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Computational modeling has been adopted in all aspects of drug research and development, from the early phases of target identification and drug discovery to the late-stage clinical trials. The different questions addressed during each stage of drug R&D has led to the emergence of different modeling methodologies. In the research phase, systems biology couples experimental data with elaborate computational modeling techniques to capture lifecycle and effector cellular functions (e.g. metabolism, signaling, transcription regulation, protein synthesis and interaction) and integrates them in quantitative models. These models are subsequently used in various ways, i.e. to identify new targets, generate testable hypotheses, gain insights on the drug's mode of action (MOA), translate preclinical findings, and assess the potential of clinical drug efficacy and toxicity. In the development phase, pharmacokinetic/pharmacodynamic (PK/PD) modeling is the established way to determine safe and efficacious doses for testing at increasingly larger, and more pertinent to the target indication, cohorts of subjects. First, the relationship between drug input and its concentration in plasma is established. Second, the relationship between this concentration and desired or undesired PD responses is ascertained. Recognizing that the interface of systems biology with PK/PD will facilitate drug development, systems pharmacology came into existence, combining methods from PK/PD modeling and systems engineering explicitly to account for the implicated mechanisms of the target system in the study of drug-target interactions. Herein, a number of popular system biology methodologies are discussed, which could be leveraged within a systems pharmacology framework to address major issues in drug development. Show less

Two series of tetrazole-containing platinum(II) and palladium(II) chlorido complexes, trans-[ML(2)Cl(2)] (M=Pt, Pd) and cis-[PtL(2)Cl(2)]·nH(2)O (n=0, 1), where L is 1- or 2-substituted 5-aminotetrazole, have been synthesized and thoroughly characterized. Configuration of platinum(II) complexes obtained from the reaction of 5-aminotetrazoles with K(2)PtCl(4) has been found to vary depending on the nature of tetrazole derivatives and reaction conditions. According to in vitro cytotoxic evaluation, only platinum complexes display noticeable antiproliferative effect, and their cytotoxicity depends strongly on their geometry and hydrophobicity of the carrier ligands. The most promising complexes are cis-[Pt(1-apt)(2)Cl(2)]·H(2)O and cis-[Pt(2-abt)(2)Cl(2)]·H(2)O, where 1-apt is 5-amino-1-phenyltetrazole and 2-abt is 5-amino-2-tert-butyltetrazole. In comparison with cisplatin, they show comparable cytotoxic potency against cisplatin-sensitive human cancer cell lines, cis-[Pt(2-abt)(2)Cl(2)]·H(2)O performing substantially higher activity against cisplatin-resistant cell lines. Cell cycle studies in H1299 cell line indicated that cis-[Pt(2-abt)(2)Cl(2)]·H(2)O induced apoptosis launched from G2 accumulations. The DNA interaction with cis-[Pt(1-apt)(2)Cl(2)]·H(2)O was followed by UV spectroscopy, circular dichroism, hydrodynamic and electrophoretic mobility studies. Both cis-[Pt(1-apt)(2)Cl(2)]·H(2)O and cis-[Pt(2-abt)(2)Cl(2)]·H(2)O complexes appeared to be significantly less toxic than cisplatin in mice, while only compound cis-[Pt(1-apt)(2)Cl(2)]·H(2)O displayed noticeable efficacy in vivo. Show less

Uncoupling proteins (UCPs) belong to a large family of mitochondrial solute carriers 25 (SLC25s) localized at the inner mitochondrial membrane. UCPs transport protons directly from the intermembrane space to the matrix. Of five structural homologues (UCP1 to 5), UCP4 and 5 are principally expressed in the central nervous system (CNS). Neurons derived their energy in the form of ATP that is generated through oxidative phosphorylation carried out by five multiprotein complexes (Complexes I-V) embedded in the inner mitochondrial membrane. In oxidative phosphorylation, the flow of electrons generated by the oxidation of substrates through the electron transport chain to molecular oxygen at Complex IV leads to the transport of protons from the matrix to the intermembrane space by Complex I, III, and IV. This movement of protons to the intermembrane space generates a proton gradient (mitochondrial membrane potential; MMP) across the inner membrane. Complex V (ATP synthase) uses this MMP to drive the conversion of ADP to ATP. Some electrons escape to oxygen-forming harmful reactive oxygen species (ROS). Proton leakage back to the matrix which bypasses Complex V resulting in a major reduction in ROS formation while having a minimal effect on MMP and hence, ATP synthesis; a process termed "mild uncoupling." UCPs act to promote this proton leakage as means to prevent excessive build up of MMP and ROS formation. In this review, we discuss the structure and function of mitochondrial UCPs 4 and 5 and factors influencing their expression. Hypotheses concerning the evolution of the two proteins are examined. The protective mechanisms of the two proteins against neurotoxins and their possible role in regulating intracellular calcium movement, particularly with regard to the pathogenesis of Parkinson's disease are discussed. Show less

