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New water-soluble bis(2-phenylazopyridine)ruthenium(II) complexes, all derivatives of the highly cytotoxic alpha-[Ru(azpy)(2)Cl(2)] (alpha denoting the coordinating pairs Cl, N(py), and N(azo) as cis, trans, cis, respectively) have been developed. The compounds 1,1-cyclobutanedicarboxylatobis(2-phenylazopyridine)ruthenium(II), alpha-[Ru(azpy)(2)(cbdca-O,O')] (1), oxalatobis(2-phenylazopyridine)ruthenium(II), alpha-[Ru(azpy)(2)(ox)] (2), and malonatobis(2-phenylazopyridine)ruthenium(II), alpha-[Ru(azpy)(2)(mal)] (3), have been synthesized and fully characterized. X-ray analyses of 1 and 2 are reported, and compound 1 is the first example in which the cbdca ligand is coordinated to a ruthenium center. The cytotoxicity of this series of water-soluble bis(2-phenylazopyridine) complexes has been determined in A2780 human ovarian carcinoma and A2780cisR, the corresponding cisplatin-resistant cell line. For comparison reasons, the cytotoxicity of the complexes alpha-[Ru(azpy)(2)Cl(2)], alpha-[Ru(azpy)(2)(NO(3))(2)], beta-[Ru(azpy)(2)Cl(2)] (beta indicating the coordinating pairs Cl, N(py), and N(azo) as cis, cis, cis, respectively), and beta-[Ru(azpy)(2)(NO(3))(2)] have been determined in this cell line. All the bis(2-phenylazopyridine)ruthenium(II) compounds display a promising cytotoxicity in the A2780 cell line (IC(50) = 0.9-10 microM), with an activity comparable to that of cisplatin and even higher than the activity of carboplatin. Interestingly, the IC(50) values of this series of ruthenium compounds (except the beta isomeric compounds) are similar in the cisplatin-resistant A2780cisR cell line compared to the normal cell line A2780, suggesting that the activity of these compounds might not be influenced by the multifactorial resistance mechanism that affect platinum anticancer agents. Show less

X-ray structures of the basic/helix-loop-helix/leucine zipper (bHLHZ) domains of Myc-Max and Mad-Max heterodimers bound to their common DNA target (Enhancer or E box hexanucleotide, 5'-CACGTG-3') have been determined at 1.9 A and 2.0 A resolution, respectively. E box recognition by these two structurally similar transcription factor pairs determines whether a cell will divide and proliferate (Myc-Max) or differentiate and become quiescent (Mad-Max). Deregulation of Myc has been implicated in the development of many human cancers, including Burkitt's lymphoma, neuroblastomas, and small cell lung cancers. Both quasisymmetric heterodimers resemble the symmetric Max homodimer, albeit with marked structural differences in the coiled-coil leucine zipper regions that explain preferential homo- and heteromeric dimerization of these three evolutionarily related DNA-binding proteins. The Myc-Max heterodimer, but not its Mad-Max counterpart, dimerizes to form a bivalent heterotetramer, which explains how Myc can upregulate expression of genes with promoters bearing widely separated E boxes. Show less

The application of pharmacokinetic (PK) and pharmacodynamic (PD) modeling in drug development has emerged during the past decades and it is has been suggested that the investigation of PK-PD relationships during drug development may facilitate and optimize the design of subsequent clinical development. Especially in oncology, well designed PK-PD modeling could be extremely useful as anticancer agents usually have a very narrow therapeutic index. This paper describes the application of the current insights in the use of PK-PD modeling to the design of clinical trials in oncology. The application of PK-PD modeling in each separate stage of (pre)clinical drug development of anticancer agents is discussed. The implementation of this approach is illustrated with the clinical development of docetaxel. Show less

Hypoxia is a stress that causes alterations in signal transduction and gene instability. In the cancer microenvironment, hypoxia plays a significant role in forming a tumor phenotype and tumor progression. We aimed to identify the genes upregulated by hypoxia in human breast cancer cell lines, a hormone-dependent MCF-7 and a hormone-independent MDA-MB-231, using microarray analysis. These cells were exposed to two oxygen concentrations such as 21% and 1% in a time-course. Out of 12625 genes, 26 genes were identified as commonly upregulated in both MCF-7 and MDA-MB-231 cells. Some of these genes were already reported as hypoxia-related, but some of those were identified newly. These commonly upregulated genes between hormone-dependent and hormone-independent cells would be a clue to study hypoxia-related events and to explore the novel therapeutic targets in human breast cancer. Show less

