Novel Piperazine Derivatives of Vindoline as Anticancer Agents.

A series of novel vindoline-piperazine conjugates were synthesized by coupling 6 N-substituted piperazine pharmacophores at positions 10 and 17 of Vinca alkaloid monomer vindoline through different types of linkers. The in vitro antiproliferative activity of the 17 new conjugates was investigated on 60 human tumor cell lines (NCI60). Nine compounds presented significant antiproliferative effects. The most potent derivatives showed low micromolar growth inhibition (GI50) values against most of the cell lines. Among them, conjugates containing [4-(trifluoromethyl)benzyl]piperazine (23) and 1-bis(4-fluorophenyl)methyl piperazine (25) in position 17 of vindoline were outstanding. The first one was the most effective on the breast cancer MDA-MB-468 cell line (GI50 = 1.00 µM), while the second one was the most effective on the non-small cell lung cancer cell line HOP-92 (GI50 = 1.35 µM). The CellTiter-Glo Luminescent Cell Viability Assay was performed with conjugates 20, 23, and 25 on non-tumor Chinese hamster ovary (CHO) cells to determine the selectivity of the conjugates for cancer cells. These compounds exhibited promising selectivity with estimated half-maximal inhibitory concentration (IC50) values of 2.54 µM, 10.8 µM, and 6.64 µM, respectively. The obtained results may have an impact on the design of novel vindoline-based anticancer compounds.

Synthesis of Novel Vindoline-Chrysin Hybrids.

Vinca alkaloids are well-known microtubule targeting agents, which are used against some types of cancer. Vindoline is one of the monomeric Vinca alkaloids which does not have anti-tumor effect, although its derivatives have serious impact on the field of these indole alkaloids. Chrysin is a secondary plant metabolite, which has broad-spectrum biological activity, among others anticancer activity. Chrysin had shown synergic effect with several antiproliferative compounds (e. g., doxorubicin, cisplatin and ciglitazone), therefore, we attempted the synthesis of a novel vindoline-chrysin hybrid molecule. However, in the first case a diphenylamine structure was isolated. The mechanism of the unexpected reaction was studied, and then the originally targeted hybrid was synthesized by a reverse route coupling. A further hybrid was produced using a different site of the molecule. The antitumor activities were determined against 60 human tumor cell lines (NCI60), where the aimed hybrid showed low micromolar GI50 values on most of the cell lines.

EFFECTS OF MATERNAL EPILEPSY AND ANTIEPILEPTIC THERAPY IN WOMEN DURING PREGNANCY.

OBJECTIVE: The aim of this study was to analyse the effects of epilepsy and antiepileptic drug (AED) treatment on pregnancy and the perinatal outcome, retrospectively. METHODS: We examined the obstetric and fetal outcomes among women with epilepsy (WWE), who were followed-up at the Department of Neurology, and who delivered at the Department of Obstetrics and Gynaecology (n=91) between 31th December 2000 and 31th March 2014. Statistical comparisons of different obstetric and fetal parameters on a sample of 91 WWE and 182 non-WWE were assessed by the chi-square-test, the independent sample t-test. RESULTS: The rate of major congenital malformations (MCMS) among the newborns of all AEDs exposed mothers was 7.69%. There were three peaks of seizures: during the third trimester, during delivery and in the puerperium. The prevalence of miscarriages, post-term birth and the rate of caesarean section were significantly higher among the WWE than among the non-WWE (p=0.001; p<0.001; p=0.02). Parameters of neonates (birth weight, birth length, head-, and chest circumference) were significantly different between the WWE group and the non-WWE group (p=0.003, p<0.001, p<0.001, p<0.001) CONCLUSIONS: In contrast with recent publications, there were significant differences in the parameters of neonates between the two groups. Our results are in accordance with those of previous studies from the aspect of AED-related MCM, the elevated risk of miscarriages and pre-existing hypertension.

Copper(II) complexes with highly water-soluble L- and D-proline-thiosemicarbazone conjugates as potential inhibitors of Topoisomerase IIα.