Mitochondrial uncoupling protein-4 (UCP4) protects against Complex I deficiency as induced by 1-methyl-4-phenylpyridinium (MPP+), but how UCP4 affects mitochondrial function is unclear. Here we investigated how UCP4 affects mitochondrial bioenergetics in SH-SY5Y cells. Cells stably overexpressing UCP4 exhibited higher oxygen consumption (10.1%, p<0.01), with 20% greater proton leak than vector controls (p<0.01). Increased ATP supply was observed in UCP4-overexpressing cells compared to controls (p<0.05). Although state 4 and state 3 respiration rates of UCP4-overexpressing and control cells were similar, Complex II activity in UCP4-overexpressing cells was 30% higher (p<0.05), associated with protein binding between UCP4 and Complex II, but not that of either Complex I or IV. Mitochondrial ADP consumption by succinate-induced respiration was 26% higher in UCP4-overexpressing cells, with 20% higher ADP:O ratio (p<0.05). ADP/ATP exchange rate was not altered by UCP4 overexpression, as shown by unchanged mitochondrial ADP uptake activity. UCP4 overexpression retained normal mitochondrial morphology in situ, with similar mitochondrial membrane potential compared to controls. Our findings elucidate how UCP4 overexpression increases ATP synthesis by specifically interacting with Complex II. This highlights a unique role of UCP4 as a potential regulatory target to modulate mitochondrial Complex II and ATP output in preserving existing neurons against energy crisis. Show less

AbstractWith the extensive use of two‐photon fluorescence microscopy (2PFM) in the biomedical field, the need for development of fluorescent probes with improved two‐photon fluorescence (2PF) properties has triggered extensive studies in the synthesis of new probes that undergo efficient two‐photon absorption (2PA). In order to provide a more comprehensive comparison of fluorophores for 2PF bioimaging, a figure of merit (FM) was developed by normalizing the 2PA action cross‐section, a commonly used parameter for characterizing bioimaging 2PF probes, by the photodecomposition quantum yield. Another important aspect of developing 2PA fluorophores is hydrophilicity. Although hydrophilic fluorophores are generally preferred in 2PFM bioimaging, hydrophobic fluorophores are typically easier to synthesize and purify, and have been used successfully in 2PFM bioimaging. The methodologies of dispersing hydrophobic fluorophores into aqueous media, such as in a DMSO/water mixture, micelles, silica nanoparticles, or forming polymer nanoparticles, are reviewed. The design and synthesis of hydrophilic 2PA fluorophores, achieved by introducing polyethylene glycol (PEG), anionic acid groups, cationic ammonium salt, and PAMAM dendrimers, is presented. Introduction of specificity to target certain biomarkers via labeling of antibodies, DNA, smallbioactive molecules, and peptides, and for the sensing of sepcific cations and pH, is also reviewed. Show less

