A strong preference for the TA/TA dinucleotide step discovered for an acridine-based, potent antitumor dsDNA intercalator, C-1305: NMR-driven structural and sequence-specificity studies.

Triazoloacridinone C-1305, a potent antitumor agent recommended for Phase I clinical trials, exhibits high activity towards a wide range of experimental colon carcinomas, in many cases associated with complete tumor regression. C-1305 is a well-established dsDNA intercalator, yet no information on its mode of binding into DNA is available to date. Herein, we present the NMR-driven and MD-refined reconstruction of the 3D structures of the d(CGATATCG)2:C-1305 and d(CCCTAGGG)2:C-1305 non-covalent adducts. In both cases, the ligand intercalates at the TA/TA site, forming well-defined dsDNA:drug 1:1 mol/mol complexes. Orientation of the ligand within the binding site was unambiguously established by the DNA/ligand proton-proton NOE contacts. A subsequent, NMR-driven study of the sequence-specificity of C-1305 using a series of DNA duplexes, allowed us to confirm a strong preference towards TA/TA dinucleotide steps, followed by the TG/CA steps. Interestingly, no interaction at all was observed with duplexes containing exclusively the AT/AT, GG/CC and GA/TC steps.

Biological responses to environmental contamination. How can metal pollution impact signal honesty in avian species?

Environmental pollution, for example with metals, can significantly affect the ecosystem balance leading to severe changes. Biologically active pigments are relevant for the appearance and condition of birds. Melanin and carotenoid particles are the most frequently deposited pigments in avian integument. They are responsible for the majority of colors of bird plumage. The phenotypic expression can be affected by metal contamination. It can be manifested as color bleaching or differences in the size of plumage badges. In this study, we performed a comprehensive review of related studies in order to estimate the underlying population effect of this potential dependency. The study is based on the review of the literature regarding several avian species. It was designed to identify an area where the effect of the exposure is still poorly known. The analysis was specifically conducted to investigate the correlation between trace element concentration and eumelanin deposition. Moreover, we searched for factors that could affect spectral properties of feathers with carotenoid-based pigmentation. As a result, we found carotenoid-based pigmentation to be of a good use in terms of visual condition assessment. Changes in melanin-based pattern should be analyzed separately for eu- and pheomelanin as well as for a range of essential and toxic elements. Comprehensive studies on the subject are still scarce. Therefore, the issue requires further investigation.

Svalbard reindeer as an indicator of ecosystem changes in the Arctic terrestrial ecosystem.

Over the years, noticeable effort has been directed towards contaminant determination in multiple biotic samples collected from the inhabitants of the Arctic. Little consideration has been given to polar herbivores, however, especially those from the European parts of the Arctic. To provide a broader perspective, we aimed to decipher trace element concentration in hairs of the key species in the Arctic, namely the Svalbard reindeer (Rangifer tarandus platyrhynchus), and to recognise whether diet variations could correspond with forward exposure. The effect of habitat and diet was investigated using the ratios of stable isotopes of carbon (δ13C) and nitrogen (δ15N), and previous literature studies on vegetation from the areas of interest. Analysis was performed for eighteen elements in total, both toxic and essential. Metals were present in a decreasing order Fe > Zn > Ba > Cu > Pb > Cr > Ni > V > Ga =La > Rb > As > Li > Co > Hg > Cd > Cs > Be. Similarity in trends in the studied subpopulations was observed for many metals. A significant log-linear correlation was observed for most of the elements, excluding nitrogen and carbon isotopes signature. Extremely high iron levels were determined in some of the samples, suggesting past iron overload. Zinc, in contrast to the remaining metals, did not correlate well with any other element. Mercury was determined at very low levels, in accordance with previous literature regarding its concentrations in moss and lichen species in Svalbard. The analysis of stable isotopes showed a high variation in nitrogen isotopes signatures. Further research is required to properly evaluate the potential health risks and ecological implications of elevated exposure.

Intercalation complex of imidazoacridinone C-1311, a potential anticancer drug, with DNA helix d(CGATCG)2: stereostructural studies by 2D NMR spectroscopy.

Imidazoacridinone C-1311 (Symadex®) is a powerful antitumor agent, which successfully made its way through the Phase I clinical trials and has been recommended for Phase II few a years ago. It has been shown experimentally that during the initial stage of its action C-1311 forms a relatively stable intercalation complex with DNA, yet it has shown no base-sequence specificity while binding to DNA. In this paper, the d(CGATCG)2:C-1311 intercalation complex has been studied by means of two-dimensional NMR spectroscopy, yielding a full assignment of the resonance lines observed in (1)H NMR spectra. The observation of the intermolecular NOE contacts between C-1311 and DNA allowed locating the ligand between the guanine and adenine moieties. Formation of a symmetric complex was pointed out on the basis of the lack of a second set of the (1)H resonances. The resulting stereostructure of the complex was then improved by means of molecular dynamics, using the CHARMM force field and GROMACS software. To this end, distance restraints derived from the NOESY cross-peak volumes were applied to the atomistic model of the d(CGATCG)2:C-1311 complex. Obtained results are in full agreement with biochemical data on the mechanism of action of C-1311, in particular with the previously postulated post-intercalation enzymatic activation of the studied drug.

