Interaction of cisplatin drug with RNase A.

cis-Pt(NH3)2Cl2 (cisplatin) is an antitumor drug with many severe toxic side effects including enzymatic structural changes associated with its mechanism of action. This study is designed to examine the interaction of cisplatin drug with ribonuclease A (RNase A) in aqueous solution at physiological pH, using drug concentration of 0.0001 mM to 0.1 mM with final protein concentration of 2% w/v. Absorption spectra and Fourier transform infrared (FTIR) spectroscopy with its self-deconvolution, second derivative resolution enhancement and curve-fitting procedures were used to characterize the drug binding mode, association constant and the protein secondary structure in the cisplatin-RNase complexes. Spectroscopic results show that at low drug concentration (0.0001 mM), no interaction occurs between cisplatin and RNase, while at higher drug concentrations, cisplatin binds indirectly to the polypeptide C=O, C-N (via H2O or NH3 group) and directly to the S-H donor atom with overall binding constant 5.66 x 10(3)M(-1). At high drug concentration, major protein secondary structural changes occur from that of the alpha-helix 29% (free enzyme) to 20% and beta-sheet 39% (free enzyme) to 45% in the cisplatin-RNase complexes. The observed structural changes indicate a partial protein unfolding in the presence of cisplatin at high drug concentration.     

Interaction of cisplatin and analogues with a Met-rich protein site

The chaperone protein CopC from Pseudomonas syringae features high-affinity binding sites (K _D ~ 10⁻¹³ M) for both Cu^I (Met-rich) and Cu^II (His-rich). When presented with these sites in the apoprotein, electrospray ionisation mass spectrometry confirmed that cis-Pt(NH₃)₂Cl₂ (cisplatin) and the fragments [Pt^IIL]²⁺ (L is 1,2-diaminoethane, 2,2′-bipyridine) occupied the Cu^I site specifically in the 1:1 Pt–CopC adducts (purified by cation-exchange chromatography). The cis-Pt(NH₃)₂ fragment was not present in these adducts (the dominant product for cisplatin was Pt–CopC in which all original ligands were displaced), while bidentate ligands L were retained in LPt–CopC adducts. In the context of the Met-rich Cu^I pump Ctr1 as a significant entry point for cisplatin into mammalian cells, the present work confirms the ability of Met-rich sites in proteins to remove all ligands from cisplatin. It focuses attention on the potential of proteins that are part of the natural copper transport pathways to sequester the drug. These pathways are worthy of further study at the molecular level. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Interaction of cisplatin and DNA-targeted 9-aminoacridine platinum complexes with DNA

Interaction of acridine- and 9-aminoacridinecarboxamide platinum complexes with DNA was investigated with respect to their DNA sequence specificity and kinetics of binding. The DNA sequence specificity of the compounds was quantitatively analyzed using a polymerase stop assay with the plasmid pUC19. The 9-aminoacridinecarboxamide platinum complexes exhibited a different sequence specificity to that of cisplatin, shifted away from runs of consecutive guanines (the main binding site for cisplatin). This alteration was dependent on chain length. Shorter chain length compounds (n = 2, 3) showed a greater difference in sequence specificity, while longer chain length compounds (n = 4, 5) more closely resembled cisplatin. An acridinecarboxamide platinum complex showed a similar sequence specificity to cisplatin, revealing that the major change of sequence specificity was due to the presence of the 9-amino substituent. A linear amplification system was used to investigate the time course of the reaction. The presence of an intercalating group (acridinecarboxamide or 9-aminoacridinecarboxamide) greatly increased the rate of reaction with DNA; this is proposed to be due to a different reaction mechanism with DNA (direct displacement by the N-7 of guanine).     