Two proline-thiosemicarbazone bioconjugates with excellent aqueous solubility, namely, 3-methyl-(S)-pyrrolidine-2-carboxylate-2-formylpyridine thiosemicarbazone [L-Pro-FTSC or (S)-H2L] and 3-methyl-(R)-pyrrolidine-2-carboxylate-2-formylpyridine thiosemicarbazone [D-Pro-FTSC or (R)-H2L], have been synthesized and characterized by elemental analysis, one- and two-dimensional (1)H and (13)C NMR spectroscopy, and electrospray ionization mass spectrometry. The complexation behavior of L-Pro-FTSC with copper(II) in an aqueous solution and in a 30% (w/w) dimethyl sulfoxide/water mixture has been studied via pH potentiometry, UV-vis spectrophotometry, electron paramagnetic resonance, (1)H NMR spectroscopy, and spectrofluorimetry. By the reaction of copper(II) acetate with (S)-H2L and (R)-H2L in water, the complexes [Cu(S,R)-L] and [Cu(R,S)-L] have been synthesized and comprehensively characterized. An X-ray diffraction study of [Cu(S,R)-L] showed the formation of a square-pyramidal complex, with the bioconjugate acting as a pentadentate ligand. Both copper(II) complexes displayed antiproliferative activity in CH1 ovarian carcinoma cells and inhibited Topoisomerase IIα activity in a DNA plasmid relaxation assay.

Identification of agents that reduce renal hypoxia-reoxygenation injury using cell-based screening: purine nucleosides are alternative energy sources in LLC-PK1 cells during hypoxia.

Acute tubular necrosis is a clinical problem that lacks specific therapy and is characterized by high mortality rate. The ischemic renal injury affects the proximal tubule cells causing dysfunction and cell death after severe hypoperfusion. We utilized a cell-based screening approach in a hypoxia-reoxygenation model of tubular injury to search for cytoprotective action using a library of pharmacologically active compounds. Oxygen-glucose deprivation (OGD) induced ATP depletion, suppressed aerobic and anaerobic metabolism, increased the permeability of the monolayer, caused poly(ADP-ribose) polymerase cleavage and caspase-dependent cell death. The only compound that proved cytoprotective either applied prior to the hypoxia induction or during the reoxygenation was adenosine. The protective effect of adenosine required the coordinated actions of adenosine deaminase and adenosine kinase, but did not requisite the purine receptors. Adenosine and inosine better preserved the cellular ATP content during ischemia than equimolar amount of glucose, and accelerated the restoration of the cellular ATP pool following the OGD. Our results suggest that radical changes occur in the cellular metabolism to respond to the energy demand during and following hypoxia, which include the use of nucleosides as an essential energy source. Thus purine nucleoside supplementation holds promise in the treatment of acute renal failure.

Copper(II)-binding ability of stereoisomeric cis- and trans-2-aminocyclohexanecarboxylic acid-L-phenylalanine dipeptides. A combined CW/pulsed EPR and DFT study.

With the aim of an improved understanding of the metal-complexation properties of alicyclic ß-amino acid stereoisomers, and their peptides, the complex equilibria and modes of coordination with copper(II) of L-phenylalanine (F) derivatives of cis/trans-2-aminocyclohexanecarboxylic acid (c/tACHC), i.e. the dipeptides F-c/tACHC and c/tACHC-F, were investigated by a combination of CW and pulsed EPR methods. For the interpretation of the experimental data, DFT quantum-chemical calculations were carried out. Simulation of a pH-dependent series of room-temperature CW-EPR spectra revealed the presence of EPR-active complexes ([Cu(aqua)](2+), [CuL](+), [CuLH(-1)], [CuLH(-2)](-), and [CuL(2)H(-1)](-)), and an EPR-inactive species ([Cu(2)L(2)H(-3)](-)) in aqueous solutions for all studied cases. [CuLH](2+) was included in the equilibrium model for the c/tACHC-F-copper(II) systems, and [CuL(2)], together with two coordination isomers of [CuL(2)H(-1)](-), were also identified in the F-tACHC-copper(II) system. Comparison of the complexation properties of the diastereomeric ligand pair F-(1S,2R)-ACHC and F-(1R,2S)-ACHC did not reveal significant differences. Considerably lower formation constants were obtained for the trans than for the cis isomers for both the F-c/tACHC and the c/tACHC-F pairs in the case of [CuLH(-1)] involving tridentate coordination by the amino, the deprotonated peptide, and the carboxylate groups. A detailed structural analysis by pulsed EPR methods and DFT calculations indicated that there was no significant destabilization for the complexes of the trans isomers. The lower stability of their complexes was explained by the limitation that only the conformer with donor groups in equatorial-equatorial ring positions can bind to copper(II), whereas both equatorial-axial conformers of the cis isomers are capable of binding. From a consideration of the proton couplings obtained with X-band (1)H HYSCORE, (2)H exchange experiments, and DFT, the thermodynamically most stable cyclohexane ring conformer was assigned for all four [CuLH(-1)] complexes. For the F-cACHC case, the conformer did not match the most stable conformer of the free ligand.

