A reactivity pattern for discrimination of ER agonism and antagonism based on 3-D molecular attributes

Various models have been developed to predict the relative binding affinity (RBA) of chemicals to estrogen receptors (ER). These models can be used to prioritize chemicals for further tiered biological testing to assess the potential for endocrine disruption. One shortcoming of models predicting RBA has been the inability to distinguish potential receptor antagonism from agonism, and hence in vivo response. It has been suggested that steroid receptor antagonists are less compact than agonists; thus, ER binding of antagonists may prohibit proper alignment of receptor protein helices preventing subsequent transactivation. The current study tests the theory of chemical bulk as a defining parameter of antagonism by employing a 3-D structural approach for development of reactivity patterns for ER antagonists and agonists. Using a dataset of 23 potent ER ligands (16 agonists, 7 antagonists), molecular parameters previously found to be associated with ER binding affinity, namely global ( E HOMO ) and local (donor delocalizabilities and charges) electron donating ability of electronegative sites and steric distances between those sites, were found insufficient to discriminate ER antagonists from agonists. However, parameters related to molecular bulk, including solvent accessible surface and negatively charged Van der Waal's surface, provided reactivity patterns that were 100% successful in discriminating antagonists from agonists in the limited data set tested. The model also shows potential to discriminate pure antagonists from partial agonist/antagonist structures. Using this exploratory model it is possible to predict additional chemicals for their ability to bind but inactivate the ER, providing a further tool for hypothesis testing to elucidate chemical structural characteristics associated with estrogenicity and anti-estrogenicity.     

Two model system of the alpha1A-adrenoceptor docked with selected ligands

In this study, we have developed a two model system to mimic the active and inactive states of a G-protein coupled receptor specifically the alpha1A adrenergic receptor. We have docked two agonists, epinephrine (phenylamine type) and oxymetazoline (imidazoline type), as well as two antagonists, prazosin and 5-methylurapidil, into two alpha1A receptor models, active and inactive. The best docking complexes for both agonists had hydrophilic interactions with D106, while neither antagonist did. Prazosin and oxymetazoline had hydrophobic interactions with F308 and F312. We predict from our study that the active state is stabilized by the interaction of F193 with I114, L197, V278, F281, and V282. The active state is further stabilized by the interaction of F312 with L75, V79, and L80. We also predict that the inactive state of the receptor is stabilized by the interaction of F312 with W102, F288, and M292.     

A reactivity pattern for discrimination of ER agonism and antagonism based on 3-D molecular attributes

Various models have been developed to predict the relative binding affinity (RBA) of chemicals to estrogen receptors (ER). These models can be used to prioritize chemicals for further tiered biological testing to assess the potential for endocrine disruption. One shortcoming of models predicting RBA has been the inability to distinguish potential receptor antagonism from agonism, and hence in vivo response. It has been suggested that steroid receptor antagonists are less compact than agonists; thus, ER binding of antagonists may prohibit proper alignment of receptor protein helices preventing subsequent transactivation. The current study tests the theory of chemical bulk as a defining parameter of antagonism by employing a 3-D structural approach for development of reactivity patterns for ER antagonists and agonists. Using a dataset of 23 potent ER ligands (16 agonists, 7 antagonists), molecular parameters previously found to be associated with ER binding affinity, namely global (E(HOMO)) and local (donor delocalizabilities and charges) electron donating ability of electronegative sites and steric distances between those sites, were found insufficient to discriminate ER antagonists from agonists. However, parameters related to molecular bulk, including solvent accessible surface and negatively charged Van der Waal's surface, provided reactivity patterns that were 100% successful in discriminating antagonists from agonists in the limited data set tested. The model also shows potential to discriminate pure antagonists from partial agonist/antagonist structures. Using this exploratory model it is possible to predict additional chemicals for their ability to bind but inactivate the ER, providing a further tool for hypothesis testing to elucidate chemical structural characteristics associated with estrogenicity and anti-estrogenicity.     