Readily synthesised and functionalised di-1,2,3-triazole “click” ligands are shown to self-assemble into coordinatively saturated, quadruply stranded helical [Pd2L4](BF4)4 cages with Pd(II) ions. The cages have been fully characterised by elemental analysis, HR-ESMS, IR, 1H, 13C and DOSY NMR, DFT calculations, and in one case by X-ray crystallography. By exploiting the CuAAC “click” reaction we were able to rapidly generate a small family of di-1,2,3-triazole ligands with different core spacer units and peripheral substituents and examine how these structural modifications affected the formation of the [Pd2L4](BF4)4 cages. The use of both flexible (1,3-propyl) and rigid (1,3-phenyl) core spacer units led to the formation of discrete [Pd2L4](BF4)4 cage complexes. However, when the spacer unit of the di-1,2,3-triazole ligand was a 1,4-substituted-phenyl group steric interactions led to the formation of an oligomeric/polymeric species. By keeping the 1,3-phenyl core spacer constant the effect of altering the “click” ligands’ peripheral substituents was also examined. It was shown that ligands with alkyl, phenyl, electron-rich and electron-poor benzyl substituents all quantitatively formed [Pd2L4](BF4)4 cage complexes. The results suggest that a wide range of functionalised palladium(II) “click” cages could be rapidly generated. These novel molecules may potentially find uses in catalysis, molecular recognition and drug delivery. Show less

Recent findings link metabolic transformation of cancer cells to aberrant functions of mitochondrial uncoupling proteins (UCPs). By inducing proton leak, UCPs interfere with mitochondrial synthesis of adenosine 5′-triphosphate, which is also a key determinant of glycolytic pathways. In addition, UCP suppress the generation of superoxide, a byproduct of mitochondrial electron transport and a major source of oxidative stress. The near ubiquitous UCP2 becomes highly abundant in some cancers and may advance metabolic reprogramming, further disrupt tumour suppression, and promote chemoresistance. Here we review current evidence to suggest that inhibition of mitochondrial uncoupling may eliminate these responses and reveal novel anti-cancer strategies. Show less

The condensation of DNA is essential for biological processes such as DNA transcription and replication, and its study receives additional impetus from an interest in gene therapy. Although many efficacious condensing agents have been discovered and investigated, little is known about the conversation of condensation-release under suitable conditions. A novel class of DNA condensing agents based on small azaheterocyclic metal-binding molecules has been discovered and described. Both linear and plasmid DNA can be condensed to nanoparticles by the title compounds with 50 °C incubation, especially in the presence of divalent metal ions. Importantly, this condensation may be released to original forms with little or no damage to the DNA under incubation at physiological temperatures. These changes in DNA morphology over time have been analyzed by gel electrophoresis, circular dichroism (CD), and atomic force microscopy (AFM). The present work might help to develop strategies for the design and synthesis of controllable condensing agents, which may also be applied to control gene expression and delivery. Show less

Mitochondria extrude protons across their inner membrane to generate the mitochondrial membrane potential (ΔΨ(m)) and pH gradient (ΔpH(m)) that both power ATP synthesis. Mitochondrial uptake and efflux of many ions and metabolites are driven exclusively by ΔpH(m), whose in situ regulation is poorly characterized. Here, we report the first dynamic measurements of ΔpH(m) in living cells, using a mitochondrially targeted, pH-sensitive YFP (SypHer) combined with a cytosolic pH indicator (5-(and 6)-carboxy-SNARF-1). The resting matrix pH (∼7.6) and ΔpH(m) (∼0.45) of HeLa cells at 37 °C were lower than previously reported. Unexpectedly, mitochondrial pH and ΔpH(m) decreased during cytosolic Ca(2+) elevations. The drop in matrix pH was due to cytosolic acid generated by plasma membrane Ca(2+)-ATPases and transmitted to mitochondria by P(i)/H(+) symport and K(+)/H(+) exchange, whereas the decrease in ΔpH(m) reflected the low H(+)-buffering power of mitochondria (∼5 mm, pH 7.8) compared with the cytosol (∼20 mm, pH 7.4). Upon agonist washout and restoration of cytosolic Ca(2+) and pH, mitochondria alkalinized and ΔpH(m) increased. In permeabilized cells, a decrease in bath pH from 7.4 to 7.2 rapidly decreased mitochondrial pH, whereas the addition of 10 μm Ca(2+) caused a delayed and smaller alkalinization. These findings indicate that the mitochondrial matrix pH and ΔpH(m) are regulated by opposing Ca(2+)-dependent processes of stimulated mitochondrial respiration and cytosolic acidification. Show less