L- and D-proline thiosemicarbazone conjugates: coordination behavior in solution and the effect of copper(II) coordination on their antiproliferative activity.

Two enantiomerically pure thiosemicarbazone-proline conjugates with enhanced aqueous solubility, namely, 2-hydroxy-3-methyl-(S)-pyrrolidine-2-carboxylate-5-methylbenzaldehyde thiosemicarbazone [L-Pro-STSC or (S)-H(2)L] and 2-hydroxy-3-methyl-(R)-pyrrolidine-2-carboxylate-5-methylbenzaldehyde thiosemicarbazone [D-Pro-STSC or (R)-H(2)L] have been synthesized and characterized by elemental analysis, spectroscopic methods (UV-vis and (1)H and (13)C NMR), and electrospray ionization mass spectrometry. The metal complexation behavior of L-Pro-STSC, stoichiometry, and thermodynamic stability of iron(II), iron(III), copper(II), and zinc(II) complexes in 30% (w/w) dimethyl sulfoxide/H(2)O solvent mixture have been studied by pH-potentiometric, UV-vis-spectrophotometric, circular dichroism, electron paramagnetic resonance, (1)H NMR spectroscopic, and spectrofluorimetric measurements. By the reaction of CuCl(2)·2H(2)O with (S)-H(2)L and (R)-H(2)L, respectively, the complexes [Cu[(S)-H(2)L]Cl]Cl and [Cu[(R)-H(2)L]Cl]Cl have been prepared and comprehensively characterized. An X-ray diffraction study of [Cu[(R)-H(2)L]Cl]Cl showed the formation of a square-planar copper(II) complex, which builds up stacks with interplanar separation of 3.3 Å. The antiproliferative activity of two chiral ligands and their corresponding copper(II) complexes has been tested in two human cancer cell lines, namely, SW480 (colon carcinoma) and CH1 (ovarian carcinoma). The thiosemicarbazone-proline conjugates L- and D-Pro-STSC show only moderate cytotoxic potency with IC(50) values of 62 and 75 µM, respectively, in CH1 cells and >100 µM in SW480 cells. However, the corresponding copper(II) complexes are 13 and 5 times more potent in CH1 cells, based on a comparison of IC(50) values, and in SW480 cells the increase in the antiproliferative activity is even higher. In both tested cell lines, L-Pro-STSC as well as its copper(II) complex show slightly stronger antiproliferative activity than the compounds with a D-Pro moiety, yielding IC(50) values of 4.6 and 5.5 µM for [Cu(L-Pro-STSC)Cl]Cl in CH1 and SW480 cells, respectively.

Highly Enhanced Curcumin Delivery Applying Association Type Nanostructures of Block Copolymers, Cyclodextrins and Polycyclodextrins.