Molecular modelling studies on the ORL1-receptor and ORL1-agonists

The ORLI (opioid receptor like 1)- receptor is a member of the family of rhodopsin-like G protein-coupled receptors (GPCR) and represents an interesting new therapeutical target since it is involved in a variety of biomedical important processes, such as anxiety, nociception, feeding, and memory. In order to shed light on the molecular basis of the interactions of the GPCR with its ligands, the receptor protein and a dataset of specific agonists were examined using molecular modelling methods. For that purpose, the conformational space of a very potent non-peptide ORL1-receptor agonist (Ro 64-6198) with a small number of rotatable bonds was analysed in order to derive a pharmacophoric arrangement. The conformational analyses yielded a conformation that served as template for the superposition of a set of related analogues. Structural superposition was achieved by employing the program FlexS. Using the experimental binding data and the superposition of the ligands, a 3D-QSAR analysis applying the GRID/GOLPE method was carried out. After the ligand-based modelling approach, a 3D model of the ORL1-receptor has been constructed using homology modelling methods based on the crystal structure of bovine rhodopsin. A representative structure of the model taken from molecular dynamics simulations was used for a manual docking procedure. Asp-130 and Thr-305 within the ORL1-receptor model served as important hydrophilic interaction partners. Furthermore, a hydrophobic cavity was identified stabilizing the agonists within their binding site. The manual docking results were supported using FlexX, which identified the same protein-ligand interaction points.     

3-D QSAR for intrinsic activity of 5-HT1A receptor ligands by the method of comparative molecular field analysis

The affinity of a ligand for a receptor is usually expressed in terms of the dissociation constant (K_i) of the drug-receptor complex, conveniently measured by the inhibition of radioligand binding. However, a ligand can be an antagonist, a partial agonist, or a full agonist, a property largely independent of its receptor affinity. This property can be quantitated as intrinsic activity (1A), which can range from 0 for a full antagonist to 1 for a full agonist. Although quantitative structure–activity relationship (QSAR) methods have been applied to the prediction of receptor affinity with considerable success, the prediction of IA, even qualitatively, has rarely been attempted. Because most traditional QSAR methods are limited to congeneric series, and there are often major structural differences between agonists and antagonists, this lack of success in predicting IA is understandable. To overcome this limitation, we used the method of comparative molecular field analysis (CoMFA), which, unlike traditional Hansch analysis, permits the inclusion of structurally dissimilar compounds in a single QSAR model. A structurally diverse set of 5-hydroxytryptamine_1A (5-HT_1A) receptor ligands, with literature IA data (determined by the inhibition of 5-HT sensitive forskolin-stimulated adenylate cyclase), was used to develop a 3-D QSAR model correlating intrinsic activity with molecular structure properties of 5HT_1A receptor ligands. This CoMFA model had a crossvalidated r² of 0.481, five components and final conventional r² of 0.943. The receptor model suggests that agonist and antagonist ligands can share parts of a common binding site on the receptor, with a primary agonist binding region that is also occupied by antagonists and a secondary binding site accommodating the excess bulk present in the sidechains of many antagonists and partial agonists. The CoMFA steric field graph clearly shows that agonists tend to be “flatter” (more coplanar) than antagonists, consistent with the difference between the 5-HT_1A agonist and antagonist pharmacophores proposed by Hibert and coworkers. The CoMFA electrostatic field graph suggests that, in the region surrounding the essential protonated aliphatic amino group, the positive molecular electrostatic potential may be weaker in antagonists as compared to agonists. Together, the steric and electrostatic maps suggest that in the secondary binding site region increased hydrophobic binding may enhance antagonist activity. These results demonstrate that CoMFA is capable of generating a statistically crossvalidated 3-D QSAR model that can successfully distinguish between agonist and antagonist 5-HT_1A ligands. To the best of our knowledge, this is the first time this or any other QSAR method has been successfully applied to the correlation of structure with IA rather than potency or affinity. The analysis has suggested various structural features associated with agonist and antagonist behaviors of 5-HT_1A ligands and thus should assist in the future design of drugs that act via 5-HT_1A receptors. © 1993 John Wiley & Sons, Inc.     

神经介素B受体与拮抗剂、激动剂的分子模拟研究

大鼠神经介素B受体(rat neuromedin B receptor, rNMBR)属于G蛋白偶联受体(G-protein coupled receptor, GPCR) A家族的成员. GPCR的结构特征和在信号传导中的重要作用决定了其可以作为很好的药物靶标. 关于rNMBR与内源性激动剂神经介素B (neuromedin B, NMB)以及与非肽类拮抗剂pd168368作用机制的研究对于合理设计受体药物分子有重要的指导意义. 在这一研究中, 我们使用同源模建, 构建受体的三维结构, 进行分子对接和分子动力学的计算. 基于受体三维结构, 通过10 ns的空载受体、激动剂-受体、拮抗剂-受体的分子动力学模拟, 探讨受体与激动剂与拮抗剂的作用机制. 研究表明rNMB-R中跨膜(transmembrane, TM)螺旋3, 5, 6, 7参与配体的结合. NMB与受体的结合, 使受体转变为活性构象, 而受体同拮抗剂pd168368恰好相反.     