Platinum-based compounds are widely used as chemotherapeutics for the treatment of a variety of cancers. The anticancer activity of cisplatin and other platinum drugs is believed to arise from their interaction with DNA. Several cellular pathways are activated in response to this interaction, which include recognition by high-mobility group and repair proteins, translesion synthesis by polymerases, and induction of apoptosis. The apoptotic process is regulated by activation of caspases, p53 gene, and several proapoptotic and antiapoptotic proteins. Such cellular processing eventually leads to an inhibition of the replication or transcription machinery of the cell. Deactivation of platinum drugs by thiols, increased nucleotide excision repair of Pt-DNA adducts, decreased mismatch repair, and defective apoptosis result in resistance to platinum therapy. The differences in cytotoxicity of various platinum complexes are attributed to the differential recognition of their adducts by cellular proteins. Cisplatin and oxaliplatin both produce mainly 1,2-GG intrastrand cross-links as major adducts, but oxaliplatin is found to be more active particularly against cisplatin-resistant tumor cells. Mismatch repair and replicative bypass appear to be the processes most likely involved in differentiating the molecular responses to these two agents. This review describes the formation of Pt-DNA adducts, their interaction with cellular components, and biological effects of this interaction. Show less

In this study, two Pt(II) and three Pt(IV) complexes with the structures of [PtL(2)Cl(2)] (1), [PtL(2)I(2)] (2), [PtL(2)Cl(2)(OH)(2)] (3), [PtL(2)Cl(2)(OCOCH(3))(2)] (4), and [PtL(2)Cl(4)] (5) (L = benzimidazole as carrier ligand) were synthesized and evaluated for their in vitro antiproliferative activities against the human MCF-7, HeLa, and HEp-2 cancer cell lines. The influence of compounds 1-5 on the tertiary structure of DNA was determined by their ability to modify the electrophoretic mobility of the form I and II bands of pBR322 plasmid DNA. The inhibition of BamH1 restriction enzyme activity of compounds 1-5 was also determined. In general, it was found that compounds 1-5 were less active than cisplatin and carboplatin against MCF-7 and HeLa cell lines (except for 1, which was found to be more active than carboplatin against the MCF-7 cell line). Compounds 1 and 3 were found to be significantly more active than cisplatin and carboplatin against the HEp-2 cell line. Show less

BACKGROUND: Macrophages of the reticuloendothelial system play a key role in recycling iron from hemoglobin of senescent or damaged erythrocytes. Heme oxygenase 1 degrades the heme moiety and releases inorganic iron that is stored in ferritin or exported to the plasma via the iron export protein ferroportin. In the plasma, iron binds to transferrin and is made available for de novo red cell synthesis. The aim of this study was to gain insight into the regulatory mechanisms that control the transcriptional response of iron export protein ferroportin to hemoglobin in macrophages. DESIGN AND METHODS: Iron export protein ferroportin mRNA expression was analyzed in RAW264.7 mouse macrophages in response to hemoglobin, heme, ferric ammonium citrate or protoporphyrin treatment or to siRNA mediated knockdown or overexpression of Btb And Cnc Homology 1 or nuclear accumulation of Nuclear Factor Erythroid 2-like. Iron export protein ferroportin promoter activity was analyzed using reporter constructs that contain specific truncations of the iron export protein ferroportin promoter or mutations in a newly identified MARE/ARE element. RESULTS: We show that iron export protein ferroportin is transcriptionally co-regulated with heme oxygenase 1 by heme, a degradation product of hemoglobin. The protoporphyrin ring of heme is sufficient to increase iron export protein ferroportin transcriptional activity while the iron released from the heme moiety controls iron export protein ferroportin translation involving the IRE in the 5'untranslated region. Transcription of iron export protein ferroportin is inhibited by Btb and Cnc Homology 1 and activated by Nuclear Factor Erythroid 2-like involving a MARE/ARE element located at position -7007/-7016 of the iron export protein ferroportin promoter. CONCLUSIONS: This finding suggests that heme controls a macrophage iron recycling regulon involving Btb and Cnc Homology 1 and Nuclear Factor Erythroid 2-like to assure the coordinated degradation of heme by heme oxygenase 1, iron storage and detoxification by ferritin, and iron export by iron export protein ferroportin. Show less