The limited bioavailability of the highly hydrophobic natural compound, curcumin with wide range of beneficial bioactivity is still a challenge. Self-association type systems of polyethylene oxide-polypropylene oxide-polyethylene oxide block copolymers (Pluronic) were applied to enhance the aqueous solubility of curcumin. Comparison of four Pluronics (94, 105, 127,108) with different compositions led to the conclusion that solubilization capacity is maximum for Pluronic 105 with intermediate polarity (hydrophilic/lipophilic balance (HLB) = 15) possessing the optimum balance between capacity of hydrophobic core of the micelle and hydrophilic stabilizing shell of the associate. Curcumin concentration in aqueous solution was managed to increase 105 times up to 1-3 g/L applying Pluronic at 0.01 mol/L. Formation of a host-guest complex of cyclodextrin as another way of increasing the curcumin solubility was also tested. Comparing the(2-hydroxypropyl)-α, ß and γ cyclodextrins (CD) with 6, 7 and 8 sugar units and their polymers (poly-α-CD, poly-ß-CD, poly-γ-CD) the γ-CD with the largest cavity found to be the most effective in curcumin encapsulation approaching the g/L range of concentration. The polymer type of the CDs presented prolonged and pH dependent release of curcumin in the gastrointestinal (GI) system modelled by simulated liquids. This retarding effect of polyCD was also shown and can be used for tuning in the combined system of Pluronic micelle and polyCD where the curcumin release was slower than from the micelle.

Complexation of 5-fluorosalicylic acid with copper(II): a pH-potentiometric, UV-vis spectroscopic, and electron spin resonance study by the two-dimensional simulation of spectra.

The copper(II)-5-fluorosalicylic acid system was investigated in water and 50 v/v% water-methanol mixture by pH potentiometry combined with UV-vis spectrophotometry, and by the two-dimensional ESR simulation method, respectively. The data revealed that the stable paramagnetic mono- and bis(salicylato) copper(II) complexes [CuLH(-1)] and [CuL2H(-2)](2-) are formed, and at low excess of ligand, the ESR-silent mixed hydroxo complex [Cu2L2H(-3)](-) is also a major species. By the two-dimensional ESR simulation method, the species [CuL]+ in the acidic region, and the minor dimer [Cu2L2H(-2)] were also identified, and the cis and trans isomers of [CuL2H(-2)](2-) were characterized. In frozen solutions, the ESR analysis revealed a slight rhombic distortion of coordination polyhedra for the latter three species.

Equilibria of 3-pyridylmethanol with copper(II). A comparative electron spin resonance study by the decomposition of spectra in liquid and frozen solutions.

The copper(II)-3-pyridylmethanol (L) system was investigated in aqueous solution by two-dimensional ESR evaluation at 298 K, and computer simulation of the individual anisotropic spectra at 77 K. The data revealed that the paramagnetic copper(II) complexes [CuL] (2+), [CuL 2] (2+), [CuL 3] (2+), and [CuL 4] (2+) are formed up to pH approximately 7 at a moderate or high excess of ligand. As compared with chelating ligands, two differences were observed for the complexation of 3-pyridylmethanol with copper(II): (1) In contrast with the well-resolved spectra in frozen solution, considerable line-broadening and distortion of the spectral shapes were seen at 298 K, which was interpreted in terms of isomeric equilibria and the medium-rate interconversion of various complexes on the ESR time-scale. (2) At low temperature, there were dramatic changes in the concentration distribution, the minor complexes with higher numbers of coordinating ligands ([CuL 3] (2+) and in particular [CuL 4] (2+)) becoming strongly favored. This phenomenon is explained by the significant differences in the formation enthalpy values of various species, shifting the equilibria according to the van't Hoff equation, and a significant undercooling in the course of fast freezing of the solution, which enhances the changes of the concentration distribution.

Oxidant-induced cardiomyocyte injury: identification of the cytoprotective effect of a dopamine 1 receptor agonist using a cell-based high-throughput assay.