Nucleotide excision repair is a major cellular defense mechanism against the toxic effects of the anticancer drug cisplatin and other platinum-based chemotherapeutic agents. In this study, mononucleosomes were prepared containing either a site-specific cis-diammineplatinum(II)-DNA intrastrand d(GpG) or a d(GpTpG) cross-link. The ability of the histone core to modulate the excision of these defined platinum adducts was investigated as a model for exploring the cellular response to platinum-DNA adducts in chromatin. Comparison of the extent of repair by mammalian cell extracts of free and nucleosomal DNA containing the same platinum-DNA adduct reveals that the nucleosome significantly inhibits nucleotide excision repair. With the GTG-Pt DNA substrate, the nucleosome inhibits excision to about 10% of the level observed with free DNA, whereas with the less efficient GG-Pt DNA substrate the nucleosome inhibited excision to about 30% of the level observed with free DNA. The effects of post-translational modification of histones on excision of platinum damage from nucleosomes were investigated by comparing native and recombinant nucleosomes containing the same intrastrand d(GpTpG) cross-link. Excision from native nucleosomal DNA is approximately 2-fold higher than the level observed with recombinant material. This result reveals that post-translational modification of histones can modulate nucleotide excision repair from damaged chromatin. The in vitro system established in this study will facilitate the investigation of platinum-DNA damage by DNA repair processes and help elucidate the role of specific post-translational modification in NER of platinum-DNA adducts at the physiologically relevant nucleosome level. Show less

Genome sequencing has revolutionized all fields of life sciences. Bioinorganic chemistry is certainly not immune to this influence, which is presenting unprecedented challenges. A new goal for bioinorganic chemistry is the investigation of the linkages between inorganic elements and genomic information. This requires new advancements andor the development of new expertise in fields such as bioinformatics and genetics but also provides a driving force to push forward the exploitation of traditional analytical techniques and spectroscopic tools. The "case study" of metal homeostasis in cells is discussed to provide a flavor of the current evolution of the field. Show less

AIM: To investigate the aquation of oxaliplatin in aqueous solution at different temperatures and gain the kinetic data. METHODS: Electronic conductometry and high performance liquid chromatography (HPLC) were used to measure the oxaliplatin content in the reaction systems at different time. RESULTS: The aquation of oxaliplatin followed a pseudo-first-order rate law. In the absence of H+, the observed rate constant kobs was 7.76 x 10(-6).min-1 and the half life t1/2 was 62 days at 25 degrees C. In the presence of H+, the aquation could be accelerated by H+ according to the equation kobs = (2.61 + 21.9 [H+]) x 10(-4).min-1. The mechanism of aquation has also been proposed in this paper. From the mechanism, the rate of aquation following to r = (k1 k2) [l-OHP]/k-1 in the absence of H+ and r = (k1 + K0k3 [H+]) [l-OHP] in the presence of H+ have been deduced, which were in perfect agreement with the experimental results. CONCLUSION: In the absence of H+, the aqueous solution of oxaliplatin is stable, which meets to the request of clinical. Show less

Antitumor effects of cis-diamminedichloroplatinum(II) (cisplatin) and the clinical inactivity of its trans isomer (transplatin) have been considered a paradigm for the classical structure-activity relationships of platinum drugs. However, several new analogues of transplatin which exhibit a different spectrum of cytostatic activity including activity in tumor cells resistant to cisplatin have been recently identified. Analogues containing the planar amine ligand of the general structure trans-[PtCl(2)(NH(3))(L)], where L = planar amine, represent an example of such compounds. DNA is believed to be the major pharmacological target of platinum compounds. To contribute to the understanding of mechanisms underlying the activation of trans geometry in transplatin analogues containing planar amine ligands, various biochemical and biophysical methods were employed in previous studies to analyze the global modifications of natural DNA by trans-[PtCl(2)(NH(3))(L)]. These initial studies have revealed some unique features of the DNA binding mode of this class of platinum drugs. As the monofunctional lesions represent a significant fraction of stable adducts formed in DNA by bifunctional antitumor trans-platinum compounds with planar ligands, we analyzed in the present work short DNA duplexes containing the single, site-specific monofunctional adduct of a representative of this class of platinum drugs, antitumor trans-[PtCl(2)(NH(3))(thiazole)]. It has been shown that, in contrast to the adducts of monodentate chlorodiethylenetriamineplatinum(II) chloride or [PtCl(NH(3))(3)]Cl, the monofunctional adduct of trans-[PtCl(2)(NH(3))(thiazole)] inhibits DNA synthesis and creates a local conformational distortion similar to that produced in DNA by the major 1,2-GG intrastrand CL of cisplatin, which is considered the lesion most responsible for its anticancer activity. In addition, the monofunctional adducts of trans-[PtCl(2)(NH(3))(thiazole)] are recognized by HMGB1 domain proteins and removed by the nucleotide excision repair system similarly as the 1,2-GG intrastrand CL of cisplatin. The results of the present work further support the view that the simple chemical modification of the structure of an inactive platinum compound alters its DNA binding mode into that of an active drug and that processing of the monofunctional DNA adducts of the trans-platinum analogues in tumor cells may be similar to that of the major bifunctional adducts of "classical" cisplatin. Show less

