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Relatively little is known about the kinetics or the pharmacological potential of organometallic complexes of osmium compared to its lighter congeners, iron and ruthenium. We report the synthesis of seven new complexes, [(eta6-arene)Os(NN)Cl]+, containing different bidentate nitrogen (N,N) chelators, and a dichlorido complex, [(eta6-arene)Os(N)Cl2]. The X-ray crystal structures of seven complexes are reported: [(eta6-bip)Os(en)Cl]PF6 (1PF6), [(eta6-THA)Os(en)Cl]BF4 (2BF4), [(eta6-p-cym)Os(phen)Cl]PF6 (5PF6), [(eta6-bip)Os(dppz)Cl]PF6 (6PF6), [(eta6-bip)Os(azpy-NMe2)Cl]PF6 (7PF6), [(eta6-p-cym)Os(azpy-NMe2)Cl]PF6 (8PF6), and [(eta6-bip)Os(NCCH3-N)Cl2] (9), where THA = tetrahydroanthracene, en = ethylenediamine, p-cym = p-cymene, phen = phenanthroline, bip = biphenyl, dppz = [3,2-a: 2',3'-c]phenazine and azpy-NMe2 = 4-(2-pyridylazo)-N,N-dimethylaniline. The chelating ligand was found to play a crucial role in enhancing aqueous stability. The rates of hydrolysis at acidic pH* decreased when the primary amine N-donors (NN = en, t1/2 = 0.6 h at 318 K) are replaced with pi-accepting pyridine groups (e.g., NN = phen, t1/2 = 9.5 h at 318 K). The OsII complexes hydrolyze up to 100 times more slowly than their RuII analogues. The pK*a of the aqua adducts decreased with a similar trend (pK*a = 6.3 and 5.8 for en and phen adducts, respectively). [(eta6-bip)Os(en)Cl]PF6/BF4 (1PF6/BF4) and [(eta6-THA)Os(en)Cl]BF4 (2BF4) were cytotoxic toward both the human A549 lung and A2780 ovarian cancer cell lines, with IC50 values of 6-10 microM, comparable to the anticancer drug carboplatin. 1BF4 binds to both the N7 and phosphate of 5'-GMP (ratio of 2:1). The formation constant for the 9-ethylguanine (9EtG) adduct [(eta6-bip)M(en)(9EtG)]2+ was lower for OsII (log K = 3.13) than RuII (log K = 4.78), although the OsII adduct showed some kinetic stability. DNA intercalation of the dppz ligand in 6PF6 may play a role in its cytotoxicity. This work demonstrates that the nature of the chelating ligand can play a crucial role in tuning the chemical and biological properties of [(eta6-arene)Os(NN)Cl]+ complexes. Show less

Potential biological and medical applications of organometallic complexes are hampered by a lack of knowledge of their aqueous solution chemistry. We show that the hydrolytic and aqueous solution chemistry of half-sandwich OsII arene complexes of the type [(eta6-arene)Os(XY)Cl] can be tuned with XY chelating ligands to achieve cancer cell cytoxicity comparable to carboplatin. Complexes containing arene = p-cymene, XY = N,O-chelating ligands glycinate (1), L-alaninate (2), alpha-aminobutyrate (3), beta-alaninate (4), picolinate (5), or 8-hydroxyquinolinate (7) were synthesized. Although, 1-4 and 7 hydrolyzed rapidly (Show less

