Characterisation and expression of β1-, β2- and β3-adrenergic receptors in the fathead minnow (Pimephales promelas)

This is the author’s version of a work that was accepted for publication in General and Comparative Endocrinology. Changes resulting from the publishing process, such as peer review, editing, corrections, structural formatting, and other quality control mechanisms may not be reflected in this document. Changes may have been made to this work since it was submitted for publication. A definitive version was subsequently published and may be accessed at the link below. Copyright © 2011 Elsevier B.V. All rights reserved.Complimentary DNAs for three beta-adrenergic receptors (βARs) were isolated and characterised in the fathead minnow. The encoded proteins of 402 (β(1)AR), 397 (β(2)AR) and 434 (β(3)AR) amino acids were homologous to other vertebrate βARs, and displayed the characteristic seven transmembrane helices of G Protein-coupled receptors. Motifs and amino acids shown to be important for ligand binding were conserved in the fathead minnow receptors. Quantitative RT-PCR revealed the expression of all receptors to be highest in the heart and lowest in the ovary. However, the β(1)AR was the predominant subtype in the heart (70%), and β(3)AR the predominant subtype in the ovary (53%). In the brain, β(1)AR expression was about 200-fold higher than that of β(2)- and β(3)AR, whereas in the liver, β(2)AR expression was about 20-fold and 100-fold higher than β(3)- and β(1)AR expression, respectively. Receptor gene expression was modulated by exposure to propranolol (0.001-1mg/L) for 21days, but not in a consistent, concentration-related manner. These results show that the fathead minnow has a beta-adrenergic receptor repertoire similar to that of mammals, with the molecular signatures required for ligand binding. An exogenous ligand, the beta-blocker propranolol, is able to alter the expression profile of these receptors, although the functional relevance of such changes remains to be determined. Characterisation of the molecular targets for beta-blockers in fish will aid informed environmental risk assessments of these drugs, which are known to be present in the aquatic environment.European Union as part of the ERAPharm project, Contract No. 511135 and NER