Binding mode similarity measures for ranking of docking poses: a case study on the adenosine A<Subscript>2A</Subscript> receptor

We report an investigation designed to explore alternative approaches for ranking of docking poses in the search for antagonists of the adenosine A_2A receptor, an attractive target for structure-based virtual screening. Calculation of 3D similarity of docking poses to crystallographic ligand(s) as well as similarity of receptor–ligand interaction patterns was consistently superior to conventional scoring functions for prioritizing antagonists over decoys. Moreover, the use of crystallographic antagonists and agonists, a core fragment of an antagonist, and a model of an agonist placed into the binding site of an antagonist-bound form of the receptor resulted in a significant early enrichment of antagonists in compound rankings. Taken together, these findings showed that the use of binding modes of agonists and/or antagonists, even if they were only approximate, for similarity assessment of docking poses or comparison of interaction patterns increased the odds of identifying new active compounds over conventional scoring.     

Binding of agonists and antagonists to muscarinic receptors.

The binding of one irreversible and two reversible radioactive antagonists to muscarinic receptors in synaptosome preparations of rat cerebral cortex has been studied. The ligands all bind to the same receptor pool and directly and competitively yield self-consistent binding constants closely similar to those obtained by pharmacological methods on intact smooth muscle. The binding process for antagonists seems to be a simple mass action-determined process with a Hill slope of 1.0. The quantitative correlations strongly support the view that the receptor studied by ligand binding corresponds to the receptor studied by pharmacological methods. Inhibition of antagonist binding by most agonists shows a reduced Hill slope which also applies to direct binding studies of [3H] acetylcholine. Mechansims that might account for the behavior of agonists are discussed but do not conclusively point to any single mechanism.     

利用肽库技术筛选成纤维细胞生长因子的短肽亲和配体

成纤维细胞生长因子(FGF)具有促进血管和神经形成的功能^[1],但它在体内作用过度则经常伴随着肿瘤的发生^[2].当前,研制和开发FGF拮抗剂,以有效地抑制FGF与细胞受体的结合,已成为国际性前沿课题.     

Rationally designed macrocyclic peptides as synergistic agonists of LPS-induced inflammatory response

Toll-like receptor 4 (TLR4) plays an important role in the regulation of the innate and adaptive immune response. Both agonists and antagonists of TLR4 are of considerable interest as drug leads for various disease indications. We herein report the rational design of two myeloid differentiation factor 2 (MD2)-derived macrocyclic peptides as TLR4 modulators, using the Rosetta Macromolecular Modeling software. The designed cyclic peptides, but not their linear counterparts, displayed synergistic activation of TLR signaling when co-administered with lipopolysaccharide (LPS). Although the understanding of the mechanism of action of these peptides remains elusive, these results underscore the utility of peptide cyclization for the discovery of biologically active agents, and also provide valuable tools for the investigation of TLR4 signaling.     

The effect of antagonists on the conformational exchange of the retinoid X receptor alpha ligand‐binding domain

The effect of retinoid X receptor (RXR) antagonists on the conformational exchange of the RXR ligand‐binding domain (LBD) remains poorly characterized. To address this question, we used nuclear magnetic resonance spectroscopy to compare the chemical shift perturbations induced by RXR antagonists and agonists on the RXRα LBD when partnered with itself as a homodimer and as the heterodimeric partner with the peroxisome proliferator‐activated receptor γ (PPARγ) LBD. Chemical shift mapping on the crystal structure showed that agonist binding abolished a line‐broadening effect caused by a conformational exchange on backbone amide signals for residues in helix H3 and other regions of either the homo‐ or hetero‐dimer, whereas binding of antagonists with similar binding affinities failed to do so. A lineshape analysis of a glucocorticoid receptor‐interacting protein 1 NR box 2 coactivator peptide showed that the antagonists enhanced peptide binding to the RXRα LBD homodimer, but to a lesser extent than that enhanced by the agonists. This was further supported by a lineshape analysis of the RXR C‐terminal residue, threonine 462 (T462) in the homodimer but not in the heterodimer. Contrary to the agonists, the antagonists failed to abolish a line‐broadening effect caused by a conformational exchange on the T462 signal corresponding to the RXRα LBD–antagonist–peptide ternary complex. These results suggest that the antagonists lack the ability of the agonists to shift the equilibrium of multiple RXRα LBD conformations in favor of a compact state, and that a PPARγ LBD‐agonist complex can prevent the antagonist from enhancing the RXRα LBD‐coactivator binding interaction. Copyright © 2009 John Wiley & Sons, Ltd.     