Abstract Phosphoglucose isomerase (PGI) is one of the glycolytic enzymes and is a multifunctional enzyme that functions in glucose metabolism inside the cell while acting as a cytokine outside the cell, with properties that include autocrine motility factor (AMF) regulating tumor cell motility. Although there are many studies indicating that PGI/AMF has been implicated in progression of metastasis, no direct studies of the significance of exogenous PGI/AMF on tumor progression have been reported. Here, we report on the mesenchymal-to-epithelial transition (MET), which is the reverse phenomenon of the epithelial-to-mesenchymal transition that is associated with loss of cell polarity, loss of epithelia markers, and enhancement of cell motility essential for tumor cell invasion and metastasis. Mesenchymal human fibrosarcoma HT1080 cells, which have naturally high levels of endogenous and exogenous PGI/AMF, were stably transfected with PGI/AMF small interfering RNA (siRNA). The siRNA targeting human PGI/AMF down-regulated the endogenous PGI/AMF expression and completely extinguished the secretion of PGI/AMF in a human fibrosarcoma HT1080, whereas the control siRNA showed no effects. The PGI/AMF siRNA caused cells to change shape dramatically and inhibited cell motility and invasion markedly. Suppression of PGI/AMF led to a contact-dependent inhibition of cell growth. Those PGI/AMF siRNA-transfected cells showed epithelial phenotype. Furthermore, tumor cells with PGI/AMF deficiency lost their abilities to form tumor mass. This study identifies that MET in HT1080 human lung fibrosarcoma cells was initiated by down-regulation of the housekeeping gene product/cytokine PGI/AMF, and the results depicted here suggest a novel therapeutic target/modality for mesenchymal cancers. [Cancer Res 2007;67(9):4236–8] Show less

The NO donor trans-[Ru(NO)(NH(3))(4)(py)](BF(4))(3).H(2)O (py=pyridine) was loaded into poly-lactic-co-glycolic acid (PLGA) microparticles using the double emulsification technique. Scanning electron microscopy (SEM) and dynamic light scattering revealed that the particles are spherical in shape, have a diameter of 1600nm, and have low tendency to aggregate. The entrapment efficiency was 25%. SEM analysis of the melanoma cell B16-F10 in the presence of the microparticles containing the complex trans-[Ru(NO)(NH(3))(4)(py)](BF(4))(3).H(2)O (pyMP) showed that the microparticles were adhered to the cell surface after 2h of incubation. The complex with concentrations lower than 1x10(-4)M did not show toxicity in B16-F10 murine cells. The complex in solution is toxic at higher concentrations (>1x10(-3)M), with cell death attributed to NO release following the reduction of the complex. pyMP is not cytotoxic due to the lower bioavailability and availability of the entrapped complex to the medium and its reducing agents. However, pyMP is phototoxic upon light irradiation. The phototoxicity strongly suggests that cell death is due to NO release from trans-[Ru(NO)(NH(3))(4)(py)](3+). This work shows that pyMP can serve as a model for a drug delivery system carrying the NO donor trans-[Ru(NO)(NH(3))(4)(py)](BF(4))(3).H(2)O, which can release NO locally at the tumor cell by irradiation with light only. Show less

Non-covalent interactions play an important role in chemistry, physics and especially in biodisciplines. They determine the structure of biomacromolecules such as DNA and proteins and are responsible for the molecular recognition process. Theoretical evaluation of interaction energies is difficult; however, perturbation as well as variation (supermolecular) methods are briefly described. Accurate interaction energies can be obtained by complete basis set limit calculations providing a large portion of correlation energy is covered (e.g. by performing CCSD(T) calculations). The role of H-bonding and stacking interactions in the stabilisation of DNA, oligopeptides and proteins is described, and the importance of London dispersion energy is shown. Show less

