Ligand-selective inhibition of the interaction of steroid receptor coactivators and estrogen receptor isoforms

Ligand-dependent nuclear hormone receptor (NR) signaling requires direct interaction between NR and the steroid receptor coactivators (SRC). Herein we utilize a library of SRC2 peptidomimetics to select for specific inhibitors of the interaction of SRC2 with the two estrogen receptor (ER) isoforms, ERalpha and ERbeta, in the presence of three different ligands: 17beta-estradiol, diethylstilbesterol, and genistein. The pattern of inhibitor selectivity for each ER isoform varied depending upon which ligand was present, thus demonstrating that the ligands exert unique allosteric effects upon the surface of the SRC binding pocket. Several of the lead compounds are highly (>100-fold) selective for blocking the binding of SRC2 to ERalpha, in preference to ERbeta, in the presence of one ligand and therefore may prove useful for decoupling ERbeta signaling from ERalpha signaling.     

New computational strategy to analyze the interactions of ERalpha and ERbeta with different ERE sequences

The importance of computational methods for the simulation and analysis of biological systems has increased during the last years. In particular, methods to predict binding energies are developing not only with the aim of ranking the affinities between two or more complexes, but also to quantify the contribution of different types of interaction. In this work, we present the application of HINT, a non Newtonian force field, to rank the affinities of complexes formed by estrogen receptors (ER) alpha and beta and different estrogen responsive elements (ERE) near the estrogen-regulated genes. We used the crystallographic coordinates of the DNA binding domain of ERalpha complexed to a consensus ERE as a starting point to simulate several complexes in which some nucleotides in the ERE sequence were mutated. Moreover, we used homology modeling methods to create the structure of the complexes between the DNA binding domain of ERbeta (for which no experimental structures are currently available) and the same ERE sequences. Our results show that HINT is able to rank the affinities of ERalpha and ERbeta for different ERE sequences, and to correctly identify the positions on the DNA sequence that are most important for binding affinity. Moreover, the HINT output gives us the opportunity to identify and quantify the role played by each single atom of amino acids and nucleotides in the binding event, as well as to predict the effect on the binding affinity for other nucleotide mutations.     

Anti-estrogenic activity of mansorins and mansonones from the heartwood of Mansonia gagei DRUMM

Through an anti-estrogenic bioassay-guided fractionation of the methanol extract of Mansonia gagei, three new coumarins, called mansorins I (1), II (2) and III (3) and a new naphthoquinone, mansonone I (4), were isolated. Their structures were determined based on their NMR data and CD spectroscopy. The anti-estrogenic activity of the fractions and the isolated compounds were investigated using a yeast two-hybrid assay method expressing estrogen receptors alpha (ERalpha) and beta (ERbeta). In addition, an ERalpha competitor screening system (ligand binding screen) was used to verify the binding affinities of the isolated compounds to the estrogen receptor. 1,2-Naphthoquinones (mansonones) showed more binding affinities to ER in both assay systems. All the tested compounds showed higher binding affinities to ERbeta than to ERalpha in the yeast two-hybrid assay. Mansonones F and S showed the most potent estrogen binding and estrogen antagonistic effects.     

Synthesis of novel nitro-substituted triaryl pyrazole derivatives as potential estrogen receptor ligands

Novel tetrasubstituted pyrazole derivatives bearing a nitro substituent on their A-phenol ring were synthesized and their binding affinity towards the estrogen receptor (ER) subtypes ERalpha and ERbeta was determined. Among compounds tested, the 2-nitrophenol derivative 5c was found to bind satisfactorily to both estrogen receptor subtypes (RBAalpha=5.17 and RBAbeta=3.27). In general, the introduction of a nitro group into the A ring of these compounds was found to benefit their ERbeta binding abilities.     

Subtype selectivity of dopamine receptor ligands: insights from structure and ligand-based methods

