Lead complexation behaviour of root exudates of salt marsh plant Salicornia europaea L

Abstract

Root exudates are considered to have an important role in mobility and bioavailability of heavy metals. High molecular weight (HMW) substances are the main components of root exudates, however, knowledge about their interactions with heavy metals is lacking. In the present study, Pb(II) complexation of the HMW fluorescent fractions in root exudates from Salicornia europaea L. was investigated using excitation emission matrix (EEM) fluorescence spectroscopy. Two protein-like fluorescence peaks were identified in the EEM spectrum of root exudates. The fluorescence of both peaks was clearly quenched by Pb(II). The values of conditional stability constants, log K_a, for these two protein-like fluorescence peaks were 4.14 and 3.79. This indicates that the fluorescent substances are strong Pb(II) complexing organic ligands.     

Displacement Reactions Employing Heterologous Tracer Ligands in Peptide Receptor Studies: A Review

Abstract

Heterologous tracer ligands used in displacement studies with peptide-receptor systems may become unsuitable to these aims for several reasons. (1) The forms of binding isotherms for the tracer and for the ligand under investigation are different. The Schild plot and similar schemes are then not applicable. Possible modifications of the computational methods are indicated. (2) The rate of dissociation from the receptor is slowed, until almost irreversible. In such cases, there is no chance for displacement studies. (3) Large discrepancy in rate constants, or dramatically different distribution coefficients between binding system and water, for the two ligands mimic irreversible binding to receptors in a pharmacological experiment. Adjustment of ligand concentrations (control of association rate) may help in some instances.     

A Computer Generated Model of Adenosine Receptors Rationalising Binding and Selectivity of Receptor Ligands

Abstract

Computer graphic analyses on a broad spectrum of adenosine receptor ligands has shown that both the A1 and A2 adenosine receptors have three binding sites. The spatial relationship of these three binding sites has been defined. Adenosine orientation at A1 and A2 is different.     

Potent and Selective Ligands for Adenosine Binding Sites

Abstract

A number of selective ligands for the different binding sites of adenosine have been synthesized and tested in several pharmacological models. The aim of these synthetic efforts is both to improve the knowledge of structure-activity relationships in the adenosine-related biological systems and to develop drugs from some of these molecules.     

Characterizing the structural variability of HIV-2 protease upon the binding of diverse ligands using a structural alphabet approach

Abstract

The HIV-2 protease (PR2) is an important target for designing new drugs against the HIV-2 infection. In this study, we explored the structural backbone variability of all available PR2 structures complexed with various inhibitors using a structural alphabet approach. 77% of PR2 positions are structurally variable, meaning they exhibit different local conformations in PR2 structures. This variability was observed all along the structure, particularly in the elbow and flap regions. A part of these backbone changes observed between the 18 PR2 is induced by intrinsic flexibility, and ligand binding putatively induces others occurring in the binding pocket. These latter changes could be important for PR2 adaptation to diverse ligands and are accompanied by changes outside the binding pocket. In addition, the study of the link between structural variability of the pocket and PR2–ligand interactions allowed us to localize pocket regions important for ligand binding and catalytic function, regions important for ligand recognition that adjust their backbone in response to ligand binding and regions important for the pocket opening and closing that have large intrinsic flexibility. Finally, we suggested that differences in ligand effectiveness for PR2 could be partially explained by different backbone deformations induced by these ligands. To conclude, this study is the first characterization of the PR2 structural variability considering ligand diversity. It provides information about the recognition of PR2 to various ligands and its mechanisms to adapt its local conformation to bound ligands that could help understand the resistance of PR2 to its inhibitors, a major antiretroviral class.

Communicated by Ramaswamy H. Sarma     

Sequestration of organometallic compounds by synthetic and naturally occurring polycarboxylate ligands. Binding of monomethylmercury(II) by polyacrylic and alginic acids

Abstract

The sequestering capacity of synthetic and naturally occurring polycarboxylate ligands towards mono-methylmercury(II) was evaluated by stability quantitative data on the interaction of CH₃Hg⁺ with different molecular weight synthetic polyacrylates (2 and 20kDa average M.wt) and alginate (70–100 kDa) extracted from brown algae Macrocystis pyrifera. The influence of ionic medium was evaluated by measurements on the CH₃Hg⁺-polyacrylate systems in NaNO₃ medium at different ionic strengths (0.10, 0.25, 0.50 and 0.75 mol L^?1), and a Debye-HiJckel type equation was used for the dependence of complex formation constants on ionic strength. Measurements on the CH3Hg⁺ - alginate system were carried out at l = 0.10 mol L^?1 in NaNO₃ medium. By using the stability data, the sequestering capacity of both ligands towards monomethylmercury(II) was determined at different pH values. Results obtained show that the binding ability of polyacrylic ligands (PAA) is stronger than the alginate (AA), following the trend PAA (20 kDa)> PAA (2kDa)>AA.     