Modelling of the binding site of the human m1 muscarinic receptor: Experimental validation and refinement

Our model of the human m1 muscarinic receptor has been refined on the basis of the recently published projection map of bovine rhodopsin. The refined model has a slightly different helix arrangement, which reveals the presence of an extra hydrophobic pocket located between helices 3, 4 and 5. The interaction of series of agonists and antagonists with the m1 muscarinic receptor has been studied experimentally by site-directed mutagenesis. In order to account for the observed results, three-dimensional models of m1 ligands docked in the target receptor are proposed. Qualitatively, the obtained models are in good agreement with the experimental observations. Agonists and partial agonists have a relatively small size. They can bind to the same region of the receptor using, however, different anchoring receptor residues. Antagonists are usually larger molecules, filling almost completely the same pocket as agonists. They can usually produce much stronger interactions with aromatic residues. Experimental data combined with molecular modelling studies highlight how subtle and diverse receptor–ligand interactions could be.     

Mechanistic Studies on the Stereoselectivity of the Serotonin 5‐HT1A Receptor

G‐protein‐coupled receptors (GPCRs) are involved in a wide range of physiological processes, and they have attracted considerable attention as important targets for developing new medicines. A central and largely unresolved question in drug discovery, which is especially relevant to GPCRs, concerns ligand selectivity: Why do certain molecules act as activators (agonists) whereas others, with nearly identical structures, act as blockers (antagonists) of GPCRs? To address this question, we employed all‐atom, long‐timescale molecular dynamics simulations to investigate how two diastereomers (epimers) of dihydrofuroaporphine bind to the serotonin 5‐HT_1A receptor and exert opposite effects. By using molecular interaction fingerprints, we discovered that the agonist could mobilize nearby amino acid residues to act as molecular switches for the formation of a continuous water channel. In contrast, the antagonist epimer remained firmly stabilized in the binding pocket.     

New imide 5-HT1A receptor ligands - modification of terminal fragment geometry

Two sets of new o-methoxyphenylpiperazine (MPP; series a) and 1,2,3,4-tetrahydroisoquinoline (THIQ; series b) derivatives, containing various imide moieties derived from NAN190, were synthesized and evaluated in vitro for their ability to bind to the serotonin 5-HT(1A) and 5-HT(2A) receptors. All new derivatives from series a demonstrated high 5-HT(1A) affinities, whereas THIQ analogues were much less active. With respect to 5-HT(2A) receptors, three MPP derivatives presented moderate activity but the rest of the investigated compounds were practically inactive. The influence of changes in terminus geometry on 5-HT(1A) receptor affinity was analyzed in regard to model compounds NAN190and MM199.     

A computational model of the nicotinic acetylcholine binding site

We have derived a model of the nicotinic acetylcholine binding site. This was accomplished by using three known agonists (acetylcholine, nicotine and epibatidine) as templates around which polypeptide side chains, found to be part of the receptor cavity from published molecular biology studies, are allowed to flow freely in molecular dynamics simulations and mold themselves around these templates. The resulting supramolecular complex should thus be a complement, both in terms of steric effects as well as electronic effects, to the agonists and it should be a good estimation of the true receptor cavity structure. The shapes of those minireceptor cavities equilibrated rapidly on the simulation time scale and their structural congruence is very high, implying that a satisfactory model of the nicotinic acetylcholine binding site has been achieved. The computational methodology was internally tested against two rigid and specific antagonists (dihydro--erytroidine and erysoidine), that are expected to give rise to a somewhat differently shaped binding site compared to that derived from the agonists. Using these antagonists as templates there were structural reorganizations of the initial receptor cavities leading to distinctly different cavities compared to agonists. This indicates that adequate times and temperatures were used in our computational protocols to achieve equilibrium structures for the agonists. Overall, both minireceptor geometries for agonists and antagonists are similar with the exception of one amino acid (ARG209).