Drugs for cancer therapy belong to different categories of chemical substances. The cellular targets for the therapeutic efficacy are often not unambiguously identified. Here, we describe the process of ribosome biogenesis as a target of a large variety of chemotherapeutic drugs. We determined the inhibitory concentration of 36 chemotherapeutic drugs for transcription and processing of ribosomal RNA by in vivo labeling experiments. Inhibitory drug concentrations were correlated to the loss of nucleolar integrity. The synergism of drugs inhibiting ribosomal RNA synthesis at different levels was studied. Drugs inhibited ribosomal RNA synthesis either at the level of (i) rRNA transcription (e.g. oxaliplatin, doxorubicin, mitoxantrone, methotrexate), (ii) early rRNA processing (e.g. camptothecin, flavopiridol, roscovitine), or (iii) late rRNA processing (e.g. 5-fluorouracil, MG-132, homoharringtonine). Blockage of rRNA transcription or early rRNA processing steps caused nucleolar disintegration, whereas blockage of late rRNA processing steps left the nucleolus intact. Flavopiridol and 5-fluorouracil showed a strong synergism for inhibition of rRNA processing. We conclude that inhibition of ribosome biogenesis by chemotherapeutic drugs potentially may contribute to the efficacy of therapeutic regimens. Show less

Cisplatin, carboplatin, and oxaliplatin are three FDA-approved members of the platinum anticancer drug family. These compounds induce apoptosis in tumor cells by binding to nuclear DNA, forming a variety of structural adducts and triggering cellular responses, one of which is the inhibition of transcription. In this report we present (i) a detailed review of the structural investigations of various Pt-DNA adducts and the effects of these lesions on global DNA geometry; (ii) research detailing inhibition of cellular transcription by Pt-DNA adducts; and (iii) a mechanistic analysis of how DNA structural distortions induced by platinum damage may inhibit RNA synthesis in vivo. A thorough understanding of the molecular mechanism of action of platinum antitumor agents will aid in the development of new compounds in the family. Show less

Nuclear factor-erythroid 2 p45-related factor 2 (Nrf2) is the primary transcription factor protecting cells from oxidative stress by regulating cytoprotective genes, including the antioxidant glutathione (GSH) pathway. GSH maintains cellular redox status and affects redox signaling, cell proliferation, and death. GSH homeostasis is regulated by de novo synthesis as well as GSH redox state; previous studies have demonstrated that Nrf2 regulates GSH homeostasis by affecting de novo synthesis. We report that Nrf2 modulates the GSH redox state by regulating glutathione reductase (GSR). In response to oxidants, lungs and embryonic fibroblasts (MEFs) from Nrf2-deficient (Nrf2(-/-)) mice showed lower levels of GSR mRNA, protein, and enzyme activity relative to wild type (Nrf2(+/+)). Nrf2(-/-) MEFs exhibited greater accumulation of glutathione disulfide and cytotoxicity compared to Nrf2(+/+) MEFs in response to t-butylhydroquinone, which was rescued by restoring GSR. Microinjection of glutathione disulfide induced greater apoptosis in Nrf2(-/-) MEFs compared to Nrf2(+/+) MEFs. In silico promoter analysis of the GSR gene revealed three putative antioxidant-response elements (ARE1, -44; ARE2, -813; ARE3, -1041). Reporter analysis, site-directed mutagenesis, and chromatin immunoprecipitation assays demonstrated binding of Nrf2 to two AREs distal to the transcription start site. Overall, Nrf2 is critical for maintaining the GSH redox state via transcriptional regulation of GSR and protecting cells against oxidative stress. Show less

AbstractA brief overview is given of platinum anticancer drugs in routine clinical use and under clinical development worldwide. Details of the binding of these drugs with nucleic acids, the preferred binding site, etc. are discussed as well. Using the mechanistic knowledge at the molecular level, in particular DNA binding, possibilities for new drugs are explored. A major part of the review deals with design, synthesis and biochemical/biophysical studies of such new compounds and their possible applications as cytostatic drugs. Special attention is given to the application of bifunctionality in such new compounds. (© Wiley‐VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2009) Show less