We report here the design of the first class of luminescent biotinylation reagents derived from rhenium(I) polypyridine complexes. These complexes [Re(N-N)(CO)(3)(py-biotin-NCS)](PF(6)) (py-biotin-NCS = 3-isothiocyanato-5-(N-((2-biotinamido)ethyl)aminocarbonyl)pyridine; N-N = 1,10-phenanthroline (phen) (1a), 3,4,7,8-tetramethyl-1,10-phenanthroline (Me(4)-phen) (2a), 4,7-diphenyl-1,10-phenanthroline (Ph(2)-phen) (3a)), containing a biotin unit and an isothiocyanate moiety, have been synthesized from the precursor amine complexes [Re(N-N)(CO)(3)(py-biotin-NH(2))](PF(6)) (py-biotin-NH(2) = 3-amino-5-(N-((2-biotinamido)ethyl)aminocarbonyl)pyridine; N-N = phen (1c), Me(4)-phen (2c), Ph(2)-phen (3c)). To investigate the amine-specific reactivity of the isothiocyanate complexes 1a-3a, they have been reacted with a model substrate ethylamine, resulting in the formation of the thiourea complexes [Re(N-N)(CO)(3)(py-biotin-TU-Et)](PF(6)) (py-biotin-TU-Et = 3-ethylthioureidyl-5-(N-((2-biotinamido)ethyl)aminocarbonyl)pyridine; N-N = phen (1b), Me(4)-phen (2b), Ph(2)-phen (3b)). All the rhenium(I) complexes have been characterized, and their photophysical properties have been studied. The avidin-binding properties of the thiourea complexes 1b-3b have been examined by the 4'-hydroxyazobenzene-2-carboxylic acid (HABA) assay. Titration results indicated that the complexes exhibited emission enhancement by ca. 1.4-1.5-fold upon binding to avidin, and the lifetimes were elongated to ca. 0.8-2.0 micros. Additionally, we have biotinylated bovine serum albumin (BSA) with the isothiocyanate complexes. All the resultant rhenium-BSA bioconjugates displayed intense and long-lived orange-yellow to greenish-yellow emission upon irradiation in aqueous buffer under ambient conditions. The avidin-binding properties of the bioconjugates have been investigated using the HABA assay. Furthermore, the cytotoxicity of the thiourea complexes 1b-3b toward the HeLa cells has been examined by the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide (MTT) assay. The IC50 values were determined to be ca. 17.5-28.5 microM, which are comparable to that of cisplatin (26.7 microM) under the same conditions. The cellular uptake of complex 3b has been investigated by fluorescence microscopy, and the results showed that the complex was localized in the perinuclear region after interiorization. Show less

Mitochondria from respiring cells were isolated under anaerobic conditions. Microscopic images were largely devoid of contaminants, and samples consumed O(2) in an NADH-dependent manner. Protein and metal concentrations of packed mitochondria were determined, as was the percentage of external void volume. Samples were similarly packed into electron paramagnetic resonance tubes, either in the as-isolated state or after exposure to various reagents. Analyses revealed two signals originating from species that could be removed by chelation, including rhombic Fe(3+) (g = 4.3) and aqueous Mn(2+) ions (g = 2.00 with Mn-based hyperfine). Three S = 5/2 signals from Fe(3+) hemes were observed, probably arising from cytochrome c peroxidase and the a(3):Cu(b) site of cytochrome c oxidase. Three Fe/S-based signals were observed, with averaged g values of 1.94, 1.90 and 2.01. These probably arise, respectively, from the [Fe(2)S(2)](+) cluster of succinate dehydrogenase, the [Fe(2)S(2)](+) cluster of the Rieske protein of cytochrome bc (1), and the [Fe(3)S(4)](+) cluster of aconitase, homoaconitase or succinate dehydrogenase. Also observed was a low-intensity isotropic g = 2.00 signal arising from organic-based radicals, and a broad signal with g (ave) = 2.02. Mössbauer spectra of intact mitochondria were dominated by signals from Fe(4)S(4) clusters (60-85% of Fe). The major feature in as-isolated samples, and in samples treated with ethylenebis(oxyethylenenitrilo)tetraacetic acid, dithionite or O(2), was a quadrupole doublet with DeltaE (Q) = 1.15 mm/s and delta = 0.45 mm/s, assigned to [Fe(4)S(4)](2+) clusters. Substantial high-spin non-heme Fe(2+) (up to 20%) and Fe(3+) (up to 15%) species were observed. The distribution of Fe was qualitatively similar to that suggested by the mitochondrial proteome. Show less