Binding of Doxorubicin-Conjugated Transferrin to U937 Cells

Abstract

Binding of transferrin (Trf) and its doxorubicin-conjugated forms (Conj) to U937 cells at 0°C were compared using ¹²⁵I-labelled Trf or Conj. The apparent binding affinity (K_a) of Conj to the surface of U937 cells was (1.9±0.4)·10⁸ 1/mol; it is about 40% of that of Trf [(5.0±1.2)· 10⁸ 1/mol]. Binding of ¹²⁵I-labelled ligands was blocked by the unlabelled ligands to the same degree, however, it was not blocked by a great excess of doxorubicin (Dox). N-ethylmaleimide caused about 10% inhibition while dithiothreitol was without effect. Dissociation of ¹²⁵I-labelled ligands in the presence of different concentrations of unlabelled ligands (Trf and Conj in the all 4 variations) resulted in different R₅₀ values (the concentration of the unlabelled ligand where 50% of the radiolabelled ligand was released). While Trf displaced Trf with an R₅₀ value close to the binding affinity, Conj displacement by Conj occurred with much lower efficiency. The heterolog displacement experiments yielded R₅₀ values inbetween the two extrema. These results suggest that 1) binding of Conj to the surface of cells is     

2-Aminopurine Fluorescence: Discrimination Between Specific and Unspecific Ligand Binding to the Kissing-Loop Dimer of the HIV-1 RNA

Abstract

The fluorescent 2-aminopurine probe (2-AP) incorporated into the loop of 23-mer RNA hairpin of HIV-1 genome dimerization initiation site (DIS) was used for discrimination of specific and unspecific binding of paromomycin and spermine to the kissing loop dimer (KD) formed in solution. While both ligands stabilized the KD RNA structure, only paromomycin binding resulted in significant increase of 2-AP fluorescence. These observations suggest that the 2-AP fluorescent RNA construct might be useful for selecting ligands specifically binding the HIV-1 kissing loop RNA dimer.     

Role of the Environment in the Interaction of Nonintercalators with Z-DNA

Abstract

Interaction of the DNA binding nonintercalators Netropsin, Distamycin and the mPD derivative with Z-DNA has been studied. It has been found that environmental factors like the solvent and added cations significantly modulate the interaction of these ligands with ZDNA However no definite Z to B transition in presence of these ligands was found in any case, in contrast to previously reported results (Ch.Zimmer, C.Marek and W.Guschlbauer, FEBS Lett. 154, 156–160(1983)).     

Characterization of free fatty acid receptors expression in an obesity rat model with high sucrose diet

Introduction/aims: In recent years, it has been shown that free fatty acids receptors (FFAR) of whose function in the cell surface plays a significant role in the regulation of cell function and nutrition as well are activated by various endogenous ligands, but mainly by fatty acids. Within FFAR of our interest are GPR 41, 43 and 120. The functions of these receptors are varied and dependent on the tissue where they are. The activation and signaling of these receptors, FFAR, are involved in many physiological processes, and currently the target of many drugs in metabolic disorders like obesity, diabetes and atherosclerosis.

Material and methods: Obesity was induced with hypercaloric diet (HD) in male Wistar rats for 20?weeks (n?=?10). At the end, adipose tissue (abdominal and subcutaneous) was taken to perform assays for relative quantification mRNA expression by end-point RT-PCR and protein level expression by Western blot.

Results: These present data have shown for the first time that total mRNA isolation and protein expression from both adipose tissues (abdominal and subcutaneous) of rat in obesity condition yield significative statistical difference among the control versus obese groups, showing that the diet high in carbohydrates modifies the total presence of mRNA and protein level expression of the receptors GPR41, 43 and 120.

Conclusions: Further comparative methods are in process to clarify whether or not the obesity changes the functional receptors in these two tissues for new pharmacological approaches.     