[Acridines as antitumor drugs]. / Akrydyny jako zwiazki aktywne przeciwnowotworowo.

Acridines belong to a group of polycyclic heteroaromatic compounds and exhibit a broad spectrum of biological activity including antiprotozoal, antibacterial, antiviral and antitumor activity. Acridine derivatives with antitumor activity have different mechanisms of action at the molecular level. The data obtained so far indicate that one of the main steps is the formation of physico-chemical complexes with DNA. Among acridine derivatives with anticancer activity we can distinguish five main classes of compounds: nitroacridines, 9-anilinoacridines, pyrazoloacridines, imidazoacridines, and triazoloacridines. Compounds from different classes differ both in mechanism of action and spectrum of antitumor activity.

[Thermodynamics aspects of interactions between small ligands and DNA]. / Termodynamiczne aspekty oddzialywania niskoczasteczkowych ligandów z DNA.

DNA is a molecular target for many anticancer and antiviral drugs. Therefore, a clear understanding of the interaction of small molecules with DNA is important in the rational design of ligands that can bind to DNA with high affinity and selectivity. There are several methods to investigate interactions between drug and DNA. Some of them measures changing into DNA structures, such as lengthening and untwisting of helix of DNA. Other techniques measure changing in drug environment. With the increasing availability of sensitive microcalorimeters, particular interest has arisen in the thermodynamics of drug-DNA interaction. Using such methods permit direct determination of enthalpy changes associated with reactions. One experiment permits to obtain also binding constant, hence an almost complete thermodynamic profile can be established. This profile offers key insights into the molecular forces that drive complex formation and permit to estimate which kind of interaction are responsible of forming these complexes.

Molecular modeling of amphotericin B-ergosterol primary complex in water II.

The work presented is a part of our continual study on the behavior of the polyene macrolide antibiotic amphotericin B (AmB) complexes with sterols on the molecular level. In contrast to the previously researched AmB-ergosterol binary complex, the AmB-ergosterol-AmB aggregates simulated of 2:1 stoichiometry retain significantly higher stability and relatively rigid, "sandwich" geometry. Van der Waals forces with a considerable share of the electrostatic interactions are responsible for such behavior. System of the intermolecular hydrogen bonds also seems to be of notable importance for the complex's structure preservation. The most energetically favored geometries match fairly close the geometric criteria and the network of interactions postulated in the contemporary hypothetical and computational models of antibiotic-sterol complexes. On the basis of works previously published and the present study novel hypotheses on the AmB selectivity towards sterols varying in chemical structure and on the possible mechanisms of channel structure formation were presented.

PCBs in fish from the southern Baltic Sea: levels, bioaccumulation features, and temporal trends during the period from 1997 to 2006.

Levels of seven marker polychlorinated biphenyls (PCBs) have been determined in five species of Baltic fish collected during 1997-2006. Downward time trends in the concentrations of heavier congeners of PCBs in different Baltic fish, with the exception of cod, have been observed between 1997 and 2001. In case of sprat and herring samples, the statistical significance of the time trends of the PCBs: 101, 118, 153, 138 and 180 concentrations has been proved. Species-specific bioaccumulation of PCBs has been indicated, and the lowest and highest levels of PCBs (expressed on the basis of lipid weight) have been observed in sprat and salmon samples, respectively. PCB profiles have been found to be similar in all the fish species tested. Sampling location has not been a crucial factor for the observed levels of various PCBs. In some fish species, PCB concentrations are negatively correlated with the fat content but have no relation with the fish length.

Organochlorine pesticides in fish from the southern Baltic Sea: levels, bioaccumulation features and temporal trends during the 1995-2006 period.

The levels of HCH isomers, HCB and summation DDTs were determined in five species of Baltic fish collected during 1995-2006. Some downward time trends of HCH isomer, pp'-DDE and pp'-DDD concentrations in Baltic fish were observed; in contrast, HCB and DDT concentrations did not exhibit any obvious trend. Concentrations of summation HCHs expressed on a lipid weight basis were very similar in all species studied. In contrast, concentrations of summation DDTs varied in tested species. The most abundant HCH isomer was beta-HCH and among DDT-related compounds, pp'-DDE prevailed. In our investigations the sampling sites were not a crucial factor for organochlorine pesticide concentrations and patterns observed in fish, but the occurrence of several inter-species differences in the bioaccumulation features of OC pesticides were observed. For cod there was a positive correlation between fish size (length) and summation DDT concentration. In herring, sprat and salmon samples, summation DDT concentrations were negatively correlated with fat content.

Glucosamine-6-phosphate synthase, a novel target for antifungal agents. Molecular modelling studies in drug design.