AbstractQM/MM calculations were employed to investigate the role of hydrogen bonding and π stacking in several single‐ and double‐stranded cisplatin–DNA structures. Computed geometrical parameters reproduce experimental structures of cisplatin and its complex with guanine–phosphate–guanine. Following QM/MM optimisation, single‐point DFT calculations allowed estimation of intermolecular forces through atoms in molecules (AIM) analysis. Binding energies of platinated single‐strand DNA qualitatively agree with myriad experimental and theoretical studies showing that complexes of guanine are stronger than those of adenine. The topology of all studied complexes confirms that platination strongly affects the stability of both single‐ and double‐stranded DNAs: PtNH⋅⋅⋅X (X = N or O) interactions are ubiquitous in these complexes and account for over 70 % of all H‐bonding interactions. The π stacking is greatly reduced by both mono‐ and bifunctional complexation: the former causes a loss of about 3–4 kcal mol−1, whereas the latter leads to more drastic disruption. The effect of platination on Watson–Crick GC is similar to that found in previous studies: major redistribution of energy occurs, but the overall stability is barely affected. The BH&H/AMBER/AIM approach was also used to study platination of a double‐stranded DNA octamer d(CCTG*G*TCC)⋅d(GGACCAGG), for which an experimental structure is available. Comparison between theory and experiment is satisfactory, and also reproduces previous DFT‐based studies of analogous structures. The effect of platination is similar to that seen in model systems, although the effect on GC pairing was more pronounced. These calculations also reveal weaker, secondary interactions of the form Pt⋅⋅⋅O and Pt⋅⋅⋅N, detected in several single‐ and double‐stranded DNA. Show less

AbstractA major problem in virtual screening concerns the accuracy of the binding free energy between a target protein and a putative ligand. Here we report an example supporting the outperformance of the AutoDock scoring function in virtual screening in comparison to the other popular docking programs. The original AutoDock program is in itself inefficient to be used in virtual screening because the grids of interaction energy have to be calculated for each putative ligand in chemical database. However, the automation of the AutoDock program with the potential grids defined in common for all putative ligands leads to more than twofold increase in the speed of virtual database screening. The utility of the automated AutoDock in virtual screening is further demonstrated by identifying the actual inhibitors of various target enzymes in chemical databases with accuracy higher than the other docking tools including DOCK and FlexX. These results exemplify the usefulness of the automated AutoDock as a new promising tool in structure‐based virtual screening. Proteins 2006. © 2006 Wiley‐Liss, Inc. Show less

Dinuclear Pt-containing compounds might be used to overcome the intrinsic and acquired cell resistance of widely used anticancer drugs such as cisplatin. Recently, the complexes [[cis-Pt(NH3)2]2(mu-OH)(mu-pz)](NO3)2 (with pz = pyrazolate) (1), [[cis-Pt(NH3)2]2(mu-OH)(mu-1,2,3-ta-N(1),N(2))](NO3)2 (with ta = 1,2,3-triazolate) (2), and the binding of 1 to d(CpTpCpTpG*pG*pTpCpTpCp) have been characterized. Here we provide the structural and electronic properties of the free drugs, of the intermediates of binding to guanine bases, and of the products, by performing DFT calculations. Our results show that in 2 an isomerization of the Pt-coordination sphere from N(2) to N(3) of the triazolate unit determines a thermodynamic stabilization of approximately 20 kcal/mol as a consequence of the formation of an allylic structure. In addition, hybrid quantum-classical molecular dynamics simulations of 1 and 2 DNA adducts have shed light on the structural distortions that the drugs induce to the DNA duplex. Our calculations show that the rise and the tilt of the two adjacent guanines are identical in the presence of 1 and 2, but they markedly increase when 2 binds in the N(1),N(3) fashion. In addition, the drugs do not provoke any kink upon binding to the double-stranded DNA, suggesting that they may act with a mechanism different than that of cisplatin. The accuracy of our calculations is established by a comparison with the NMR data for the corresponding complex with 1. Show less

AbstractThe early phases of commercial drug discovery programs are increasingly guided by information extracted from three‐dimensional structures of the target proteins and in silico design techniques. This review addresses key issues of docking and scoring, a popular technique in structure‐based drug design. The pros and cons of computational tools currently used will be outlined as well as the integration of these methods in the lead finding and lead optimization process. Show less

The sulforhodamine B (SRB) assay is used for cell density determination, based on the measurement of cellular protein content. The method described here has been optimized for the toxicity screening of compounds to adherent cells in a 96-well format. After an incubation period, cell monolayers are fixed with 10% (wt/vol) trichloroacetic acid and stained for 30 min, after which the excess dye is removed by washing repeatedly with 1% (vol/vol) acetic acid. The protein-bound dye is dissolved in 10 mM Tris base solution for OD determination at 510 nm using a microplate reader. The results are linear over a 20-fold range of cell numbers and the sensitivity is comparable to those of fluorometric methods. The method not only allows a large number of samples to be tested within a few days, but also requires only simple equipment and inexpensive reagents. The SRB assay is therefore an efficient and highly cost-effective method for screening. Show less

