Selectivity fields: comparative molecular field analysis (CoMFA) of the glycine/NMDA and AMPA receptors

An approach for evaluation of binding selectivity was suggested and exemplified using glycine/NMDA and AMPA receptors. For analyzing the pairwise selectivity, we propose to use the difference between biological activities (expressed as -log Ki) of ligands with respect to different receptor subtypes as a dependent variable for building comparative molecular field analysis (CoMFA) models. The resulting fields (which will be referred to as the "selectivity fields") indicate the ways of increasing selectivity of binding, inhibition, etc. As an example, CoMFA of a set of pyrazolo[1,5-c]quinazolines and triazolo[1,5-c]quinazolines was used for considering the binding selectivity with respect to glycine/NMDA and AMPA receptors. In addition, the mapping of these fields onto the molecular models of the corresponding receptors makes it possible to reveal the reasons for experimentally observed selectivity as well as to suggest additional ways of increasing selectivity.

Artificial receptors for the extraction of nucleoside metabolite 7-methylguanosine from aqueous media made by molecular imprinting

A series of imprinted polymers targeting nucleoside metabolites, prepared using a template analogue approach, are presented. These were prepared following selection of the optimum functional monomer by solution association studies using 1H-NMR titrations whereby methacrylic acid was shown to be the strongest receptor with and affinity constant of 621 ± 51 L mol-1 vs. 110 ± 16 L mol-1 for acrylamide. The best performing polymers were prepared using methanol as porogenic co-solvent and although average binding site affinities were marginally reduced, 2.3×104 L mol-1 vs. 2.7×104 L mol-1 measured for a polymer prepared in acetonitrile, these polymers contained the highest number of binding sites, 5.27 μmol g-1¬¬ vs. 1.64 μmol g-1, while they also exhibited enhanced selectivity for methylated guanosine derivatives. When applied as sorbents in the extraction of nucleoside derivative cancer biomarkers from synthetic urine samples, significant sample clean-up and recoveries of up to 90% for 7-methylguanosine were achieved.

Molecular characterization and phenotypic expression of mutations in genes for gonadotropins and their receptors in humans.

Gonadotropin hormones undergo important dynamic changes during life. Their rise during puberty stimulates gonadal steroid secretion, triggering the development of secondary sexual characteristics and the acquisition of fertility. The full spectrum of possible mutations and polymorphisms in the human gonadotropins and in their receptor genes has been described in recent years. Patients harboring these mutations display a very wide range of phenotypes affecting all aspects of the reproductive axis. An important insight provided by the careful study of these patients lies in the striking gender differences in the phenotypes associated with a given mutation. As a result, the careful study of these rare patients has allowed us to better define the respective roles of luteinizing hormone and follicle-stimulating hormone in normal human pubertal development and in the achievement of full fertility potential in either males or females. In this work, we describe briefly the known mutations in the genes for both gonadotropins and their receptors, and discuss their genotype/phenotype correlations in light of these important gender differences.

Functional and molecular characterization of Tachykinins and tachykinin receptors in the mouse uterus

The aim of this study was to analyze the function and expression of tachykinins, tachykinin receptors, and neprilysin (NEP) in the mouse uterus. A previous study showed that the uterotonic effects of substance P (SP), neurokinin A (NKA), and neurokinin B (NKB) in estrogen-treated mice were mainly mediated by the tachykinin NK, receptor. In the present work, further contractility studies were undertaken to determine the nature of the receptors mediating responses to tachykinins in uteri of late pregnant mice. Endpoint and real-time quantitative RTPCR were used to analyze the expression of the genes that encode the tachykinins SP/NKA, NKB, and hemokinin-1 (HK-1) (Tac1, Tac2, and Tac4); and the genes that encode tachykinin NK1 (Tacr1), NK2 (Tacr2), and NK3 (Tacr3) receptors in uteri from pregnant and nonpregnant mice. The data show that the mRNAs of tachykinins (particularly NKB and HK-1), tachykinin receptors, and NEP are locally expressed in the mouse uterus, and their expression changes during the estrous cycle and during pregnancy. The tachykinin INK, receptor is the predominant tachykinin receptor in the nonpregnant and early pregnant mouse and may mediate tachykinin-induced uterine contractions in the nonpregnant mouse. The tachykinin NK, receptor is predominant in the late pregnant mouse and is the main receptor mediating uterotonic responses to tachykinins at late pregnancy. The tachykinin NK, receptor is expressed in considerable amounts only in uteri from nonpregnant diestrous animals, and its physiological significance remains to be clarified.

Only subtle protein conformational adaptations are required for ligand binding to thyroid hormone receptors: Simulations using a novel multipoint steered molecular dynamics approach

Thyroid hormone receptors (TR) are hormone-dependent transcription regulators that play a major role in human health, development, and metabolic functions. The thyroid hormone resistance syndrome, diabetes, obesity, and some types of cancer are just a few examples of important diseases that are related to TR malfunctioning, particularly impaired hormone binding. Ligand binding to and dissociation from the receptor ultimately control gene transcription and, thus, detailed knowledge of binding and release mechanisms are fundamental for the comprehension of the receptor`s biological function and development of pharmaceuticals. In this work, we present the first computational study of ligand entry into the ligand binding domain (LBD) of a nuclear receptor. We report molecular dynamics simulations of ligand binding to TRs using a generalization of the steered molecular dynamics technique designed to perform single-molecule pulling simulations along arbitrarily nonlinear driving pathways. We show that only gentle protein movements and conformational adaptations are required for ligand entry into the LBDs and that the magnitude of the forces applied to assist ligand binding are of the order of the forces involved in ligand dissociation. Our simulations suggest an alternative view for the mechanisms ligand binding and dissociation of ligands from nuclear receptors in which ligands can simply diffuse through the protein surface to reach proper positioning within the binding pocket. The proposed picture indicates that the large-amplitude protein motions suggested by the apo- and holo-RXR alpha crystallographic structures are not required, reconciling conformational changes of LBDs required for ligand entry with other nuclear receptors apo-structures that resemble the ligand-bound LBDs.