Subtype selective dopamine receptor ligands have long been sought after as therapeutic and/or imaging agents for the treatment and monitoring of neurologic disorders. We report herein on a combined structure- and ligand-based approach to explore the molecular mechanism of the subtype selectivity for a large class of D?-like dopamine receptor ligands (163 ligands in total). Homology models were built for both human D(?L) and D? receptors in complex with haloperidol. Other ligands, which included multiple examples of substituted phenylpiperazines, were aligned against the binding conformations of haloperidol, and three-dimensional quantitative structure activity relationship (3D-QSAR) analyses were carried out. The receptor models show that although D? and D? share highly similar folds and 3D conformations, the slight sequence differences at their extracellular loop regions result in the binding cavity in D? being comparably shallower than in D?, which may explain why some larger ligands bind with greater affinity at D? compared to D? receptors. The QSAR models show excellent correlation and high predictive power even when evaluated by the most stringent criteria. They confirm that the origins of subtype selectivity for the ligands arise primarily due to differences in the contours of the two binding sites. The predictive models suggest that while both steric and electrostatic interactions contribute to the compounds' binding affinity, the major contribution arises from hydrophobic interactions, with hydrogen bonding conferring binding specificity. The current work provides clues for the development of more subtype selective dopamine receptor ligands. Furthermore, it demonstrates the possibility of being able to apply similar modeling methods to other subtypes or classes of receptors to study GPCR receptor-ligand interactions at a molecular level.     

Supraphos: a supramolecular strategy to prepare bidentate ligands

We report a new strategy for the preparation of chelating bidentate ligands, which involves just the mixing of two monodentate ligands functionalized with complementary binding sites. In the current example, the assembly process is based on selective metal-ligand interactions, using phosphite zinc(II) porphyrins 1-6 and the nitrogen donor ligands b-i. From only 16 monodentate ligands, a library of 60 palladium catalysts based on 48 bidentate ligand assemblies has been prepared. The relatively small catalyst library gave a large variety in the selectivity of the alkylation of rac-1,3-diphenyl-2-propenyl acetate. Importantly, small variations in the building blocks lead to large differences in the enantioselectivity imposed by the catalyst (up to 97% ee).     

Use of 13C NMR spectrometric data to produce a predictive model of estrogen receptor binding activity

We have developed a spectroscopic data-activity relationship (SDAR) model based on 13C NMR spectral data for 30 estrogenic chemicals whose relative binding affinities (RBA) are available for the alpha (ERalpha) and beta (ERbeta) estrogen receptors. The SDAR models segregated the 30 compounds into strong and medium binding affinities. The SDAR model gave a leave-one-out (LOO) cross-validation of 90%. Two compounds that were classified incorrectly in the SDAR model were in the transition zone between classifications. Real and predicted 13C NMR chemical shifts were used with test compounds to evaluate the predictive behavior of the SDAR model. The 13C NMR SDAR model using predicted 13C NMR data for the test compounds provides a rapid, reliable, and simple way to screen whether a compound binds to the estrogen receptors.     

A computational tool to optimize ligand selectivity between two similar biomacromolecular targets

Summary Algorithms for a new computer program designed to increase ligand--receptor selectivity between two proteins are described. In this program ligand--receptor selectivity is increased by functional modifications to the ligand so as to increase the calculated binding affinity of it to one protein and/or decrease the calculated binding affinity of it to the other protein. The structure of the ligand is modified by selective replacement of atoms and/or functional groups in silico based on a specific set of steric and/or hydropathic complementarity rules involving atoms and functional groups. Relative binding scores are calculated with simple grid-based steric penalty, hydrogen bond complementarity, and with the HINT score model. Two examples are shown. First, modifying the structure of the ligand CB3717 is illustrated in a number of ways such that the binding selectivity to wild type L. casei thymidylate synthase or its E60Q mutant may be improved. Second, starting with a non-selective lead compound that had been co-crystallized with both plant and mammalian 4-hydroxyphenylpyruvate dioxygenases, new compounds (similar to selective ligands discovered by screening) to improve the selectivity of (herbicidal) inhibitors for the plant enzyme were designed by the program.     

Efficient Separation of Light Lanthanides(III) by Using Bis-Lactam Phenanthroline Ligands

Due to the ever-increasing demand for high-purity individual rare-earth elements, novel and highly selective separation processes are increasingly sought after. Herein, we report a separation protocol that employs shape-persistent 2,9-bis-lactam-1,10-phenanthroline (BLPhen) ligands exhibiting unparalleled selectivity for light trivalent lanthanides. The highly preorganised binding pockets of the ligands allowed for the separation of lanthanides with high fidelity, even in the presence of competing transition metals, in a biphasic separation system. Notably, the selectivity trends of the BLPhen ligands towards metal ions across the lanthanide series can be chemically modulated by altering the molecular rigidity of the extractant.     

