed.

Cisplatin and DNA repair in cancer chemotherapy.

Cisplatin, a DNA-damaging agent, is one of the most widely used anticancer drugs. As with all members of this class of chemotherapeutic compounds, the clinical success of cisplatin is compromised if tumor cells become resistant by various mechanisms, including enhanced DNA repair. In addition to its role in resistance, DNA repair has been linked to the cytotoxic mechanism of cisplatin. DNA damaged by the drug has proved to be a valuable tool for exploring the details of the nucleotide excision repair pathway.

Direct ratiometric detection of nitric oxide with Cu(ii)-based fluorescent probes.

We report the first Cu(ii)-based fluorescent sensors for ratiometric detection of nitric oxide. The two probes operate by energy transfer between hydroxycoumarin and fluorescein chromophores, contain polyproline or piperazine as rigid spacers, and elicit a rapid and selective ratiometric response upon direct reaction with nitric oxide.

Formation of cis-diamminedichloroplatinum(II) 1,2-intrastrand cross-links on DNA is flanking-sequence independent.

Mapping of cis-diamminedichloroplatinum(II) (cis-DDP, cisplatin) DNA adducts over >3000 nucleotides was carried out using a replication blockage assay. The sites of inhibition of modified T4 DNA polymerase, also referred to as stop sites, were analyzed to determine the effects of local sequence context on the distribution of intrastrand cisplatin cross-links. In a 3120 base fragment from replicative form M13mp18 DNA containing 24.6% guanine, 25.5% thymine, 26.9% adenine and 23.0% cytosine, 166 individual stop sites were observed at a bound platinum/nucleotide ratio of 1-2 per thousand. The majority of stop sites (90%) occurred at G(n>2) sequences and the remainder were located at sites containing an AG dinucleotide. For all of the GG sites present in the mapped sequences, including those with Gn(>)2, 89% blocked replication, whereas for the AG sites only 17% blocked replication. These blockage sites were independent of flanking nucleotides in a sequence of N(1)G*G*N(2) where N(1), N(2) = A, C, G, T and G*G* indicates a 1,2-intrastrand platinum cross-link. The absence of long-range sequence dependence was confirmed by monitoring the reaction of cisplatin with a plasmid containing an 800 bp insert of the human telomere repeat sequence (TTAGGG)(n). Platination reactions monitored at several formal platinum/nucleotide ratios or as a function of time reveal that the telomere insert was not preferentially damaged by cisplatin. Both replication blockage and telomere-insert plasmid platination experiments indicate that cisplatin 1,2-intrastrand adducts do not form preferentially at G-rich sequences in vitro.

Cisplatin: from DNA damage to cancer chemotherapy.

Cisplatin [cis-DDP, cis-diamminedichloroplatinum(II)] is a potent anticancer drug that has been used successfully to treat tumors of the head, neck, lungs, and genitourinary tract. The biological activity of cisplatin was discovered serendipitously more than 30 years ago, and since that time research efforts have focused on elucidating its mechanism of action. The present review provides a historical perspective of our attempts to understand this complex phenomenon and the results of recent work that guides our current activities in this field. Continued efforts to understand the mechanism of genotoxicity of cisplatin are expected to lead to the discovery of new drugs and combinations for the improvement of cancer chemotherapy.

Mechanistic studies of a novel class of trisubstituted platinum(II) antitumor agents.