Fungal infections are a growing problem in contemporary medicine, yet only a few antifungal agents are used in clinical practice. In our laboratory we proposed the enzyme L-glutamine: D-fructose-6-phosphate amidotransferase (EC 2.6.1.16) as a new target for antifungals. The structure of this enzyme consists of two domains, N-terminal and C-terminal ones, catalysing glutamine hydrolysis and sugar-phosphate isomerisation, respectively. In our laboratory a series of potent selective inhibitors of GlcN-6-P synthase have been designed and synthesised. One group of these compounds, including the most studied N3-(4-methoxyfumaroyl)-l-2,3-diaminopropanoic acid (FMDP), behave like glutamine analogs acting as active-site-directed inactivators, blocking the N-terminal, glutamine-binding domain of the enzyme. The second group of GlcN-6-P synthase inhibitors mimic the transition state of the reaction taking place in the C-terminal sugar isomerising domain. Surprisingly, in spite of the fact that glutamine is the source of nitrogen for a number of enzymes it turned out that the glutamine analogue FMDP and its derivatives are selective against GlcN-6-P synthase and they do not block other enzymes, even belonging to the same family of glutamine amidotransferases. Our molecular modelling studies of this phenomenon revealed that even within the family of related enzymes substantial differences may exist in the geometry of the active site. In the case of the glutamine amidotransferase family the glutamine binding site of GlcN-6-P synthase fits a different region of the glutamine conformational space than other amidotransferases. Detailed analysis of the interaction pattern for the best known, so far, inhibitor of the sugar isomerising domain, namely 2-amino-2-deoxy-D-glucitol-6-phosphate (ADGP), allowed us to suggest changes in the structure of the inhibitor that should improve the interaction pattern. The novel ligand was designed and synthesised. Biological experiments confirmed our predictions. The new compound named ADMP is a much better inhibitor of glucosamine-6-phosphate synthase than ADGP.

Molecular basis of the low activity of antitumor anthracenediones, mitoxantrone and ametantrone, in oxygen radical generation catalyzed by NADH dehydrogenase. Enzymatic and molecular modelling studies.

Synthetic antitumor anthracenedione drugs, in contrast to anthracycline antibiotics, are ineffective in free radical formation in NADH dehydrogenase system. Our results have indicated that neither the reduction potential nor the side chain conformation and the energies of border orbitals (HOMO and LUMO) determine the ability of anthracenediones to stimulate reactive oxygen species formation in NADH dehydrogenase system. It was shown that the distribution of the molecular electrostatic potential (MEP), around the quinone system was crucial for this ability. We have found for non-stimulating anthracenediones that the clouds of positive MEP cover the quinone carbon atoms while for agents effective in stimulating reactive oxygen species formation the clouds of negative MEP cover continuously the aromatic core together with the quinone system.

Studies of the effects of antifungal cationic derivatives of amphotericin B on human erythrocytes.

The novel group of amphotericin B (AmB) cationic derivatives has been examined in terms of relationship between self-association and selective toxicity. In all determinations AmB has been used as reference compound. In vitro toxic effects of the compounds on human erythrocytes were determined by measuring leakage of intracellular potassium ions and hemolysis. Antifungal effects were determined as MIC and intracellular potassium loss. The compounds self-association was followed by UV-Vis spectroscopy. The results suggested that: i) unlike AmB the monomer/self-associated species ratio is not an essential in governing the selective toxicity of the derivatives studied; ii) the presence of a bulky substituent in the AmB molecule, preferably located at the amino group of mycosamine moiety is the structural factor essential for the selective toxicity improvement.

Molecular modelling of membrane sterols with the use of the GROMOS 96 force field.

Membrane located sterols determine the structure and function of eucariotic cell membranes. Moreover, they are targets for important antifungal antibiotic amphotericin B. Knowledge about the geometry and dynamics of sterols in the environment of lipidic membranes is necessary to understand their functions. However, due to the dynamic character of the membrane, no experimental data about sterol behaviour on the molecular level is available. Hence molecular modelling simulations could be a source of useful information. The main goal of this paper is to prove the adequacy of the GROMOS 96 force field for molecular simulations of membrane sterols. We focused our attention on the reproduction of characteristic geometrical features observed in the crystal of cholesterol hemiethanolate by molecular dynamics simulations. The results presented clearly indicate that the GROMOS 96 force field can be a useful tool to simulate the highly lipophilic systems. Moreover, interactions responsible for the stability of such systems can also be recognised.

Molecular modelling of amphotericin B-ergosterol primary complex in water.

The properties of amphotericin B-ergosterol primary complex have been studied with the use of the molecular dynamics simulation. Possible geometries of the complex were tested first in order to find the structures with the most favourable values of the intermolecular interactions energy. The molecules studied possessed a tendency to fit each other's shapes, which favours intermolecular van der Waals interactions. The main simulations were performed for the best structures found. Presence of hydrogen bonds between the sterol hydroxyl group and polar fragments of mycosamine (most frequently 2'OH) was coupled with a relatively high level of the intermolecular energy values. The structures obtained are hardly comparable to the hypothetical and 'computational' models of the antibiotic-sterol complex. The geometries found are not suitable to assemble the presupposed structure of the water channel, however, the existence of the complex in the shape anticipated is not in contradiction to the results of biophysical experiments on the complexation in water and in hydroalcoholic media.