Oxaliplatin undergoes extensive non-enzymatic chemical transformation in the body. Complexes with sulphur-containing compounds have previously been found in plasma from patients treated with oxaliplatin. We have studied the kinetics for the reactions between oxaliplatin and cysteine, methionine, and glutathione, by determination of the degradation of oxaliplatin using liquid chromatography with UV-detection. We also studied the degradation of oxaliplatin in plasma ultrafiltrate (PUF). For the degradation of oxaliplatin in the presence of glutathione, methionine, and cysteine, the second-order rate constants were 4.7M(-1)min(-1) (95% confidence interval [C.I.], 4.4-5.0M(-1)min(-1)), 5.5M(-1)min(-1) (95% C.I., 5.2-5.7M(-1)min(-1)), and 15M(-1)min(-1) (95% C.I., 14-17M(-1)min(-1)), respectively. The reaction rate was much faster than previously reported kinetics for cisplatin. The degradation rate of oxaliplatin in PUF was biphasic. The rate constant for the first phase varied from 9.5x10(-3) to 0.13min(-1) and for the second phase from (1.7 to 1.8)x10(-3)min(-1) in PUF from five healthy volunteers. The first hours of the degradation of oxaliplatin in PUF are accounted for by the degradation of oxaliplatin in a cocktail of sodium chloride and sulphur-containing compounds at physiological plasma concentrations. In conclusion, the rate of the reaction of oxaliplatin with three sulphur-containing compounds was faster for oxaliplatin than what is previously known for cisplatin. This may be important with respect to differences in the cellular effects of cisplatin and oxaliplatin treatment. Show less

Metal-thiolate active sites play major roles in bioinorganic chemistry. The M--S(thiolate) bonds can be very covalent, and involve different orbital interactions. Spectroscopic features of these active sites (intense, low-energy charge transfer transitions) reflect the high covalency of the M--S(thiolate) bonds. The energy of the metal-thiolate bond is fairly insensitive to its ionic/covalent and pi/sigma nature as increasing M--S covalency reduces the charge distribution, hence the ionic term, and these contributions can compensate. Thus, trends observed in stability constants (i.e., the Irving-Williams series) mostly reflect the dominantly ionic contribution to bonding of the innocent ligand being replaced by the thiolate. Due to high effective nuclear charges of the Cu(II) and Fe(III) ions, the cupric- and ferric-thiolate bonds are very covalent, with the former having strong pi and the latter having more sigma character. For the blue copper site, the high pi covalency couples the metal ion into the protein for rapid directional long range electron transfer. For rubredoxins, because the redox active molecular orbital is pi in nature, electron transfer tends to be more localized in the vicinity of the active site. Although the energy of hydrogen bonding of the protein environment to the thiolate ligands tends to be fairly small, H-bonding can significantly affect the covalency of the metal-thiolate bond and contribute to redox tuning by the protein environment. Show less

We describe a 1.2 A X-ray structure of a double-stranded B-DNA dodecamer (the Dickerson Dodecamer, DDD, [d(CGCGAATTCGCG)]2) associated with a cytotoxic platinum(II) complex, [{trans-Pt(NH3)2(NH2(CH2)6(NH3+)}2-mu-{trans-Pt(NH3)2(NH2(CH2)6NH2)2}] (TriplatinNC). TriplatinNC is a multifunctional DNA ligand, with three cationic Pt(II) centers, and directional hydrogen bonding functionalities, linked by flexible hydrophobic segments, but without the potential for covalent interaction. TriplatinNC does not intercalate nor does it bind in either groove. Instead, it binds to phosphate oxygen atoms and thus associates with the backbone. The three square-planar tetra-am(m)ine Pt(II) coordination units form bidentate N...O...N complexes with OP atoms, in a motif we call the Phosphate Clamp. The geometry is conserved among the 8 observed phosphate clamps in this structure. The interaction appears to prefer O2P over O1P atoms (frequency of interaction is O2P > O1P, base and sugar oxygens > N). The high repetition and geometric regularity of the motif suggests that this type of Pt(II) center can be developed as a modular nucleic acid binding device with general utility. TriplatinNC extends along the phosphate backbone, in a mode of binding we call "Backbone Tracking" and spans the minor groove in a mode of binding we call "Groove Spanning". Electrostatic forces appear to induce modest DNA bending into the major groove. This bending may be related to the direct coordination of a sodium cation by a DNA base, with unprecedented inner-shell (direct) coordination of penta-hydrated sodium at the O6 atom of a guanine. Show less