Experiments were done to study the dynamic structural motions that determine protein hydrogen exchange (HX) behavior. The replacement of a solvent-exposed lysine residue with glycine (Lys8Gly) in a helix of recombinant cytochrome c does not perturb the native structure, but it entropically potentiates main-chain flexibility and thus can promote local distortional motions and large-scale unfolding. The mutation accelerates amide hydrogen exchange of the mutated residue by about 50-fold, neighboring residues in the same helix by less, and residues elsewhere in the protein not at all, except for Leu98, which registers the change in global stability. The pattern of HX changes shows that the coupled structural distortions that dominate exchange can be several residues in extent, but they expose to exchange only one amide NH at a time. This "local fluctuation" mode of hydrogen exchange may be generally recognized by disparate near-neighbor rates and a low dependence on destabilants (denaturant, temperature, pressure). In contrast, concerted unfolding reactions expose multiple neighboring amide NHs with very similar computed protection factors, and they show marked destabilant sensitivity. In both modes, ionic hydrogen exchange catalysts attack from the bulk solvent without diffusing through the protein matrix. Show less

Platinum anticancer drugs, such as cisplatin, are thought to exert their activity by DNA damage. Oxaliplatin, a clinically active diaminocyclohexane platinum compound, however, requires fewer DNA-Pt adducts than cisplatin to achieve cell growth inhibition. Here we investigated whether secondary DNA damage and apoptotic responses to oxaliplatin compensate for the reduced formation of DNA adducts. Oxaliplatin treatment of leukemic CEM and ovarian A2780 cancer cells resulted in early (4 hr) induction of DNA single-strand breaks measured by nucleoid sedimentation. These infrequent early lesions progress with time into massive double-stranded DNA fragmentation (fragments >50k bp) paralleled by characteristic apoptotic changes revealed by cell morphology and multivariate flow cytometry. Profound oxaliplatin-induced apoptotic DNA fragmentation was detectable following a 24 hr treatment of A2780 and CEM cells with 2 and 10 microM oxaliplatin, respectively. This DNA fragmentation was inhibited completely by the broad-spectrum caspase inhibitor Z-VAD-fmk. Cisplatin, which forms markedly more DNA-Pt adducts in CEM and A2780 cells than equimolar oxaliplatin, was similarly potent as oxaliplatin in terms of early strand breaks and later apoptotic responses. Oxaliplatin was also profoundly apoptotic in several other tumor cell lines of prostate origin but had only a marginal effect in normal prostate PrEC cells. Collectively, the results demonstrate that, relative to the magnitude of the primary DNA-Pt lesions, oxaliplatin is disproportionately more potent than cisplatin in the induction of apoptosis. Apoptosis induction, possibly enhanced by a contribution of targets other than DNA, seems to be an important factor in the mechanism of action of oxaliplatin. Show less

Transcription inhibition by DNA adducts of cisplatin is considered to be one of the major routes by which this anticancer drug kills cancer cells. Stalled RNA polymerases at platinum-DNA lesions evoke various cellular responses such as nucleotide excision repair, polymerase degradation, and apoptosis. T7 RNA polymerase and site-specifically platinated DNA templates immobilized on a solid support were used to study stalled transcription elongation complexes. In vitro transcription studies were performed in both a promoter-dependent and -independent manner. An elongation complex is strongly blocked by cisplatin 1,2-intrastrand d(GpG) and 1,3-intrastrand d(GpTpG) cross-links located on the template strand. Polymerase action is inhibited at multiple sites in the vicinity of the platinum lesion, the nature of which can be altered by the choice and concentration of NTPs. The [(1R,2R-diaminocyclohexane)Pt]2+ DNA adducts formed by oxaliplatin, which carries a stereochemically more demanding spectator ligand than the ammine groups in cisplatin, also strongly block the polymerase with measurable differences compared with cis-[(NH3)2Pt]2+ lesions. Elongation complexes stopped at sites of platinum damage were isolated and characterized. The stalled polymerase can be dissociated from the DNA by subsequent polymerases initiated from the same template. We also discovered that a polymerase stalled at the platinum-DNA lesion can resume transcription after the platinum adduct is chemically removed from the template. Show less