Mitochondria are cell substructures (organelles) critical for cell life, because biological fuel production, the ATP synthesis by oxidative phosphorylation, occurs in them driven by acidity (pH) gradients. Mitochondria play a key role as well in the cell death and in various fatigue and exercise intolerance syndromes. It is clear now that mitochondria present an astonishing variety of inner membrane morphologies, dynamically correlated with their functional state, coupled with the rate of the ATP synthesis, and characteristic for normal as well as for pathological cases. Our work offers some original insights into the factors that determine the dynamical tubular structures of the inner membrane cristae. We show the possibility to induce, by localized proton flow, a macroscopic cristae-like shape remodeling of an only-lipid membrane. We designed a minimal membrane system (GUV) and experimentally showed that the directional modulation of local pH gradient at membrane level of cardiolipin-containing vesicles induces dynamic cristae-like membrane invaginations. We propose a mechanism and theoretical model to explain the observed tubular membrane morphology and suggest the underlying role of cardiolipin. Our results support the hypothesis of localized bioenergetic transduction and contribute to showing the inherent capacity of cristae morphology to become self-maintaining and to optimize the ATP synthesis. Show less

The cytotoxicity of platinum compounds is thought to be determined primarily by their DNA adducts. Cisplatin and oxaliplatin are structurally distinct, but form the same types of adducts at the same sites on DNA. However, the DNA adducts are differentially recognized by a number of cellular proteins. For example, mismatch repair proteins and some damage-recognition proteins bind to cisplatin-GG adducts with higher affinity than to oxaliplatin-GG adducts, and this differential recognition of cisplatin- and oxaliplatin-GG adducts is thought to contribute to the differences in cytotoxicity and tumor range of cisplatin and oxaliplatin. A detailed kinetic analysis of the insertion and extension steps of dNTP incorporation in the vicinity of the adduct shows that both DNA polymerase beta (pol beta) and DNA polymerase eta (pol eta) catalyze translesion synthesis past oxaliplatin-GG adducts with greater efficiency than past cisplatin-GG adducts. In the case of pol eta, the efficiency and fidelity of translesion synthesis in vitro is very similar to that previously observed with cyclobutane TT dimers, suggesting that pol eta is likely to be involved in error-free bypass of Pt adducts in vivo. This has been confirmed for cisplatin by comparing the cisplatin-induced mutation frequency in human fibroblast cell lines with and without pol eta. Thus, the greater efficiency of bypass of oxaliplatin-GG adducts by pol eta may explain the lower mutagenicity of oxaliplatin compared to cisplatin. The ability of these cellular proteins to discriminate between cisplatin and oxaliplatin adducts suggest that there exist significant conformational differences between the adducts, yet the crystal structures of the cisplatin- and oxaliplatin-GG adducts were very similar. We have recently solved the solution structure of the oxaliplatin-GG adduct and have shown that it is significantly different from the previously published solution structures of the cisplatin-GG adducts. Furthermore, the observed differences in conformation provide a logical explanation for the differential recognition of cisplatin and oxaliplatin adducts by mismatch repair and damage-recognition proteins. Show less

Oxaliplatin is a relatively new platinum analogue that is currently used in pharmacotherapy of metastatic colorectal cancer. Its dose-limiting toxicity is sensory neuropathy, which can be modulated by infusion of calcium and magnesium. Oxaliplatin exerts its anti-tumour effects by platinum-adduct formation, binding to cellular proteins and possibly interfering with RNA synthesis as well. If they are not removed from DNA, oxaliplatin adducts are lethal. Cellular defense mechanisms prevent adduct formation (e.g., glutathione-S-transferase) or remove DNA adducts (e.g., nucleotide excision repair). Depending on the activity of necessary enzymes in these cellular defense pathways, oxaliplatin induced damage varies from one individual to another. There is growing evidence that polymorphisms in genes coding for DNA repair enzymes and metabolic inactivation routes contribute to the interindividual differences in anti-tumour efficacy and toxicity of oxaliplatin. Single nucleotide polymorphisms (SNPs) may yield inactive enzymes, or increased gene transcription and hence increased enzyme production. This review covers findings of recent investigations on the associations of SNPs and clinical outcome after oxaliplatin chemotherapy in metastatic colorectal cancer. 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

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