Molecular orbital calculation for the model compounds of kainoid amino acids, agonists of excitatory amino acid receptors. Does the kainoid C4-substituent directly interact with the receptors?

Kainoid amino acids are agonists of the AMPA/kainate receptors and exhibit highly potent neuroexcitatory activity. From the results of extensive structure-activity relationship studies, we previously postulated that the C4-substituent of the kainoid amino acids interacts with an allosteric site of the glutamate receptor with electron-donating character. In order to investigate the mode of action in more detail, molecular orbital calculation for model compounds of the kainoid were performed. The results indicated that the HOMO energy level of the C4-substituent is involved in the potent neuroexcitatory activity, thus supporting our hypothesis. (C) 2002 Elsevier B.V. Ltd. All rights reserved.

Molecular analysis of the site for 2-arachidonylglycerol (2-AG) on the β 2 subunit of GABA A receptors

2-arachidonyl glycerol (2-AG) allosterically potentiates GABAA receptors via a binding site located in transmembrane segment M4 of the β2 subunit. Two amino acid residues have been described that are essential for this effect. With the aim to further describe this potential drug target, we performed a cysteine scanning of the entire M4 and part of M3. All four residues in M4 affecting the potentiation here and the two already identified residues locate to the same side of the α-helix. This side is exposed to M3, where further residues were identified. From the fact that the important residues span > 18 Å, we conclude that the hydrophobic tail of the bound 2-AG molecule must be near linear and that the site mainly locates to the inner leaflet but stretches far into the membrane. The influence of the structure of the head group of the ligand molecule on the activity of the molecule was also investigated. We present a model of 2-AG docked to the GABAA receptor.

Molecular basis of retinoid action: The role of retinoic acid receptors in retinoic acid-induced tissue transglutaminase expression

Retinoic acid has profound effects on the cellular growth and differentiation of a variety of cells. However, the molecular basis of retinoic acid action has, until recently, not been well understood. The identification of retinoic acid receptors which bear a high degree of homology to members of the steroid receptor super-family has dramatically altered our understanding of the biology of retinoids. The focus of this dissertation has been toward identification of retinoic acid binding proteins responsible for the effects of this molecule on gene expression.^ We have characterized in detail the retinoic acid-dependent induction of tissue transglutaminase gene expression in a myeloid cell line, human promyelocytic leukemia cells (HL-60 cells). Using cDNA probes specific for tissue transglutaminase, we have determined that the retinoic acid induced increase in enzyme level is due to an increase in the level of tissue transglutaminase mRNA. We have used this model as a probe to investigate the molecular basis of retinoid regulated gene expression.^ This thesis demonstrates that retinoic acid receptors are expressed in cells which induce tissue transglutaminase expression in response to retinoic acid. In Hl-60 cells retinoic acid-induced transglutaminase expression is associated with saturable nuclear retonic acid binding. Transcripts for both the alpha and beta forms of the retinoic acid receptors can be detected in these cells. Pretreatment of HL-60 cells with agents that potentiate retinoic acid-induced transglutaminase expression also modestly induced the alpha form of the retinoic acid receptor. Studies in macrophages and umbilical vein endothelial cells have also associated expression of the beta form of the retinoic acid with retinoic acid induced tissue transglutaminase expression.^ To investigate directly if retinoic acid receptors regulate retinoic acid-induced tissue transglutaminase expression we developed a series of stably transfected Balb-c 3T3 cells expressing different levels of the beta or gamma form of the retinoic acid receptor. These studies indicated that either the beta or gamma receptor can stimulate endogenous tissue transglutaminase expression in response to retinoic acid. These are among the first studies in the steroid field to describe regulation of an endogenous gene by a transfected receptor. ^

Development of small molecular tools for the cellular study of adenosine receptors

The adenosine receptors are members of the G-protein coupled receptor (GPCR) family which represents the largest class of cell-surface proteins mediating cellular communication. As a result, GPCRs are formidable drug targets and it is estimated that approximately 30% of the marketed drugs act through members of this receptor class. There are four known subtypes of adenosine receptors: A1, A2A, A2B and A3. The adenosine A1 receptor, which is the subject of this presentation, mediates the physiological effects of adenosine in various tissues including the brain, heart, kidney and adipocytes. In the brain for instance, its role in epilepsy and ischemia has been the focus of many studies. Previous attempts to study the biosynthesis, trafficking and agonist-induced internalisation of the adenosine A1 receptor in neurons using fluorescent protein-receptor fusion constructs have been hampered by the sheer size of the fluorescent protein (GFP) that ultimately affected the function of the receptor. We have therefore initiated a research programme to develop small molecule fluorescent agonists that selectively activate the adenosine A1 receptor. Our probe design is based on the endogenous ligand adenosine and the known unselective adenosine receptor agonist NECA. We have synthesised a small library of non-fluorescent adenosine derivatives that have different cyclic and bicyclic moieties at the 6 position of the purine ring and have evaluated the pharmacology of these compounds using a yeast-based assay. This analysis revealed compounds with interesting behaviour, i.e. exhibiting subtype-selectivity and biased signalling, that can be potentially used as tool compounds in their own right for cellular studies of the adenosine A1 receptor. Furthermore, we have also linked fluorescent dyes to the purine ring and discovered fluorescent compounds that can activate the adenosine A1 receptor.