Myocyte injury due to myocardial reperfusion injury plays a crucial role in the pathogenesis of acute myocardial infarction even after successful coronary revascularization. Identification of compounds that reduce reperfusion-associated myocyte death is important. Therefore, we developed an in vitro model of myocardial reperfusion injury in H9c2 rat cardiomyocytes and applied a cell-based high-throughput approach to screen a standard library of pharmacologically active compounds (LOPAC) in order to identify drugs with cardioprotective effects. Oxidative stress was induced with hydrogen peroxide (H2O2) treatment, which resulted in approximately 50% reduction in cell viability. Test compounds were added at a 3-microM final concentration as a pretreatment or in a delayed fashion (30 min after the peroxide challenge in order to imitate pharmacological treatment following angioplasty). Cells were cultured for 3 or 24 h. Viability was quantitated with the methylthiazolyldiphenyl-tetrazolium bromide method. Cytotoxicity and cytoprotection were also evaluated by measuring the lactate dehydrogenase activity in the cell culture supernatant. The screening identified a number of compounds with cytoprotective action, including molecules that are known to interfere with components of DNA repair and cell cycle progression, e.g. poly(ADP-ribose) polymerase (PARP) inhibitors, topoisomerase inhibitors, and cyclin dependent kinase inhibitors, or reduce energy consumption by interfering with cardiac myofilament function. A number of dopamine D1 receptor agonists also provided significant cytoprotection at 3 h, but only three of them showed a similar effect at 24 h: chloro- and bromo-APB and chloro-PB hydrobromide. Chloro-APB hydrobromide significantly reduced peroxide-induced PARP activation in the myocytes independently of its action on dopamine D1 receptors, but lacked PARP inhibitor capacity in a cell-free PARP assay system. In conclusion, the pattern of cytoprotective drugs identified in the current assay supports the overall validity of our model system. The findings demonstrate that cytoprotective agents, including novel indirect inhibitors of cellular PARP activation can be identified with the method, chloro-APB hydrobromide being one such compound. The current experimental setting can be employed for cell-based high-throughput screening of various compound libraries.

A novel 1,3,5-triaminocyclohexane-based tripodal ligand forms a unique tetra(pyrazolate)-bridged tricopper(ii) core: solution equilibrium, structure and catecholase activity.

Copper(ii) complexes of a polydentate tripodal ligand L × 3HCl (L = N,N',N''-tris(5-pyrazolylmethyl)-cis,cis-1,3,5-triaminocyclohexane) were characterized in both solution and solid states. Combined evaluation of potentiometric, UV-VIS, and EPR data indicated the formation of two mononuclear (CuHL, CuL) and three trinuclear (Cu3H-xL2, x = 2, 3, 4) complexes. The high stability and spectroscopic properties of the CuL species indicate a coordination of two pyrazole rings in addition to the three secondary amino groups of L in a square pyramidal geometry. In parallel with the formation of trinuclear species, intense charge transfer bands appear at around 400-500 nm, which indicate the formation of pyrazolate-bridged complexes. The crystal structure of [Cu3H-4L2](ClO4)2·5H2O (1) reveals the formation of a unique trinuclear complex that features a tetra(pyrazolate)-bridged linear tricopper(ii) core. The CuCu interatomic distances are around 3.8 Å. The two peripheral copper(ii) ions have a slightly distorted square pyramidal geometry. The four pyrazole rings bound to the peripheral copper(ii) ions are deprotonated and create a flattened tetrahedral environment for the central copper(ii), i.e. the formation of the trinuclear complexes is under the allosteric control of the two peripheral copper(ii) ions. The triply deprotonated trinuclear complex is an efficient catechol oxidase mimic with a surprisingly low pH optimum at pH = 5.6. Since the mononuclear CuL species is not able to promote the oxidation of 3,5-di-tert-butylcatechol, we assume that the central copper(ii) ion of the trinuclear complex with an unsaturated coordination sphere has a fundamental role in the binding and oxidation of the substrate. The experimental and structural details were further elaborated by a series of hybrid density functional theory calculations that support the presence of an antiferromagnetically coupled ground state. However, the magnitude and the pattern of spin coupling are dependent on the composition of the functionals. The optimized theoretical structures highlight the role of the crystal packing effects in inducing asymmetry between the two peripheral copper(ii) sites.