Interaction of cisplatin with planar model membranes - dose dependent change in electrical characteristics

The drug cisplatin has broad antineoplastic activity against advanced testicular and ovarian cancers, epithelial malignancies, cancers of the head, neck, bladder, oesophagus and lungs. Peripheral neurotoxicity, ototoxicity and nephrotoxicity are its major side effects. The nonspecific action of this drug on the lipid bilayer architecture of membranes has been studied by following the effects produced on the electrical characteristics of model planar bilayer lipid membranes (BLM). The results confirm that the drug has a strong surface interaction with the zwitterionic polar head groups of the amphipathic phospholipids constituting the BLM. The permeability characteristics of cisplatin through the hydrophobic core are limited. Cisplatin does not fluidise the membrane sufficiently to cause its breakdown but creates small ion conducting defects on the membrane bilayer resulting in a marginal increase in ion conductivity. These results indicate that cisplatin exhibits a non-specific action on the lipid bilayer component of the membrane which might be partly responsible for its neurotoxic side effects.     

Interaction of the anti-cancer drug cisplatin with phosphatidylserine in intact and semi-intact cells.

The anti-cancer drug cisplatin (cis-diamminedichloroplatinum(II)) forms a stable coordination complex with phosphatidylserine (PS) in model membrane systems (Speelmans et al., Biochemistry 36 (1997) 10545-10550). Because a similar interaction in vivo would be expected to have important physiological implications we studied cisplatin-PS interaction in human erythrocytes and tumor cell lines. Although cisplatin was efficiently taken up by intact erythrocytes, a cisplatin-PS complex was only detected in cells which had lysed as a result of prolonged storage or hypotonic shock. Despite the use of highly sensitive detection methods, and despite efficient cellular uptake of cisplatin, a complex could also not be detected in four human tumor cell lines, unless cells were permeabilized. In experiments in which cisplatin was incubated with PS-containing liposomes in the presence of an alternative cellular substrate, such as reduced glutathione, the relative affinity of cisplatin for PS was found to be low. Moreover, loading erythrocyte ghosts with physiological concentrations of glutathione strongly reduced cisplatin-PS complexation. Thus, in intact (tumor) cells a complex is not detected, most likely, because of the presence of higher affinity substrates. Though a transient complexation of cisplatin to PS cannot be excluded, our data suggest that cisplatin-PS does not play a direct role in the cellular (cyto)toxicity of cisplatin.     

Targeting biomolecules with reversible covalent chemistry

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Cell-selective metabolic labeling of biomolecules with bioorthogonal functionalities

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Interaction of cisplatin drug with Na,K-ATPase: drug binding mode and protein secondary structure

cis-Pt(NH(3))(2)Cl(2) (cisplatin) is an antitumor drug with many severe toxic side effects including enzymatic changes associated with its mechanism of action. This study was designed to examine the interaction of cisplatin drug with the Na(+), K(+)-dependent adenosine triphosphatase (Na,K-ATPase) in H(2)O and D(2)O solutions at physiological pH, using drug concentrations of 0.1 microM to 1 mM. UV absorption spectra and Fourier transform infrared difference spectroscopy with its self-deconvolution, second derivative resolution enhancement and curve-fitting procedures were applied to characterize the drug binding mode, the drug binding constant and the protein secondary structure in the cisplatin-ATPase complexes. Spectroscopic evidence showed that at low drug concentration (0.1 microM), cisplatin binds mainly to the lipid portion of the enzyme, whereas at higher drug contents, the Pt cation interaction is through the polypeptide C==O and C-N groups with overall binding constant of K=1.93 x 10(4) M(-1). At high cisplatin concentration (1 mM), drug binding results in protein secondary structural changes from that of the alpha-helix 19.8%; beta-pleated 25.6%; turn 9.1%; beta-antiparallel 7.5% and random 38%, in the free Na,K-ATPase to that of the alpha-helix 22.2%; beta-pleated 23.2%; turn 9.4%; beta-antiparallel 2.2% and random 43%, in the cis-Pt-ATPase complexes.     