Chemical and biological studies are presented for a new series of platinum(II) antitumor agents that violate the classical structure-activity relationships established for platinum complexes. These new agents, which have demonstrated activity against murine and human tumor systems, are cis-[Pt(NH3)2(Am)Cl]+ cations, in which Am is a derivative of pyridine, pyrimidine, purine, or aniline. Members from this series block simian virus 40 DNA replication in vitro and inhibit the action of DNA polymerases at individual guanine residues in replication mapping experiments. Monoclonal antibodies that bind selectively to cisplatin lesions on calf thymus DNA were used in a competitive enzyme-linked immunosorbent assay study to show that the platinum-triamine complexes do not produce the type of intrastrand cross-links on DNA that are characteristics of cisplatin and analogues with the general formula cis-[Pt(amine)2X2]. These results indicate that cis-[Pt(NH3)2(Am)Cl]+ cations form monofunctional adducts on DNA rather than eliminate NH3 or Am to afford bifunctional lesions. This conclusion is further supported by nuclear magnetic resonance spectroscopic and enzymatic digestion analyses of the products of the reactions of these triamine complexes with d(GpG) and dG, which also reveal monofunctional binding. When cis-[Pt(NH3)2(4-Br-pyridine)Cl]+ was allowed to stand in phosphate-buffered saline at 37 degrees C for 14 days, however, NH4+ was released and trans-[Pt(NH3)(4-Br-pyridine)Cl2] formed concomitantly. This compound was characterized by a single crystal X-ray diffraction study, the details of which are reported. The fact that trans-[Pt(NH3)(4-Br-pyridine)Cl2] displays no anticancer activity, however, indicates that its formation from cis-[Pt(NH3)2(4-Br-pyridine)Cl]+ is not a significant component of the mechanism of action of this platinum-triamine complex. Taken together, these findings indicate that the cytotoxicity of cis-[Pt(NH3)2(Am)Cl]+ complexes most likely arises from the formation of monofunctional adducts. The DNA binding properties associated with this new class of antitumor agents suggest that they may display an activity profile different from that of cisplatin and related analogues.

13C magnetic resonance investigation of mercury (II) binding to nucleosides and thiolated nucleosides in dimethyl sulfoxide.

Natural abundance, proton-decoupled 13C magnetic resonance spectroscopy is shown to be a useful technique for identifying the mercury (II) binding sites on nucleosides and especially thiolated nucleosides. Measurements made on dimethyl sulfoxide-d6 solutions, 0.5 M in nucleoside and 0.15 M in mercury, reveal that both CH3 HgCl and HgCl2 bind principally to the sulfur atoms of s6 Guo and s8 Guo. The 13C NMR spectra of the unthiolated nucleosides in the presence of excess (4:1) mercury reveal that HgCl2 binds to N-3 of cytidine, to more than one site on adenosine and guanosine, but not strongly to uridine. Excess HgCl2 shifts the thiocarbonyl carbon atoms in s6 Guo and s8 Guo approx. 16 ppm upfield compared to the free nucleosides, and there is evidence for additional coordination to N-7 of s6 Guo. Binding to the ribose hydroxyl groups is clearly ruled out. At least in these instances, 13C NMR proves to be useful for assigning the mercury (II) binding sites, complementing the results of proton magnetic resonance studies. Proton NMR data for the binding of CH3 HgCl and HgCl2 to s6 Guo and s8 Guo are also presented.

Crystallization and preliminary X-ray analysis of the methane monooxygenase hydroxylase protein from Methylococcus capsulatus (Bath).

Methane monooxygenase is a multicomponent enzyme system that catalyzes the conversion of methane to methanol in methanotrophic bacteria. Catalysis occurs at non-heme dinuclear iron centers contained in the hydroxylase component of the system, a dimer of composition alpha 2 beta 2 gamma 2. The hydroxylase protein from Methylococcus capsulatus (Bath) has been crystallized from aqueous solutions containing polyethylene glycol, lithium sulfate, and ammonium acetate. The crystals are orthorhombic, space group P2(1)2(1)2(1), with one dimer of relative molecular mass M(r) = 252,000 in the asymmetric unit. The unit cell dimensions are a = 62.6 A, b = 110.1 A, c = 333.5 A. The crystals diffract uniformly beyond 2.5 A resolution. Crystals of the related hydroxylase from Methylosinus trichosporium OB3b have also been obtained.

Multiple heavy-atom reagents for macromolecular X-ray structure determination. Application to the nucleosome core particle.