A Method for Dissecting Two Site Receptor Interactions in Ligand Binding Studies

Abstract

A general model has been developed describing the relationship between the measured (IC₅₀) and absolute affinities (K_I), observed in radioligand binding studies when two ligands, one radioactive, interact with two receptors or binding sites. The model shows the dependence of the IC₅₀'s upon the concentration of radioligand for any combinations of the absolute affinities of the radioligand (K_d's) and the displacing ligand (K_I's). By constraining the affinities of the two ligands for the sites, five special cases of the general model can be described that model all possible 'selectivities' the ligands may have for the sites. The properties of these five cases can be exploited experimentally to probe the nature of the ligand/site interactions by the simple expedient of constructing a number of displacement curves at different radioligand concentrations. The method has been tested experimentally in three situations where two ligand/two site interactions occur, and is shown to be a useful technique to qualitatively examine the underlying binding reactions.     

Revealing binding selectivity of ligands toward murine double minute 2 and murine double minute X based on molecular dynamics simulations and binding free energy calculations

Abstract

It is well known that the interactions of p53 with murine double minute 2 and murine double minute X, namely MDM2 and MDMX, have been significant targets of efficient anti-cancer drug design. In this study, molecular dynamics (MD) simulations, principal component (PC) analysis and binding free energy calculations are combined to recognize binding selectivity of three ligands to MDM2 and MDMX. The binding free energies were estimated by using molecular mechanics generalized Born surface area (MM-GBSA) method and the obtained results display that the increase in the binding enthalpy of three ligands to MDM2 relative to MDMX mainly drives the binding selectivity of them toward MDM2 and MDMX. The information obtained from PC analysis shows that the associations of ligands exert important impacts on internal dynamics of MDM2 and MDMX. Meanwhile, the calculations of residue-based free energy decomposition not only identify the hot interaction spots of ligands with MDM2 and MDMX, but also show the residues (L54, M53), (Y67, Y66), (V93, V92), (H96, P95), (I99, I98) and (Y100, Y99) in (MDM2, MDMX) are responsible for most contributions to the binding selectivity of three ligands toward MDM2 and MDMX. It is believed that this work can provide useful information for design of highly selective and dual inhibitors targeting MDM2 and MDMX.

Communicated by Ramaswamy H. Sarma     

Prediction,docking study and molecular simulation of 3D DNA aptamers to their targets of endocrine disrupting chemicals

Abstract

Typical endocrine disrupting chemicals, including BPA (Bisphenol A), E₂ (17-β-Estradiol) and PCB 72 (polychlorinated biphenyl 72), are commonly and widely present in the environment with good chemical stability that are difficult to decompose in vitro and in vivo. Most of the high-qualified antibodies are required as the key biomaterials to fabricate the immunosensor for capturing and detecting. As an ideal alternative, the short-chain oligonucleotides (aptamer) are essentially and effectively employed with the advantages of small size, chemical stability and high effectiveness for monitoring these environmental contaminants. However, the molecular interaction, acting site and mode are still not well understood. In this work, we explored the binding features of the aptamers with their targeting ligands. The molecular dynamics simulations were performed on the aptamer–ligand complex systems. The stability of each simulation system was evaluated based on its root-mean-square deviation. The affinities of these proposed ligands and the predicted binding sites are analyzed. According to the binding energy analysis, the affinities between ligands and aptamers and the stability of the systems are BPA?>?PCB 72 >E₂. Trajectory analysis for these three complexes indicated that these three ligands were able to steadily bind with aptamers at docking site from 0 to 50?ns and contributed to alteration of conformation of aptamers.     

Impacts of hydrophobicity and ionicity of phendione-based cobalt(II)/(III) complexes on binding with bovine serum albumin

Abstract

For efficient designing of metallodrugs, it is imperative to analyse the binding affinity of those drugs with drug-carrying serum albumins to comprehend their structure–activity correlation for biomedical applications. Here, cobalt(II) and cobalt(III) complexes comprising three phendione ligands, [Co(phendione)₃]Cl₂ (1) and [Co(phendione)₃]Cl₃ (2), where, phendione = 1,10-phenanthroline-5,6-dione, has been chosen to contrast the impact of their hydrophobicity and ionicity on binding with bovine serum albumin (BSA) through spectrophotometric titrations. The attained hydrophobicity values using octanol/water partition coefficient method manifested that complex 1 is more hydrophobic than complex 2, which could be attributed to lesser charge on its coordination sphere. The interaction of complexes 1 and 2 with BSA using steady state fluorescence studies revealed that these complexes quench the intrinsic fluorescence of BSA through static mechanism, and the extent of quenching and binding parameters are higher for complex 2. Further thermodynamics of BSA-binding studies revealed that complexes 1 and 2 interact with BSA through hydrophobic and hydrogen bonding/van der Waals interactions, respectively. Further, UV–visible absorption, circular dichroism and synchronous fluorescence studies confirmed the occurrence of conformational and microenvironmental changes in BSA upon binding with complexes 1 and 2. Molecular docking studies have also shown that complex 2 has a higher binding affinity towards BSA as compared to complex 1. This sort of modification of ionicity and hydrophobicity of metal complexes for getting desirable binding mode/strength with drug transporting serum albumins will be a promising pathway for designing active and new kind of metallodrugs for various biomedical applications.