Copper(II) directs formation of toxic amorphous aggregates resulting in inhibition of hen egg white lysozyme fibrillation under alkaline salt-mediated conditions.

Hen egg white lysozyme (HEWL) adopts a molten globule-like state at high pH (~12.75) and is found to form amyloid fibrils at alkaline pH. Here, we report that Cu(II) inhibits self-association of HEWL at pH 12.75 both at 37 and 65 °C. A significant reduction in Thioflavin T fluorescence intensity, attenuation in ß-sheet content and reduction in hydrophobic exposure were observed with increasing Cu(II) stoichiometry. Electron paramagnetic resonance spectroscopy suggests a 4N type of coordination pattern around Cu(II) during fibrillation. Cu(II) is also capable of altering the cytotoxicity of the proteinaceous aggregates. Fibrillar species of diverse morphology were found in the absence of Cu(II) with the generation of amorphous aggregates in the presence of Cu(II), which are more toxic compared to the fibrils alone.

Evidence of two oxidation states of copper during aggregation of hen egg white lysozyme (HEWL).

In vitro fibrillation of hen egg white lysozyme (HEWL) causes complete reduction of Cu(II) to Cu(I) at pH 7. Here in the present article, we have shown the presence of both Cu(II) and Cu(I) at pH 11 during fibrillation of HEWL using electron paramagnetic resonance and Raman spectroscopy. Our results suggest the existence of a partially reducing environment during fibrillation of hen egg white lysozyme at pH 11. The fibrillation process is governed by the pH of the solution and maximum fibrillation is found to occur at pH 11. Fibrils formed in the absence of Cu(II) were also found to cause significant hemolysis of RBC.

Vanadium(IV/V) complexes of Triapine and related thiosemicarbazones: Synthesis, solution equilibrium and bioactivity.

The stoichiometry and thermodynamic stability of vanadium(IV/V) complexes of Triapine and two related α(N)-heterocyclic thiosemicarbazones (TSCs) with potential antitumor activity have been determined by pH-potentiometry, EPR and (51)V NMR spectroscopy in 30% (w/w) dimethyl sulfoxide/water solvent mixtures. In all cases, mono-ligand complexes in different protonation states were identified. Dimethylation of the terminal amino group resulted in the formation of vanadium(IV/V) complexes with considerably higher stability. Three of the most stable complexes were also synthesized in solid state and comprehensively characterized. The biological evaluation of the synthesized vanadium complexes in comparison to the metal-free ligands in different human cancer cell lines revealed only minimal influence of the metal ion. Thus, in addition the coordination ability of salicylaldehyde thiosemicarbazone (STSC) to vanadium(IV/V) ions was investigated. The exchange of the pyridine nitrogen of the α(N)-heterocyclic TSCs to a phenolate oxygen in STSC significantly increased the stability of the complexes in solution. Finally, this also resulted in increased cytotoxicity activity of a vanadium(V) complex of STSC compared to the metal-free ligand.

Strong effect of copper(II) coordination on antiproliferative activity of thiosemicarbazone-piperazine and thiosemicarbazone-morpholine hybrids.