The X-ray structure of the nucleosome core particle was solved at 7 A resolution using the method of multiple isomorphous replacement based on two isomorphous derivatives, each containing a different multiple heavy-atom compound. The preparation of these heavy-atom compounds and their application to this macromolecular structure determination are described. The first of these reagents, TAMM (tetrakis(acetoxymercuri)methane), was solubilized by the addition of an excess of glycylglycine and, when added to crystals of the nucleosome core particle, produced a derivative with a single major site. Despite the large mass of 206,000 daltons per asymmetric unit, the position of the TAMM molecule was found in these crystals using the difference Patterson technique. This compound was sufficiently electron-dense to produce a unique solution, whereas the mono-mercurial, methylmercury nitrate had been inadequate. The second reagent, PIP (di-mu-iodobis(ethylenediamine)diplatinum(II) nitrate), is freely soluble in aqueous solution and, on addition to the crystals, labelled the histone proteins at several sites. The locations of the PIP groups were determined from difference Fourier and Patterson maps. The X-ray structure and solution characterization of this compound are reported. These multiple heavy-atom compounds appear to be generally applicable to X-ray structure determination, and are particularly useful in conjunction with crystals having asymmetric units of large volume but lacking non-crystallographic symmetry elements.

Effects of DNA adduct structure and distribution on the mutagenicity and genotoxicity of two platinum anticancer drugs.

cis-Diamminedichloroplatinum(II) (cis-DDP) and cis,trans,cis-ammine(cyclohexylamine)-dibutyratodichloroplatinu m(IV) (ACDDP) are anticancer drugs that bind to DNA, forming replication blocking adducts. ACDDP, probably manifests its cytotoxicity through the metabolite cis-ammine(cyclohexylamine)dichloroplatinum(II) (ACDP). The biological effects of ACDP and cis-DDP were compared by studying polymerase inhibition in vitro and mutagenesis and genotoxicity in vivo in the duplex genome of bacteriophage M13mp18 replicated in Escherichia coli. cis-DDP and ACDP adducts were equally genotoxic within the statistical error limits of the analysis. Survival of genomes platinated by either drug, increased by threefold in cells pretreated with u.v. irradiation to induce the SOS functions of the host. In the M13mp18 lacZ' gene fragment, the mutagenicity of ACDP was lower than that of cis-DDP; the difference was approximately twofold at a dose of two adducts per 370 base-pair mutational target. Mutagenesis by both compounds was SOS-dependent. The structural basis for lower mutagenicity of ACDP is proposed to be its reduced reactivity at d(ApG) sites. This effect is attributed to an orientational isomerism that precludes the formation of one of two possible DNA adducts at d(ApG) residues. The types of mutations induced for both drugs were similar, but they occurred with different distributions. Both compounds induced primarily G-->T transversions at d(GpG) sites whereas G-->A transitions and A-->T transversions, many at d(ApG), d(GpNpG), and d(GpG) sites, were also well represented. The mapping of DNA adducts by DNA synthesis inhibition revealed excellent correlation between the location of DNA lesions and the sites of mutations. Analysis of the distribution of mutations and the distribution of potential platination sites revealed no sequence-dependent mutation hotspots; i.e. mutagenesis was random throughout the lacZ' region of the M13mp18 bacteriophage genome. These results offer insights into the molecular mechanism of mutagenicity of platinum anticancer drugs.

DNA sequence context modulates the impact of a cisplatin 1,2-d(GpG) intrastrand cross-link on the conformational and thermodynamic properties of duplex DNA.