Theoretical study of the interaction of agonists with the 5-HT2A receptor

The 5-HT2A receptor (5-HT2AR) is a biogenic amine receptor that belongs to the class A of G protein coupled receptors. It is characterized by a low affinity for serotonin (5-HT) and for other primary amines. Introduction of an ortho-methoxybenzyl substituent at the amine nitrogen increases the partial agonistic activity by a factor of 40 to 1400 compared with 5-HT. The present study was to analyse the QSAR of a series of 51 5-HT2AR partial agonistic arylethylamines, tested in vascular in-vitro assays on rats, at a structure-based level and to suggest ligand binding sites. The compounds belong to three different structural classes, (1) indoles, (2) methoxybenzenes and (3) quinazolinediones. Following a hierarchical strategy, different methods have been applied which all contribute to the investigation of ligand-receptor interactions: fragment regression analysis (FRA), receptor modeling, docking studies and 3D QSAR approaches (comparative molecular field analysis, CoMFA, and comparative molecular similarity index analysis, CoMSIA). An initial FRA indicated that methoxy substituents at indole and phenyl derivatives increase the activity and may be involved in polar interactions with the 5-HT2AR. The large contribution of lipophilic substituents in p position of phenethylamines suggests fit to a specific hydrophobic pocket. Secondary benzylamines are more than one order of magnitude more active than their NH2 analogs. An ortho-OH or -OMe substituent at the benzyl moiety further increases activity. Homology models of the human and rat 5-HT2AR were generated using the crystal structure of bovine rhodopsin and of the beta2-adrenoceptor as templates. The derivation of the putative binding sites for the arylethylamines was based on the results from FRA and on mutagenesis data. Both templates led to 5-HT2AR models with similar topology of the binding pocket within the transmembrane domains TM3, TM5, TM6 and TM7. Docking studies with representative members of the three structural classes suggested that the aryl moieties and particularly para-substituents in phenyl derivatives fit into a hydrophobic pocket formed by Phe2435.47, Phe2445.48 and Phe3406.52. The 5-methoxy substituents in indole and phenyl compounds form H bonds with Ser2395.43. In each case, an additional H bond with Ser1593.36 may be assumed. The cationic amine interacts with the conserved Asp1553.32. The benzyl group of secondary arylethylamines is inserted into another hydrophobic pocket formed by Phe3396.51, Trp3677.40 and Tyr3707.43. In this region, the docking poses depend on the template used for model generation, leading to different interactions especially of ortho- substituents. The docking studies with the beta2-adrenoceptor based rat 5-HT2AR model provided templates for a structure-based alignment of the whole series which was used in 3D QSAR analyses of the partial agonistic activity. Both approaches, CoMFA and CoMSIA, led to highly predictive models with low complexity (cross-validated q2 of 0.72 and 0.81 at 4 and 3 components, respectively). The results were largely compatible with the binding site and confirm the docking studies and the suggested ligand-receptor interactions. Steric and hydrophobic field effects on the potency indicate a hydrophobic pocket around the aryl moiety and near the para position of phenyl derivatives and account for the increased activity of secondary benzylamines. The effects of electrostatic and H-bond acceptor fields suggest a favourable influence of negative charges around the aryl moiety, corresponding to the increase in potency caused by methoxy substituents in 2-, 4-, 5- and 6-position of phenethylamines and by the quinazolinedione oxygens. This is in accord with the role of Ser1593.36 and Ser2395.43 as H bond donors. At the benzyl moiety, the negative charge and the acceptor potential of 2-hydroxy and -methoxy substituents is of advantage. Agonists stabilize or induce active receptor states not reflected by the existing crystal structures. Based on models of different rhodopsin states, a homology modeling and ligand docking study on corresponding 5-HT2AR states suggested to be specific to agonist and partial agonist binding, respectively, was performed. The models indicate collective conformational changes of TM domains during activation. The different 5-HT2AR states are similar with respect to the amino acids interacting with the arylethylamines, but show individual topologies of the binding sites. The interconversion of states by TM movements may be accompanied by co-translations and rotations of the ligands. In the case of the secondary amines considered, the tight fit of the benzyl substituent into a hydrophobic pocket containing key residues in TM6 probably impedes the complete receptor activation due to inhibiting the rotation of this helix. High affinity of a partial agonist is therefore often at the expense of its ability to fully activate a receptor

Enhancing the fight against malaria : from genome to structure and activity of a G-protein coupled receptor from the mosquito, Anopheles Gambiae

Includes abstract.Includes bibliographical references (leaves 183-184).G-proton coupled receptors (GPCRs) are excellent drug targets that occupy a central position in the physiology of insects and are involved in transmission of signal from the extracellular to the intracellular side of the cell. Adipokinetic hormone receptors (AKHRs) are GPCRs that mediate physiological functions of the neurohormones, adipokinetic hormones (AKHs) that regulate mobilisation of energy reserves during mosquito flight. Ligand binding to GPCRs depends on the three dimensional (3D) structures of the receptors but to date no crystal structures of insect GPCRs are available. This work focused on building molecular models of AKHR from the genome of the malaria mosquito, identifying its binding site and studying the conformational and structural changes during molecular dynamics of the active and inactive receptor

Formation of Multiple Dimer Interfaces in the Active and Inactive States of a Model G Protein-Coupled Receptor