Interaction of anticancer drug cisplatin with guanine: density functional theory and surface-enhanced Raman spectroscopy study

Surface-enhanced Raman spectroscopy (SERS) in a silver sol assisted by density functional theory (DFT) calculations is shown to be a promising tool in the characterization of platinum complexes and their interaction with nucleic acid bases. This is demonstrated using cisplatin and guanine as a model. The energies and geometric parameters of cisplatin, guanine, and their reaction products are calculated at Becke's nonlocal three parameter exchange and correlation functional and the Lee-Yang-Parr correlation functional level using the 6-31++G(d,p) basis set on the light elements and the effective core potential by Hay and Wadt on platinum. Available X-ray crystallography data are mostly in agreement with predictions within the experimental precision level, although Pt-N bond lengths tend to be systematically overestimated. The normal Raman spectrum of cisplatin is assigned. The SERS spectra of cisplatin and its reaction product with guanine are measured from 10(-6) M aqueous solution. The observed spectral changes in the SERS spectrum of guanine upon cisplatin binding are modeled by DFT calculations. The best agreement between theory and experiment is achieved when the adsorbed reaction product is assumed to be the 1:1 adduct cis-Pt(NH3)2ClG in which Pt is bound to N7 and guanine is deprotonated at N9.     

Ultrasensitive detection of biomolecules with fluorescent dye-doped nanoparticles

Fluorescent-labeled molecules have been used extensively for a wide range of applications in biological detection and diagnosis. A new form of highly luminescent and photostable nanoparticles was generated by doping the fluorescent dye tris(2'2-bipyridyl)dichlororuthenium(II)hexahydrate (Rubpy) inside silica material. Because thousands of fluorescent dye molecules are encapsulated in the silica matrix that also serves to protect Rubpy dye from photodamaging oxidation, the Rubpy-dye-doped nanoparticles are extremely bright and photostable. We have used these nanoparticles successfully in various fluorescence labeling techniques, including fluorescent-linked immunosorbent assay, immunocytochemistry, immunohistochemistry, DNA microarray, and protein microarray. By combining the high-intensity luminescent nanoparticles with the specificity of antibody-mediated recognition, ultrasensitive target detection has been achieved. In all cases, assay results clearly demonstrated the superiority of the nanoparticles over organic fluorescent dye molecules and quantum dots in probe labeling for sensitive target detection. These results demonstrate the potential to apply these newly developed fluorescent nanoparticles in various biodetection systems.     

Weak acid-induced release of liposome-encapsulated carboxyfluorescein

Leakage of the entrapped anionic fluorophore carboxyfluorescein was used as a measure of the permeability of liposomes to several different acids. Carboxyfluorescein leakage increased with increasing buffer concentration at a given pH and depended on its chemical nature: apolar weak acids such as acetic or pyruvic acids induced fast leakage at relatively high pH (4 to 5), while glycine, aspartic, citric and hydrochloric acids induced leakage only at lower pH. Fluorescence leakage measurements reflected the acidification of the liposomes' aqueous spaces, which was primarily caused by the diffusion of undissociated acid molecules across the lipid bilayer. A simple mathematical model in accord with this hypothesis and assuming that carboxyfluorescein leakage was directly related to the proportion of its neutral lactone form, described satisfactorily the carboxyfluorescein leakage kinetics and allowed rough estimation of permeability coefficients for carboxyfluorescein (neutral lactone form; 9 · 10^?9 cm · s^?1), acetic acid ($\text{>1 · 10}{}^{\mathrm{?7}}\text{cm}\text{·}\text{s}{}^{\mathrm{?1}}$) and glycine (cation: 6 · 10^?9 cm · s^?1). These results are consistent with low effective proton permeability of liposomes ($\text{<5 · 10}{}^{\mathrm{?12}}\text{cm}\text{·}\text{s}{}^{\mathrm{?1}}$) and with the permeability coefficient of HCl (3 · 10^?3 cm · s^?1) reported by Nozaki and Tanford ((1981) Proc. Natl. Acad. Sci. U.S.A. 78, 4324–4328). Diffusion of weak acid molecules across lipid membranes has implications for drug encapsulation and delivery, and may be of biological significance.     