Evaluation of binding of perylene diimide and benzannulated perylene diimide ligands to dna by electrospray ionization mass spectrometry

Electrospray ionization mass spectrometry (ESI-MS) and spectroscopic studies in solution were used to evaluate the self-association, G-quadruplex DNA binding, and selectivity of a series of perylene diimides (PDIs) (PIPER, Tel01, Tel11, Tel12, and Tel18) or benzannulated perylene diimide ligands (Tel34 and Tel32). Fluorescence and resonance light scattering spectra of Tel01, Tel12, Tel32, and Tel34 reveal that these analogs undergo self-association in solution. UV-Vis and fluorescence titrations with G-quadruplex, duplex, or single-stranded DNA demonstrate that all the analogs, with the exception of Tel32, bind to G-quadruplex DNA, with those PDIs that are self-associated in solution showing the highest degree of selectivity for binding G-quadruplex DNA. Parallel ESI-MS analysis of the stoichiometries demonstrates the ability of the ligands, with the exception of Tel32, to bind to G-quadruplex DNA. While most ligands show major 1:1 and 2:1 binding stoichiometries as expected in the case of end-stacking, interestingly, three of the most quadruplex-selective ligands show a different behavior. Tel01 forms 3:1 complexes, while Tel12 and Tel32 only form 1:1 complexes. Collisional activation dissociation patterns are compatible with ligand binding to G-quadruplex DNA via stacking on the ends of the terminal G-tetrads. Experiments with duplex and single strand DNA were performed to assess the binding selectivities of the ligands. PIPER, Tel11, and Tel18 demonstrated extensive complexation with duplex DNA, while Tel11 and Tel18 bound to single strand DNA, confirming the lack of selectivity of these two ligands. Our results indicate that Tel01, Tel12, and Tel34 are the most selective for G-quadruplex DNA.     

Electrophoretic mobility shift assay on poly(ethylene glycol)-modified glass microchips for the study of estrogen responsive element binding

The binding of estrogen receptor (ER) to estrogen response element (ERE) is essential for genomic pathways of estrogens and gel-based electrophoretic mobility shift assay (EMSA) is commonly used for analyzing ERE binding. Gel-based EMSA, however, requires the use of hazard radio isotopes and they are slow, labor-intensive and difficult to quantify. Here, we present quantitative affinity assays based on microchip electrophoresis using PEG-modified glass microchannels, which bear neutral surfaces against the adsorption of acidic DNA molecules and basic ER proteins. We first demonstrated the feasibility of the method by measuring binding constants of recombinant ERalpha and ERbeta with a consensus ERE sequence (cERE, 5'-GGTCAGAGTGACC-3') as well as with an ERE-like sequence (ERE 1576, 5'-GACCGGTCAGCGGACTCAC-3'). Changes in mobility as a function of protein-DNA molar ratios were plotted and the dissociation constants were determined based on non-linear curve fitting. The minimum amount of ER proteins required for one assay was around 0.2 ng and the run time for one chip analysis was less than 2 min. We further measured the estrogenic compound-mediated dissociation constants with recombinant ER proteins as well as with the extracted ERbeta from treated and untreated A549 bronchioloalveolar carcinoma cells. Dissociation constants determined by this method agree with the fact that agonist compounds such as 17beta-estradiol (1.70 nM), diethylstilbestrol (0.14 nM), and genistein (0.80 nM) assist ERE binding by decreasing the constants; while antagonist compounds such as testosterone (140.4 nM) and 4-hydroxytamoxifen (10.5 nM) suppress the binding by increasing the dissociation constant.     

Photo‐Cross‐Linking Probes for Trapping G‐Quadruplex DNA

We have developed a straightforward synthetic pathway to a set of six photoactivatable G‐quadruplex ligands with a validated G4‐binding motif (the bisquinolinium pyridodicarboxamide PDC‐360A) tethered through various spacers to two different photo‐cross‐linking groups: benzophenone and an aryl azide. The high quadruplex‐versus‐duplex selectivity of the PDC core was retained in the new derivatives and resulted in selective alkylation of two well‐known G‐quadruplexes (human telomeric G4 and oncogene promoter c‐myc G4) under conditions of harsh competition. The presence of two structurally different photoactivatable functions allowed the selective alkylation of G‐quadruplex structures at specific nucleobases and irreversible G4 binding. The topology and sequence of the quadruplex matrix appear to influence strongly the alkylation profile, which differs for the telomeric and c‐myc quadruplexes. The new compounds are photoactive in cells and thus provide new tools for studying G4 biology.     