In this study, 2-formylpyridine thiosemicarbazones and three different heterocyclic pharmacophores were combined to prepare thiosemicarbazone­piperazine mPip-FTSC (HL1) and mPip-dm-FTSC (HL2), thiosemicarbazone­morpholine Morph-FTSC (HL3) and Morph-dm-FTSC (HL4), thiosemicarbazone­methylpyrrole-2-carboxylate hybrids mPyrr-FTSC (HL5) and mPyrr-dm-FTSC (HL6) as well as their copper(II) complexes [CuCl(mPipH-FTSC-H)]Cl (1 + H)Cl, [CuCl(mPipH-dm-FTSC-H)]Cl (2 + H)Cl, [CuCl(Morph-FTSC-H)] (3), [CuCl(Morph-dm-FTSC-H)] (4), [CuCl(mPyrr-FTSC-H)(H2O)] (5) and [CuCl(mPyrr-dm-FTSC-H)(H2O)] (6). The substances were characterized by elemental analysis, one- and two-dimensional NMR spectroscopy (HL1­HL6), ESI mass spectrometry, IR and UV­vis spectroscopy and single crystal X-ray diffraction (1­5). All compounds were prepared in an effort to generate potential antitumor agents with an improved therapeutic index. In addition, the effect of structural alterations with organic hybrids on aqueous solubility and copper(II) coordination ability was investigated. Complexation of ligands HL2 and HL4 with copper(II) was studied in aqueous solution by pH-potentiometry, UV­vis spectrophotometry and EPR spectroscopy. Proton dissociation processes of HL2 and HL4 were also characterized in detail and microscopic constants for the Z/E isomers were determined. While the hybrids HL5, HL6 and their copper(II) complexes 5 and 6 proved to be insoluble in aqueous solution, precluding antiproliferative activity studies, the thiosemicarbazone­piperazine and thiosemicarbazone­morpholine hybrids HL1­HL4, as well as copper(II) complexes 1­4 were soluble in water enabling cytotoxicity assays. Interestingly, the metal-free hybrids showed very low or even a lack of cytotoxicity (IC50 values > 300 µM) in two human cancer cell lines HeLa (cervical carcinoma) and A549 (alveolar basal adenocarcinoma), whereas their copper(II) complexes were cytotoxic showing IC50 values from 25.5 to 65.1 µM and 42.8 to 208.0 µM, respectively in the same human cancer cell lines after 48 h of incubation. However, the most sensitive for HL4 and complexes 1­4 proved to be the human cancer cell line LS174 (colon carcinoma) as indicated by the calculated IC50 values varying from 13.1 to 17.5 µM.

Copper(II) complexes of some N-substituted bis(aminomethyl)phosphinate ligands. An integrated EPR study of microspeciation and coordination modes by the two-dimensional simulation method.

Copper(II) complexes of bis(aminomethyl)phosphinic acid (L1), bis(N-glycino-N-methyl)phosphinic acid (L2), bis(N-benzylglycino-N-methyl)phosphinic acid (L3), bis(l-prolino-N-methyl)phosphinic acid (L4) and bis(iminodicarboxymethyl-N-methyl)phosphinic acid (L5) were studied in aqueous solution by pH-potentiometric and electron paramagnetic resonance (EPR) spectroscopic methods. The EPR spectrum packages recorded at various ligand-to-metal concentration ratios and pH's were analyzed (after matrix rank analysis by the method of residual intensities as a complementary method) by the two-dimensional computer simulation method, which simultaneously determines the formation constants and the EPR parameters of the various (micro)species. L1 forms mono and bis complexes in different protonation states; for the other ligands, the mono complexes are always prevalent. For steric reasons, the formation of CuL is shifted to increasingly higher pH regions in the sequence L2, L3 and L4. CuLH was identified for L3, L4 and L5, and also CuLH(2) for L4 and L5. Cu(2)L(2) was found in small amounts for L3 and L4, while it predominates at pH>4 for L5. For L5, Cu(2)L(2)H(2) was also detected. For the ligands that form dimeric metal complexes in equimolar solution or at a ligand excess, Cu(2)L is formed at a metal ion excess. Ligation of the phosphinate O was suggested by indirect proofs in the protonated complexes of L1. For the ligands L2, L3 and L4, the copper(II) coordination in various species in different protonation states is reminiscent of that in the mono and bis complexes of simple amino acids. For the bis(aminomethyl)phosphinates, however, the cis positions of the amino groups in CuL are ensured by the structure of the ligand, and the isomers differ from each other in the (equatorial or axial) position of the second carboxylate group.

Fibrillation of human serum albumin shows nonspecific coordination on stoichiometric increment of Copper(II).