The RAS proteins control signalling pathways that are key regulators of several aspects of normal cell growth and malignant transformation. They are aberrant in most human tumours due to activating mutations in the RAS genes themselves or to alterations in upstream or downstream signalling components. Rational therapies that target the RAS pathways might inhibit tumour growth, survival and spread. Several of these new therapeutic agents are showing promise in the clinic and many more are being developed. Show less

Advances in bioinorganic chemistry since the 1970s have been driven by three factors: rapid determination of high-resolution structures of proteins and other biomolecules, utilization of powerful spectroscopic tools for studies of both structures and dynamics, and the widespread use of macromolecular engineering to create new biologically relevant structures. Today, very large molecules can be manipulated at will, with the result that certain proteins and nucleic acids themselves have become versatile model systems for elucidating biological function. Show less

Alkaline hydrolysis of the platinum anticancer drug oxaliplatin gives the oxalato monodentate complex and the dihydrated oxaliplatin complex in two consecutive steps. The acid dissociation constant for the oxalato monodentate intermediate was determined by a kinetic approach. The pK(a) value was estimated as 7.23. The monodentate intermediate is assumed to rapidly react with endogenous compounds, resulting in a continuous conversion of oxaliplatin via the monodentate form. Show less

The alkaline degradation of the chemotherapeutic agent oxaliplatin has been studied using liquid chromatography. The oxalato ligand is lost in two consecutive steps. First, the oxalato ring is opened, forming an oxalato monodentate intermediate, as identified by electrospray ionization mass spectrometry. Subsequently, the oxalato ligand is lost and the dihydrated oxaliplatin complex is formed. The observed rate constants for the first step (k(1)) and the second step (k(2)) follow the equation k(1) or k(2) = k(0) + k(OH(-) )[OH(-)], where k(0) is the rate constant for the degradation catalyzed by water and k(OH(-) ) represents the second-order rate constant for the degradation catalyzed by the hydroxide ion. At 37 degrees C the rate constants for the first step are k(OH(-) ) = 5.5 x 10(-2) min(-1) M(-1) [95% confidence interval (CI), 2.7 x 10(-2) to 8.4 x 10(-2) min(-1) M(-1)] and k(0) = 4.3 x 10(-2) min(-1) (95% CI, 4.0 x 10(-2) to 4.7 x 10(-2) min(-1)). For the second step the rate constants are k(OH(-) ) = 1.1 x 10(-3) min(-1) M(-1) (95% CI, -1.1 x 10(-3) to 3.3 x 10(-3)) min(-1) M(-1) and k(0) = 7.5 x 10(-3) min(-1) (95% CI, 7.2 x 10(-3) to 7.8 x 10(-3) min(-1)). Thus, the ring-opening step is nearly six times faster than the step involving the loss of the oxalato ligand. Show less

We have studied the effect of N-(4-hydroxyphenyl)retinamide on either malignant human leukaemia cells or normal cells and investigated its mechanism of action. We demonstrate that 4HPR induces reactive oxygen species increase on mitochondria at a target between mitochondrial respiratory chain complex I and II. Such oxidative stress causes cardiolipin peroxidation which in turn allows cytochrome c release to cytosol, caspase-3 activation and therefore apoptotic consumption. Moreover, this apoptotic pathway seems to be bcl-2/bax independent and count only on malignant cells but not normal nor activated lymphocytes. Show less