G protein-coupled receptors (GPCRs) are a class of integral membrane receptor proteins that are characterized by seven-transmembrane (7TM) domains connected by intracellular and extracellular loops, an extracellular N-terminus, and an intracellular Cterminus. GPCRs recognize neurotransmitters, sensory molecules and chemotactic agents and are involved in the control of many aspects of metabolism. Since GPCRs play important roles in diverse processes such as pain perception, growth and blood pressure regulation, and viral pathogenesis, GPCRs became important target for therapeutic agents. The tridecapeptide α-factor pheromone (W1H2W3L4Q5L6K7P8G9Q10P11M12Y13) of Saccharomyces cerevisiae and Ste2p, its cognate GPCR, have been used extensively as a model for peptide ligand-GPCR structure and function. The power of yeast genetics has been used to examine the structure and function of Ste2p. Recently, GPCR homodimerization has been demonstrated for many GPCRs, although the role(s) of dimerization in receptor function is disputed. In this dissertation, Ste2p has been used to investigate GPCR dimerization. Part I of this dissertation is an overview of the GPCR structure and its ligandinduced conformational change with specific emphasis on the peptide pheromone α- factor and its receptor Ste2p. Part II of this dissertation is a study originally designed to probe inter-helical interaction between TM1 and TM7 of Ste2p. Site-directed mutagenesis and cysteine cross-linking with targeted residues of Ste2p were carried out. Although the anticipated inter-helical interactions were not identified from this study, the results provided strong evidence for Ste2p dimerization. Part III of this dissertation describes dimer interfaces including TM1 and TM7 of Ste2p. By using the disulfide cross-linking methodology, we studied the participation of specific residues at the intracellular boundary between TM1 and intracellular loop one and the entire TM7 in Ste2p dimerization. The final part of this dissertation contains a study of the participation of the Ste2p N-terminus in homo-dimer formation and the effect of ligand binding on this interaction. This part also includes overall conclusions and suggestions for future experiments that could contribute to an understanding of the dimer interfaces in Ste2p and the role of dimerization in the function of this receptor

Predicted Structures and Dynamics for Agonists and Antagonists Bound to Serotonin 5-HT2B and 5-HT2C Receptors

Subtype 2 serotonin (5-hydroxytryptamine, 5-HT) receptors are major drug targets for schizophrenia, feeding disorders, perception, depression, migraines, hypertension, anxiety, hallucinogens, and gastrointestinal dysfunctions.' We report here the predicted structure of 5-HT2B and 5-HT2C receptors bound to highly potent and selective 5-HT2B antagonist PRX-08066 3, (pKi: 30 nM), including the key binding residues [V103 (2.53), L132 (3.29), V190 (4.60), and L347 (6.58)] determining the selectivity of binding to 5-HT2B over 5-HT2A. We also report structures of the endogenous agonist (5 HT) and a HT2B selective antagonist 2 (1-methyl-1-1,6,7,8-tetrahydro-pyrrolo [2,3-g]quinoline-5-carboxylic acid pyridine-3-ylamide). We examine the dynamics for the agonist-and antagonist-bound HT2B receptors in explicit membrane and water finding dramatically different patterns of water migration into the NPxxY motif and the binding site that correlates with the stability of ionic locks in the D(E)RY region

ABCC2 transporter and α2 adrenoceptors : Identification of novel compounds and their mode of action