Protein aggregation is related to a series of pathological disorders the main cause of which are the fibrillar species generated during the process. Human serum albumin (HSA) undergoes rapid fibrillation in the presence of Cu(II) at pH 7.4 in 60% ethanol after 6-h incubation (∼65 °C) followed by room temperature incubation. Here, we have investigated the effect of a stoichiometric variation of Cu(II) on the self-assembly of HSA using Congo red and thioflavin T dye-binding studies, circular dichroism spectroscopy, Fourier transform infrared spectroscopy, electron paramagnetic resonance spectroscopy, fluorescence microscopy and transmission electron microscopy. The simulation of EPR spectra suggests that with the increment in Cu(II) ion concentration, there is a change in ligand field coordination. Kinetic parameters indicate reduced cooperativity that may be related to the nonspecific coordination on increment of Cu(II) concentration. Cu(II) is also able to direct the accumulation of a large number of fibers along with a formation of dense fibrillar network which is evident from microscopic images.

A comparative study on the possible zinc binding sites of the human ZnT3 zinc transporter protein.

The brain specific zinc transporter protein ZnT3 can be related to the amyloid neuropathology of Alzheimer's disease. In order to analyze the metal binding ability of human ZnT3 protein, here we report a potentiometric and solution structural (UV-Vis, CD, EPR, NMR) study of nickel(II), copper(II) and zinc(II) complexes of three peptides mimicking the possible metal binding sequences of this protein. The peptide L¹ (Ac-RHQAGPPHSHR-NH2) is a minimalist, the cyclic peptide L² (cyclo(Ac-CKLHQAGPPHSHGSRGAEYAPLEEGPEEKC-NH2) is a more complete model of the intracellular His-rich loop, which is widely accepted as a putative metal binding site. The peptide L³ (Ac-PFHHCHRD-NH2) is the model of the conserved cytoplasmic N-terminal -HHCH- sequence. In the physiological pH-range, the ZnL¹, ZnH3L² and ZnL³ complexes are the major species in the corresponding binary systems, with {3N(im)}, {3N(im),2/3O(amide)} and {3N(im),S(-)} coordination environments, respectively. The species ZnL³ has 3-4 orders of magnitude higher stability than the other two complexes, indicating the presence of a high-affinity zinc-binding site at the N-terminal tail of the human ZnT3 transporter. Moreover, L³ shows preferred zinc binding as compared to nickel (log ß(ZnL³) - log ß(NiL³) = 2.3), probably due to the higher preference of zinc(II) for tetrahedral geometry. These facts suggest that zinc binding to the N-terminal -HHCH- sequence of human ZnT3 may be involved in the biological activity of this zinc transporter protein in zinc sensing, binding or translocation processes.

A minimalist chemical model of matrix metalloproteinases--can small peptides mimic the more rigid metal binding sites of proteins?

In order to mimic the active center of matrix metalloproteinases (MMPs), we synthesized a pentadecapeptide (Ac-KAHEFGHSLGLDHSK-NH2) corresponding to the catalytic zinc(II) binding site of human MMP-13. The multi-domain structural organization of MMPs fundamentally determines their metal binding affinity, catalytic activity and selectivity. Our potentiometric, UV-visible, CD, EPR, NMR, mass spectrometric and kinetic studies are aimed to explore the usefulness of such flexible peptides to mimic the more rigid metal binding sites of proteins, to examine the intrinsic metal binding properties of this naked sequence, as well as to contribute to the development of a minimalist, peptide-based chemical model of MMPs, including the catalytic properties. Since the multiimidazole environment is also characteristic for copper(II), and recently copper(II) containing variants of MMPs have been identified, we also studied the copper(II) complexes of the above peptide. Around pH 6-7 the peptide, similarly to MMPs, offers a {3Nim} coordination binding site for both zinc(II) and copper(II). In the case of copper(II), the formation of amide coordinated species at higher pH abolished the analogy with the copper(II) containing MMP variant. On the other hand, the zinc(II)-peptide system mimics some basic features of the MMP active sites: the main species around pH7 (ZnH2L) possesses a {3Nim,H2O} coordination environment, the deprotonation of the zinc-bound water takes place near the physiological pH, it forms relatively stable ternary complexes with hydroxamic acids, and the species ZnH2L(OH) and ZnH2L(OH)2 have notable hydrolytic activity between pH7 and 9.