AbstractFollowing phagocytosis in vivo, acidification of extracellular pH (pHo) and intracellular metabolic acid generation contribute to cytosolic proton loading in neutrophils. Cytosolic pH (pHi) affects neutrophil function, although its regulation is incompletely understood. Its effect on mechanisms of neutrophil death is also uncertain. Thus, we investigated pHi regulation in Escherichia coli–exposed neutrophils, at various pathogen-to-phagocyte ratios (0:1-50:1), under conditions simulating the inflammatory milieu in vivo and correlated pHi changes with mechanisms of neutrophil death. Following phagocytosis, proton extrusion was dominated early by passive proton conductance channels, and later by Na+/H+ exchange (NHE). H+-translocating adenosine triphosphatase (V-ATPase) pHi regulation was evident primarily at lower bacterial densities. At physiologic pHo, lower pathogen-to-phagocyte ratios alkalinized pHi and inhibited apoptosis, whereas higher ratios acidified pHi (despite proton extrusive mechanisms) and promoted apoptosis. Necrosis was induced by high-density bacterial exposure at reduced pHo. Following phagocytosis, targeted inhibition of NHEs, proton conductance channels, or V-ATPases (amiloride, ZnCl2, or bafilomycin, respectively) moderately hyperacidified pHi and accelerated apoptosis. However, in combination they profoundly acidified pHi and induced necrosis. Proinflammatory mediators in vivo might affect both pHi regulation and cell death, so we tested the effects of bronchoalveolar lavage (BAL) fluid from patients with cystic fibrosis (CF) and healthy subjects. Only CF BAL fluid alkalinized pHi and suppressed apoptosis at physiologic pHo, but failed to prevent necrosis following phagocytosis at low pHo. Thus, a precarious balance between cytosolic proton loading and extrusion after phagocytosis dictates the mode of neutrophil cell death. pHi/pHo might be therapeutically targeted to limit neutrophil necrosis and protect host tissues during necrotizing infections. Show less

The role of specific lipid structures in biological membranes has been elusive. There are hundreds of them in nature. Why has nature made them? How do they aid in the functioning of membrane proteins? Genetics with its 'knock out' organisms declares that functions persist in the absence of any particular lipid. Nonetheless some lipids, such as cardiolipin (CL), are associated with particular functions in the cell. It may merely expand the variety of culture conditions (pH, temperature, etc.) under which the wild-type organism survives. This article explores a unique role of CL as a proton trap within membranes that conduct oxidative phosphorylation and therefore the synthesis of ATP. CL's pK(2) (above 8.0) provides a role for it as a headgroup proton trap for oxidative phosphorylation. It suggests why CL is found in membranes that pump protons. The high pK(2) also indicates that the headgroup has but one negative charge in the neutral pH range. Data on the binding of CL to all of the oxidative phosphorylation proteins suggest that the CL may aggregate the oxidative phosphorylation proteins into a patch while it restricts pumped protons within its headgroup domain - supplying protons to the ATP synthase with minimal changes in the bulk phase pH. Show less

Oxaliplatin (Eloxatine) is a third-generation platinum compound which has shown a wide antitumour effect both in vitro and in vivo, a better safety profile than cisplatin and a lack of cross-resistance with cisplatin and carboplatin. In this scenario, oxaliplatin may represent an innovative and challenging drug extending the antitumour activity in diseases such as gastrointestinal cancer that are not usually sensitive to these coordination complexes. Oxaliplatin has a non-hydrolysable diaminocyclohexane (DACH) carrier ligand which is maintained in the final cytotoxic metabolites of the drug. Like cisplatin, oxaliplatin targets DNA producing mainly 1,2-GG intrastrand cross-links. The cellular and molecular aspects of the mechanism of action of oxaliplatin have not yet been fully elucidated. However, the intrinsic chemical and steric characteristics of the DACH-platinum adducts appear to contribute to the lack of cross-resistance with cisplatin. To date, mismatch repair and replicative bypass appear to be the processes most likely involved in differentiating the molecular responses to these agents. Show less

AbstractProtein‐based virtual screening of chemical libraries is a powerful technique for identifying new molecules that may interact with a macromolecular target of interest. Because of docking and scoring limitations, it is more difficult to apply as a lead optimization method because it requires that the docking/scoring tool is able to propose as few solutions as possible and all of them with a very good accuracy for both the protein‐bound orientation and the conformation of the ligand. In the present study, we present a consensus docking approach (ConsDock) that takes advantage of three widely used docking tools (Dock, FlexX, and Gold). The consensus analysis of all possible poses generated by several docking tools is performed sequentially in four steps: (i) hierarchical clustering of all poses generated by a docking tool into families represented by a leader; (ii) definition of all consensus pairs from leaders generated by different docking programs; (iii) clustering of consensus pairs into classes, represented by a mean structure; and (iv) ranking the different means starting from the most populated class of consensus pairs. When applied to a test set of 100 protein–ligand complexes from the Protein Data Bank, ConsDock significantly outperforms single docking with respect to the docking accuracy of the top‐ranked pose. In 60% of the cases investigated here, ConsDock was able to rank as top solution a pose within 2 Å RMSD of the X‐ray structure. It can be applied as a postprocessing filter to either single‐ or multiple‐docking programs to prioritize three‐dimensional guided lead optimization from the most likely docking solution. Proteins 2002;47:521–533. © 2002 Wiley‐Liss, Inc. Show less