The main goal of this dissertation is to identify novel modulators acting on ATP Binding Cassette subfamily C member 2 (ABCC2) transporters and α2-adrenoceptors subtypes. With the purpose of identifying novel modulators and their mode of action, a combination of experimental and computational approaches have been used. The first protein presented in this dissertation is the ABCC2 transporter, also known as the multidrug resistance associated protein 2 (MRP2), an efflux transporter expressed in polarized cells where it effluxes a variety of both endogenous and exogenous molecules out of the cell. The most common way to study the interactions between small molecules and ABCC2 transporter is by a vesicle transport assay. Three assays are commercially available, which use different probes to define the ABCC2- transport. With the intent to define the different assays and identify the effect that small molecules have on the ABCC2-transport, a small set of eight compounds and, subsequently a larger library of compounds were tested with the different assays. Additionally, the aim was to identify and characterise novel ABCC2 inhibitors, 16 inhibitors have been identified from the larger library and classification models were built to identify important descriptors that were able to discriminate inhibitors from inactive molecules. Instant structure-activity relationships (SAR) of four scaffolds of ABCC2 modulators are also presented. In addition, some unpublished results are presented, the homology model of ABCC2 and further insights into the SAR of ABCC2 modulators. The other proteins included in this dissertation are the three subtypes of the α2-adrenoceptors, G-protein coupled receptors, involved in the signalling pathway of adrenaline and noradrenaline. A clear subtype characterization/profile of these proteins is not available. Selective molecules could be used in treatment of high blood pressure, in the alleviation of withdrawal symptoms, and as anaesthetic with fewer side effects than the current drugs. To define the affinity of a small set of antagonists and outline the involvement of the first transmembrane helix in ligand binding, a competition binding assay has been used with chimera receptors where the first transmembrane helix has been swapped between the three subtypes. Molecular modelling has been used to explain the different binding affinities to the chimera receptors. Additionally, the aim was to identify novel α2B-adrenoceptor selective compounds, thus a mid-sized library has been screened using a miniaturized binding assay. Hierarchical classification and chemoinformatics analysis has been used to visualize and analyse the screening results.Väitöskirja käsittelee uusien ABCC2-kuljetinproteiinin modulaattoreiden ja α2 adrenoseptorialatyyppien inhibiittoreiden tunnistusta sekä kokeellisia että laskennallisia menetelmiä käyttäen. Väitöskirjan aluksi käsittelen ABBCC2-kuljetinproteiinia (ATP Binding Casette -proteiiniperhe, ryhmä C, alatyyppi 2), joka tunnetaan myös MRP2-proteiinina (monilääkeresistenssiin liittyvä proteiini 2). Se on polarisoituneissa soluissa ilmentyvä efflux-kuljetinproteiini, joka pumppaa monia endogeenisiä ja eksogeenisiä molekyylejä ulos soluista. Yhdisteiden ABCC2-vuorovaikutuksia tutkitaan yleisesti vesikkelikuljetuskokeella. Kaupallisesti on saatavilla kolmeen eri testiyhdisteeseen perustuvia ABCC2-kuljetuskokeita. Arvioidakseni näitä kokeita ja pienmolekyylien vaikutusta ABCC2-kuljetukseen testasin eri koeasetelmissa ensin kahdeksan yhdistettä ja sitten suuremman yhdistekirjaston. Tunnistin 16 uutta ABCC2-inhibiittoria, ja rakensin deskriptoripohjaisen luokittelumallin erottelemaan estäjät inaktiivisista yhdisteistä. Esitän myös ABCC2-moduloinnin rakenne-aktiivisuussuhteet neljälle ydinrakenteelle ja käsittelen lisäksi joitakin julkaisemattomia tuloksia, kuten ABCC2-proteiinista rakennettua homologimallia ja jatkotutkimusta ABCC2-modulaattorien rakenne-aktiivisuussuhteista. Käsittelen väitöskirjassani myös kolmea α2-adrenoseptorin alatyyppiä, jotka kuuluvat G proteiinikytkentäisiin reseptoreihin ja osallistuvat adrenaliinin ja noradrenaliinin signalointiin. Näiden reseptorialatyyppien karakterisointi on toistaiseksi puutteellinen. Alatyyppiselektiivisiä molekyylejä voitaisiin hyödyntää verenpainetaudin hoidossa, lievittämään vieroitusoireita sekä nykyisiä lääkkeitä vähemmän haittavaikutuksia aiheuttavana anesteettina. Määrittääkseni pienen antagonistijoukon affiniteetin ja tutkiakseni ensimmäisen kalvon läpäisevän heliksin (TM1) osuutta sitoutumiseen käytin kilpailevaa sitoutumiskoetta ja kimeerisiä reseptoreita, joissa ensimmäistä kalvon läpäisevää heliksiä (TM1) vaihdettiin kolmen reseptorialatyypin välillä. Selitän havaittuja affiniteettieroja molekyylimallinuksen avulla. Lisäksi seuloin keskikokoisen yhdistekirjaston miniatyrisoidulla sitoutumiskokeella tunnistaakseni uusia α2B adrenoseptoriselektiivisiä yhdisteitä. Hyödynnän hierarkista luokittelua ja kemoinformatiikkaa seulontatulosten analysoinnissa ja esittämisessä

Understanding ligand binding, selectivity and functions on the G protein-coupled receptors: A molecular modeling approach