Depletion of cellular cholesterol interferes with intracellular trafficking of liposome-encapsulated ovalbumin

Cholesterol is a major constituent of plasma cell membranes and influences the functions of proteins residing in the membrane. To assess the role of cholesterol in phagocytosis and intracellular trafficking of liposomal antigen, macrophages were treated with inhibitors of cholesterol biosynthesis for various time periods and levels of cholesterol depletion were assessed by thin layer chromatography. In control macrophages, cholesterol was present in the plasma membrane and in intracellular stores, as visualised by staining with the cholesterol-binding compound filipin, whereas macrophages treated with cholesterol inhibitors failed to stain with filipin. However, these macrophages were still capable of phagocytosis as evidenced by their internalisation of fluorescent-labelled bacteria and liposome-encapsulated Texas red labelled-ovalbumin, L(TR-OVA). While fluorescent ovalbumin (OVA) was consistently transported to the Golgi in macrophages incubated with L(TR-OVA), in cells treated with cholesterol inhibitors, OVA remained spread diffusely throughout the cytoplasm. Even though the mean fluorescence intensity of MHC class I molecules on cholesterol inhibitor-treated macrophages was equivalent to that of the control macrophages, the amount of MHC class I-liposomal OVA-peptide complex detected on the cell surface of cholesterol inhibitor-treated macrophages, was only 45.6 +/- 7.4% (n = 4, mean +/- SEM) of control levels after intracellular processing of L(OVA). We conclude that cholesterol depletion does not eliminate phagocytosis or MHC class I surface expression, but does affect the trafficking and consequently the MHC class I antigen-processing pathway.     

Interleukin 1 release by human monocytes treated with liposome-encapsulated lipopolysaccharide

Liposome therapy offers a unique method of delivering antifungals, antiprotozoans, and macrophage-activating factors directly to reticuloendothelial cells. Although liposomes are entering clinical trials, their effect on Il 1 synthesis and release has yet to be determined. The beneficial, as well as possible pathological, effects of liposome therapy may be due to IL 1 released by reticuloendothelial cells. This study examined the effects of multilamellar phospholipid vesicles on the synthesis and release of IL 1 from human peripheral blood monocytes. Liposomes consisting of phosphatidylcholine and phosphatidylserine in molar ratio of 7:3 did not stimulate IL 1 release, and did not diminish the level of IL 1 release when monocytes were subsequently stimulated with LPS or Staphylococcus aureus. Surprisingly, LPS encapsulated in liposomes failed to stimulate IL 1 release from monocytes. This defect was limited to the release of IL 1, because monocytes stimulated with liposomes containing LPS had control levels of intracellular (cytosolic) and membrane IL 1. By contrast, LPS associated with lyophilized liposomes induced the secretion of IL 1 and behaved similarly to free LPS. These findings indicate that LPS activation of monocytes may involve not only the synthesis of IL 1 but activation of the secretory mechanism for IL 1, and that these two events could be independent, that the density of LPS molecules at the surface of the liposome may influence the degree of monocyte activation as measured by intracellular IL 1 synthesis, membrane accumulation, and secretion into the medium, and that intracellular lipids may not interfere with the secretory mechanism, because liposomes made with phospholipids differing in acyl chain length or the headgroup behaved identically.     

Comparative analyses of biomolecules

Systematic examination of the gene encoding CYP2B6, a human cytochrome P450, has characterized genetic polymorphisms that might account for its variability in expression and function between individuals.     

Adjuvant effect of liposome-encapsulated natural phosphodiester CpG-DNA

Immunostimulatory CpG-DNA targeting TLR9 is one of the most extensively evaluated vaccine adjuvants. Previously, we found that a particular form of natural phosphodiester bond CpG-DNA (PO-ODN) encapsulated in a phosphatidyl-Β-oleoyl- γ-palmitoyl ethanolamine (DOPE) : cholesterol hemisuccinate (CHEMS) (1 : 1 ratio) complex (Lipoplex(O)) is a potent adjuvant. Complexes containing peptide and Lipoplex(O) are extremely useful for B cell epitope screening and antibody production without carriers. Here, we showed that IL-12 production was increased in bone marrow derived dendritic cells in a CpG sequence-dependent manner when PO-ODN was encapsulated in Lipoplex(O), DOTAP or lipofectamine. However, the effects of Lipoplex(O) surpassed those of PO-ODN encapsulated in DOTAP or lipofectamine and also other various forms of liposome-encapsulated CpG-DNA in terms of potency for protein antigen-specific IgG production and Th1- associated IgG2a production. Therefore, Lipoplex(O) may have a unique potent immunoadjuvant activity which can be useful for various applications involving protein antigens as well as peptides.     

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.