Communicated by Ramaswamy H. Sarma     

Ligand Binding by Fibroblast Growth Factor Receptors Investigated Using Chimeric Receptor Molecules

Abstract

In order to map in detail the ligand binding sites of fibroblast growth factor receptor 2 (FGFR2) and keratinocyte growth factor receptor (KGFR), we have generated receptor molecules that are chimeric within the membrane proximal sequence that varies between them. The chimeric molecules are found to bind aFGF with a greater than 5-fold difference in affinity, indicating that there is coupling between the chimeric regions with respect to aFGF binding. Further, binding of bFGF and KGF is abolished in the chimeras, showing that the binding site for these ligands requires the whole of the 48- or 50- amino acid variable sequence to be intact. Direct interactions between the different regions exchanged in the chimeras are most probably involved in forming KGF or bFGF binding sites.     

The IN-VIVO Occurrence of Z DNA

Abstract

The energetics of the B-Z transition of two different types of cloned alternating purine/pyrimidine DNA sequences have been analysed by a two dimensional electrophoretic technique. Since the transition between right handed and left handed forms of these polymers is detected by alterations of electrophoretic mobilities of topoisomers of the plasmid DNA molecules, the method is not dependent on Z-DNA binding ligands. The measurements reflect intrinsic properties of the DNA unperturbed by the free energy of binding such a ligand.

Direct evidence from the analysis of topoisomer distributions is presented which shows that d(GC)n.d(GC)n sequence elements within an E. coli plasmid will adopt a Z conformation in-vivo under conditions of blocked protein synthesis. Evidence for the in-vivo occurrence of Z-DNA was not detected in plasmid DNA isolated from bacterial cells growing in the absence of protein synthesis inhibitors.

A model is proposed for a function for the B-Z transition in ensuring the correct pairing of homologous chromosomes during meiosis.     

Multiple Interactions of Unsaturated Fatty Acids with Opiate and Ouabain Binding Sites and β-Adrenergic Sensitive Adenylate Cyclase System

Abstract

The unsaturated fatty acids oleic, linoleic and arachidonic inhibited binding of ligands to the ouabain, opiate, and β-adrenergic plasma membrane receptors. Low concentrations of fatty acids slightly increased the binding of ouabain to its binding sites. The effect of these fatty acids on β-adrenergic sensitive adenylate cyclase was more complex. 0.2–0.3 mM fatty acids increased adenylate cyclase activity, while higher concentrations of arachidonic and linoleic acids, but not oleic acid     

Quantitative Receptor Autoradiography: Application to the Characterization of Multiple Receptor Subtypes

Abstract

In vitro autoradiographic techniques combined with computer assisted microdensitometry were used to analyze the characteristics and distribution of multiple recognition sites for the neurotransmitters acetylcholine (M₁ and M₂) and serotonin (5-HT_1A and 5-HT_1B). For this purpose, binding competition experiments were performed using non-subtype selective ³H-labeled ligands and selective unlabeled compounds. Consecutive tissue sections were incubated in the presence of increasing concentrations of displacers. By using this approach, maximal densities of binding sites, as well as competition profiles of several drugs could be analyzed and quantified in microscopic brain areas. Our results reveal the presence of brain structures enriched in one class of muscarinic or serotonergic-1 recognition sites. This provides a tool for better characterization of the proposed “subtype-selective” ligands and suggests physiological functions for these receptor subtypes. It is concluded that quantitative autoradiographic techniques provide a level of anatomical and pharmacological information on neurotransmitter receptor subtypes, which is difficult to attain using membrane binding studies.     

Interactions of Trimeric Purine Nucleoside Phosphorylases with Ground State Analogues—Calorimetric and Fluorimetric Studies

Abstract

Binding enthalpies, dissociation constants and stoichiometry of binding for interaction of trimeric calf spleen and Cellulomonas sp. purine nucleoside phosphorylases with their ground state analogues (substrates and inhibitors) were studied by calorimetric and spectrofluorimetric methods. Data for all ligands, with possible exception of hypoxanthine, are consistent with three identical non-interacting binding sites.