The assessment of target protein molecular structure provides a distinct advantage in the rational drug design process. The increasing number of available G protein-coupled receptor crystal structures has enabled utilization of a varied number of computational approaches for understanding the ligand-receptor interactions, ligand selectivity and even receptor response upon ligand binding. The following dissertation examines the results from three different projects with varied objectives – i) structural modeling of human C-C chemokine receptor type 5 (CCR5) and assessment of the ligand binding pocket of the receptor, ii) assessment of the selectivity profile of naltrexone derivatives on the three opioid receptors (μ-opioid, κ-opioid, δ-opioid) with an aim towards designing selective μ-opioid receptor antagonists, and iii) structural modeling of the ‘active’ state conformation of the κ-opioid receptor in response to agonist binding and determination of a plausible molecular mechanism involved in activation ‘switch’ of the κ-opioid receptor. In absence of a crystal-based molecular structure of CCR5, a homology model of the receptor was built and the ligand binding pocket was validated. On the basis of evaluation of the ligand-receptor interactions on the validated binding pocket, structural and chemical modifications to anibamine, a natural plant product, were proposed to enhance its receptor binding. The selectivity of naltrexone (a universal antagonist) was assessed with respect to the three opioid receptors by employing ligand docking studies and the ‘message-address’ concept. Multiple address sites were identified on the opioid receptors and structural modifications were proposed for the naltrexone derivatives for their enhanced selectivity. In the third project, structural modeling of the active state conformation of the κ-opioid receptor covalently bound to a salvinorin A derivative (agonist) was attempted via molecular dynamics simulations. Although the obtained molecular model lacked the signature ‘agonist-like’ conformations, the result provides a template for such studies in the future

Characterisation of novel alpha1-adrenoceptor ligands

α1A-adrenoceptor (AR) antagonists used clinically to treat urogenital conditions, such as benign prostatic hyperplasia, have side effects due to a lack of subtype selectivity and the 5-HT1A-R off-target affinity. In this study, computer-aided drug design, structure-activity relationship, and molecular pharmacology methods were used to identify α1A-AR subtype selective compounds with reduced 5-HT1A-R off-target affinity. In silico database screening identified 2 structurally novel compounds with nanomolar affinity for the α1A-AR, and 4 compounds with selectivity for the α1A-AR over the 5-HT1A-R. Homobivalent 4-aminoquinoline compounds with increasing carbon linker lengths (C2-C12) were examined for α1A-AR selectivity. C2 and C9 provided the optimal linker length to achieve the highest affinity among this series of compounds. C2 to C6 and C12 showed selectivity for the α1A-AR over α1D-AR and the 5-HT1A-R. To improve selectivity of these compounds for α1A over α1B-AR, methyl (Me) and methoxyl (OMe) substituted analogues of C2 and C7 were produced. The substituted compound, 8-Me C2 showed selectivity for the α1A-AR over all other tested receptors, while 6-Me C2, 6-Me C7, and 7-OMe C7 were selective for the α1B-AR over all other tested receptors. Docking studies suggested that C2 bound within the orthosteric endogenous ligand binding pocket, but that C7-C11 interacted with a second allosteric site in a bitopic manner. 4-aminoquinoline, C9, C10, and C11 increased the dissociation rate of [3H] prazosin from the α1A-AR, suggesting allosteric modulation. C9 was further characterised and demonstrated to be a negative allosteric modulator of orthosteric ligands of the α1A-AR. The allosteric site was shown by docking of 4-aminoquinoline, C9, C10 and C11 to be composed of S832.61, F862.64 and E872.65, located on the extracellular part of transmembrane helix II. In further support of this region’s role in modulating orthosteric ligand affinity, F862.64 was shown to be involved in the process of the dissociation of [3H] prazosin from the α1A-AR. In summary, this study has identified both α1A, and α1B-AR subtype selective compounds. Furthermore, it has identified a new negative allosteric modulator and proposed an allosteric site of the α1A-AR. These findings provide the basis for the future development of highly selective α1-AR drugs

Allosteric sodium in class A GPCR signaling

Despite their functional and structural diversity, G protein-coupled receptors (GPCRs) share a common mechanism of signal transduction via conformational changes in the seven-transmembrane (7TM) helical domain. New major insights into this mechanism come from the recent crystallographic discoveries of a partially hydrated sodium ion that is specifically bound in the middle of the 7TM bundle of multiple class A GPCRs. This review discusses the remarkable structural conservation and distinct features of the Na+ pocket in this most populous GPCR class, as well as the conformational collapse of the pocket on receptor activation. New insights help to explain allosteric effects of sodium on GPCR agonist binding and activation, and sodium’s role as a potential co-factor in class A GPCR function