The anticancer activity of cisplatin derives from its ability to bind and cross-link DNA, with the major adduct being the 1,2-d(GpG) intrastrand cross-link. Here, the consequences of this adduct on the conformation, thermal stability, and energetics of duplex DNA are assessed, and the modulation of these parameters by the sequence context of the adduct is evaluated. The properties of a family of 15-mer DNA duplexes containing a single 1,2-d(GpG) cis-¿Pt(NH(3))(2)¿(2+) intrastrand cross-link are probed in different sequence contexts where the flanking base-pairs are systematically varied from T.A to C.G to A.T. By using a combination of spectroscopic and calorimetric techniques, the structural, thermal, and thermodynamic properties of each duplex, both with and without the cross-link, are characterized. Circular dichroism spectroscopic data reveal that the cross-link alters the structure of the host duplex in a manner consistent with a shift from a B-like to an A-like conformation. Thermal denaturation data reveal that the cross-link induces substantial thermal and thermodynamic destabilization of the host duplex. Significantly, the magnitudes of these cross-link-induced effects on duplex structure, thermal stability, and energetics are influenced by the bases that flank the adduct. The presence of flanking A.T base-pairs, relative to T.A or C.G base-pairs, enhances the extent of cross-link-induced alteration to an A-like conformation and dampens the extent of cross-link-induced duplex destabilization. These results are discussed in terms of available structural data, and in terms of the selective recognition of cisplatin-DNA adducts by HMG-domain proteins.

Mutational and structural analyses of the regulatory protein B of soluble methane monooxygenase from Methylococcus capsulatus (Bath).

BACKGROUND: The soluble methane monooxygenase (sMMO) system in methanotrophic bacteria uses three protein components to catalyze the selective oxidation of methane to methanol. The coupling protein B (MMOB) both activates the carboxylate-bridged diiron center in the hydroxylase (MMOH) for substrate oxidation and couples the reaction to electron transfer from NADH through the sMMO reductase. Although the X-ray structure of the hydroxylase is known, little structural information is available regarding protein B. RESULTS: Wild-type protein B from Methylococcus capsulatus (Bath) is very susceptible to degradation. The triple mutant protein B, Gly10-->Ala, Gly13-->Gln, Gly16-->Ala is resistant to degradation. Analyzing wild-type and mutant forms of protein B using size exclusion chromatography and circular dichroism spectroscopy suggests that the amino terminus of MMOB (Ser1-Ala25) is responsible for the proteolytic sensitivity and unusual mobility of the protein. We used the stable triple glycine protein B mutant to generate an affinity column for the hydroxylase and investigated the interaction between MMOH and MMOB. These results suggest the interaction is dominated by hydrophobic contacts. CONCLUSIONS: A structural model is presented for protein B that explains both its proclivity for degradation and its anomalous behavior during size exclusion chromatography. The model is consistent with previously published biophysical data, including the NMR structure of the phenol hydroxylase regulatory protein P2. Furthermore, this model allows for detailed and testable predictions about the structure of protein B and the role of proposed recognition sites for the hydroxylase.

Rapid fluorescence-based reporter-gene assays to evaluate the cytotoxicity and antitumor drug potential of platinum complexes.

BACKGROUND: The need for new platinum antitumor drugs is underscored by the usefulness of cisplatin and carboplatin in chemotherapy and the resistance of many tumors to these compounds. Combinatorial chemistry could aid in the search for cisplatin analogs if fast, high-throughput assays were available. Our goal was to develop rapid cell-based assays suitable for high-throughput screening that accurately predict the cytotoxicity of platinum complexes. We examined the effects of platinum complexes and other agents on reporter-gene expression in cancer cells. RESULTS: HeLa Tet-On cells with inducible enhanced green fluorescent protein (EGFP) were prepared. Cisplatin and other cis-disubstituted platinum complexes inhibited EGFP expression, with a strong positive correlation between EGFP inhibition and cytotoxicity. By contrast, trans-[Pt(NH(3))(2)Cl(2)], other trans-platinum complexes, methyl methanesulfonate or heat shock stimulated EGFP expression. Northern and nuclear run-on analyses revealed that the changes in EGFP expression were at the level of transcription. In another reporter-gene assay in Jurkat cells, cisplatin, but not trans-[Pt(NH(3))(2)Cl(2)] or K(2)[PtCl(4)], inhibited beta-lactamase expression, as measured by hydrolysis of the fluorescent substrate CCF2. CONCLUSIONS: The EGFP results indicate that cytotoxic stress enhances transcription from the inducible promoter, whereas compounds able to form the 1,2-intrastrand platinum-DNA cross-links repress transcription. Both fluorescence-based reporter-gene assays afford promising new approaches to platinum anticancer drug discovery.