The US National Cancer Institute (NCI) 60 human tumour cell line anticancer drug screen (NCI60) was developed in the late 1980s as an in vitro drug-discovery tool intended to supplant the use of transplantable animal tumours in anticancer drug screening. This screening model was rapidly recognized as a rich source of information about the mechanisms of growth inhibition and tumour-cell kill. Recently, its role has changed to that of a service screen supporting the cancer research community. Here I review the development, use and productivity of the screen, highlighting several outcomes that have contributed to advances in cancer chemotherapy. Show less

We report structure-activity relationships for organometallic RuII complexes of the type [(eta6-arene)Ru(XY)Cl]Z, where XY is an N,N- (diamine), N,O- (e.g., amino acidate), or O,O- (e.g., beta-diketonate) chelating ligand, the arene ranges from benzene derivatives to fused polycyclic hydrocarbons, and Z is usually PF6. The X-ray structures of 13 complexes are reported. All have the characteristic "piano-stool" geometry. The complexes most active toward A2780 human ovarian cancer cells contained XY=ethylenediamine (en) and extended polycyclic arenes. Complexes with polar substituents on the arene or XY=bipyridyl derivatives exhibited reduced activity. The activity of the O,O-chelated complexes depended strongly on the substituents and on the arene. For arene=p-cymene, XY=amino acidate complexes were inactive. Complexes were not cross-resistant with cisplatin, and cross-resistance to Adriamycin was circumvented by replacing XY=en with 1,2-phenylenediamine. Some complexes were also active against colon, pancreatic, and lung cancer cells. Show less

The aim of this study was to investigate cellular response to several ruthenium(III), chromium(III) and rhodium(III) compounds carrying bidentate beta-diketonato ligands: [(acac)--acetylacetonate ligand, (tfac)--trifluoroacetylacetonate ligand]. Cell sensitivity studies were performed on several cell lines (A2780, cisplatin-sensitive and -resistant U2-OS and U2-OS/Pt, HeLa, B16) using growth-inhibition assay. Effect of intracellular GSH depletion on cell sensitivity to the agents was analyzed in A2780 cells. Flow cytometry was used to assess apoptosis by Annexin-V-FITC/PI staining, and to analyze induction of caspase-3 activity. Possible DNA binding/damaging affinity was investigated, by inductively coupled mass spectrometry, and by 14C-thymidine / 3H-uridine incorporation assay. Cell sensitivity studies showed that the pattern of sensitivity to Ru(tfac)3 complex of the two cisplatin-sensitive/-resistant osteosarcoma cell lines, U2-OS and U2-OS/Pt, was similar to that of A2780 cells (72 h exposure), with the IC50 being around 40 microM. The growth-inhibitory effect of Ru(acac)3 ranged over 100 microM, while Cr(III) and Rh(III) complexes were completely devoid of antitumor action in vitro. Ru(tfac)3 exhibited strong potential for apoptosis induction on A2780 cells (up to 40%) and caused cell cycle arrest in the S phase as well as decrease of the percent of G1 and G2 cells. Ru(acac)3-induced apoptosis was slightly higher than 10%, whereas activation of caspase-3 in HeLa cells was moderate. DNA binding study revealed that only Cr(acac)3 was capable of binding DNA, while Cr(III) and Ru(III) compounds possess potential to inhibit DNA/RNA synthesis. In conclusion, only Ru(III) complexes showed potential for antitumor action. Show less