The selective mono and difunctionalization of carbocyclic cleft molecules with pyridyl groups and X-ray crystallographic analysis

The diesterification and selective mono and dialkylation of carbocyclic analogues of Tröger’s base with pyridyl groups has been achieved in high yield and good selectivity giving access to a novel range of cleft molecules capable of binding events. Reaction conditions for the selective functionalization of this carbocyclic cleft molecule are discussed as well as the solid state structures of these newly synthesized ligands.     

Linkage effects on binding affinity and activation of GPR30 and estrogen receptors ERalpha/beta with tridentate pyridin-2-yl hydrazine tricarbonyl-Re/(99m)Tc(I) chelates

We describe a new structural class of neutral tridentate pyridin-2-yl hydrazine chelates for labeling with tricarbonyl Re/99mTc(I) under aqueous conditions and investigate the receptor binding of synthetic estradiol derivatives with the novel G-protein-coupled receptor GPR30 and estrogen receptors ERalpha/beta. The steroid linkage affected the affinity and selectivity of estrogen binding with these receptors. Fluorescence assays based on calcium signaling demonstrate that membrane-permeable chelates 2 and 3 interact with the receptors in whole cells. These results suggest that in vitro assays will facilitate the development of targeted imaging agents for intracellular receptors and the feasibility of targeting GPR30 and ERalpha/beta for diagnostic tumor imaging.     

Study of binding affinity and selectivity of perylene and coronene derivatives towards duplex and quadruplex DNA by ESI‐MS

In this paper, we report an extensive electrospray ionization mass spectrometry (ESI‐MS) study of the noncovalent interactions between different intermolecular and intramolecular G‐quadruplex structures and several perylene and coronene ligands. The selectivity of these compounds toward quadruplex structures with respect to duplex DNA, a fundamental topic for the biological evaluation and the pharmacological application of these ligands as potential chemotherapeutic agents, has also been investigated. After exploring this topic according to the classical approach based on the very simple duplex model of an autocomplementary dodecamer, we extended our analysis reporting for the first time a competition ESI‐MS experiment in the presence of genomic DNA fragments. Whereas those ligands showing a high level of selectivity between quadruplex and duplex oligonucleotides, in terms of binding constants and percentage of bound DNA, confirmed their selectivity in the competition experiment, the contrary was not always true: some ligands showing poor selectivity with the autocomplementary dodecamer resulted selective in the presence of genomic DNA fragments. This result suggests that physiologically nonrelevant interactions are possible with a short duplex oligonucleotide. This means that the dodecamer can fail in representing a biologically significant structural model, or, better, that it can be used to quickly screen potentially selective molecules, but bearing in mind the high probability of false negative results. Copyright © 2008 John Wiley & Sons, Ltd.     

αvβ3‐ or α5β1‐Integrin‐Selective Peptidomimetics for Surface Coating

Engineering biomaterials with integrin‐binding activity is a very powerful approach to promote cell adhesion, modulate cell behavior, and induce specific biological responses at the surface level. The aim of this Review is to illustrate the evolution of surface‐coating molecules in this field: from peptides and proteins with relatively low integrin‐binding activity and receptor selectivity to highly active and selective peptidomimetic ligands. In particular, we will bring into focus the difficult challenge of achieving selectivity between the two closely related integrin subtypes αvβ3 and α5β1. The functionalization of surfaces with such peptidomimetics opens the way for a new generation of highly specific cell‐instructive surfaces to dissect the biological role of integrin subtypes and for application in tissue engineering and regenerative medicine.     

Identification of Compounds that Selectively Stabilize Specific G‐Quadruplex Structures by Using a Thioflavin T‐Displacement Assay as a Tool

Small molecules are used in the G‐quadruplex (G4) research field in vivo and in vitro, and there are increasing demands for ligands that selectively stabilize different G4 structures. Thioflavin T (ThT) emits an enhanced fluorescence signal when binding to G4 structures. Herein, we show that ThT can be competitively displaced by the binding of small molecules to G4 structures and develop a ThT‐displacement high‐throughput screening assay to find novel and selective G4‐binding compounds. We screened approximately 28 000 compounds by using three different G4 structures and identified eight novel G4 binders. Analysis of the structural conformation and stability of the G4 structures in presence of these compounds demonstrated that the four compounds enhance the thermal stabilization of the structures without affecting their structural conformation. In addition, all four compounds also increased the G4‐structure block of DNA synthesis by Taq DNA polymerase. Also, two of these compounds showed selectivity between certain Schizosaccharomyces pombe G4 structures, thus suggesting that these compounds or their analogues can be used as selective tools for G4 DNA studies.