Publications by authors named "Ad P IJzerman"

47 Publications

The RESOLUTE consortium: unlocking SLC transporters for drug discovery.

Authors:
Giulio Superti-Furga Daniel Lackner Tabea Wiedmer Alvaro Ingles-Prieto Barbara Barbosa Enrico Girardi Ulrich Goldmann Bettina Gürtl Kristaps Klavins Christoph Klimek Sabrina Lindinger Eva Liñeiro-Retes André C Müller Svenja Onstein Gregor Redinger Daniela Reil Vitaly Sedlyarov Gernot Wolf Matthew Crawford Robert Everley David Hepworth Shenping Liu Stephen Noell Mary Piotrowski Robert Stanton Hui Zhang Salvatore Corallino Andrea Faedo Maria Insidioso Giovanna Maresca Loredana Redaelli Francesca Sassone Lia Scarabottolo Michela Stucchi Paola Tarroni Sara Tremolada Helena Batoulis Andreas Becker Eckhard Bender Yung-Ning Chang Alexander Ehrmann Anke Müller-Fahrnow Vera Pütter Diana Zindel Bradford Hamilton Martin Lenter Diana Santacruz Coralie Viollet Charles Whitehurst Kai Johnsson Philipp Leippe Birgit Baumgarten Lena Chang Yvonne Ibig Martin Pfeifer Jürgen Reinhardt Julian Schönbett Paul Selzer Klaus Seuwen Charles Bettembourg Bruno Biton Jörg Czech Hélène de Foucauld Michel Didier Thomas Licher Vincent Mikol Antje Pommereau Frédéric Puech Veeranagouda Yaligara Aled Edwards Brandon J Bongers Laura H Heitman Ad P IJzerman Huub J Sijben Gerard J P van Westen Justine Grixti Douglas B Kell Farah Mughal Neil Swainston Marina Wright-Muelas Tina Bohstedt Nicola Burgess-Brown Liz Carpenter Katharina Dürr Jesper Hansen Andreea Scacioc Giulia Banci Claire Colas Daniela Digles Gerhard Ecker Barbara Füzi Viktoria Gamsjäger Melanie Grandits Riccardo Martini Florentina Troger Patrick Altermatt Cédric Doucerain Franz Dürrenberger Vania Manolova Anna-Lena Steck Hanna Sundström Maria Wilhelm Claire M Steppan

Nat Rev Drug Discov 2020 07;19(7):429-430

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http://dx.doi.org/10.1038/d41573-020-00056-6DOI Listing
July 2020

LUF7244 plus Dofetilide Rescues Aberrant K11.1 Trafficking and Produces Functional I.

Mol Pharmacol 2020 06 2;97(6):355-364. Epub 2020 Apr 2.

Department of Medical Physiology, University Medical Center Utrecht, Utrecht, The Netherlands (M.Q., Y.J., M.J.C.H., F.R., D.F., W.B.H., M.A.G.H.); Department of Physiology, University of Kentucky, Lexington, Kentucky (T.D.G., B.P.D.); Department of Pharmacology and Toxicology, University of Vienna, Vienna, Austria (W.B.H., A.S.-W.); Leiden Academic Centre for Drug Research, Division of Drug Discovery and Safety, Leiden, The Netherlands (A.P.I., L.H.H.); and Department of Medicine, University of Wisconsin, Madison, Wisconsin (C.T.J.)

Voltage-gated potassium 11.1 (K11.1) channels play a critical role in repolarization of cardiomyocytes during the cardiac action potential (AP). Drug-mediated K11.1 blockade results in AP prolongation, which poses an increased risk of sudden cardiac death. Many drugs, like pentamidine, interfere with normal K11.1 forward trafficking and thus reduce functional K11.1 channel densities. Although class III antiarrhythmics, e.g., dofetilide, rescue congenital and acquired forward trafficking defects, this is of little use because of their simultaneous acute channel blocking effect. We aimed to test the ability of a combination of dofetilide plus LUF7244, a K11.1 allosteric modulator/activator, to rescue K11.1 trafficking and produce functional K11.1 current. LUF7244 treatment by itself did not disturb or rescue wild type (WT) or G601S-K11.1 trafficking, as shown by Western blot and immunofluorescence microcopy analysis. Pentamidine-decreased maturation of WT K11.1 levels was rescued by 10 μM dofetilide or 10 μM dofetilide + 5 μM LUF7244. In trafficking defective G601S-K11.1 cells, dofetilide (10 μM) or dofetilide + LUF7244 (10 + 5 μM) also restored K11.1 trafficking, as demonstrated by Western blot and immunofluorescence microscopy. LUF7244 (10 μM) increased I despite the presence of dofetilide (1 μM) in WT K11.1 cells. In G601S-expressing cells, long-term treatment (24-48 hour) with LUF7244 (10 μM) and dofetilide (1 μM) increased I compared with nontreated or acutely treated cells. We conclude that dofetilide plus LUF7244 rescues K11.1 trafficking and produces functional I Thus, combined administration of LUF7244 and an I trafficking corrector could serve as a new pharmacological therapy of both congenital and drug-induced K11.1 trafficking defects. SIGNIFICANCE STATEMENT: Decreased levels of functional K11.1 potassium channel at the plasma membrane of cardiomyocytes prolongs action potential repolarization, which associates with cardiac arrhythmia. Defective forward trafficking of K11.1 channel protein is an important factor in acquired and congenital long QT syndrome. LUF7244 as a negative allosteric modulator/activator in combination with dofetilide corrected both congenital and acquired K11.1 trafficking defects, resulting in functional K11.1 current.
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http://dx.doi.org/10.1124/mol.119.118190DOI Listing
June 2020

LUF7244, an allosteric modulator/activator of K 11.1 channels, counteracts dofetilide-induced torsades de pointes arrhythmia in the chronic atrioventricular block dog model.

Br J Pharmacol 2019 10 30;176(19):3871-3885. Epub 2019 Aug 30.

Department of Medical Physiology, University Medical Centre Utrecht, Utrecht, The Netherlands.

Background And Purpose: K 11.1 (hERG) channel blockade is an adverse effect of many drugs and lead compounds, associated with lethal cardiac arrhythmias. LUF7244 is a negative allosteric modulator/activator of K 11.1 channels that inhibits early afterdepolarizations in vitro. We tested LUF7244 for antiarrhythmic efficacy and potential proarrhythmia in a dog model.

Experimental Approach: LUF7244 was tested in vitro for (a) increasing human I and canine I and (b) decreasing dofetilide-induced action potential lengthening and early afterdepolarizations in cardiomyocytes derived from human induced pluripotent stem cells and canine isolated ventricular cardiomyocytes. In vivo, LUF7244 was given intravenously to anaesthetized dogs in sinus rhythm or with chronic atrioventricular block.

Key Results: LUF7244 (0.5-10 μM) concentration dependently increased I by inhibiting inactivation. In vitro, LUF7244 (10 μM) had no effects on I , I , I , and I , doubled I , shortened human and canine action potential duration by approximately 50%, and inhibited dofetilide-induced early afterdepolarizations. LUF7244 (2.5 mg·kg ·15 min ) in dogs with sinus rhythm was not proarrhythmic and shortened, non-significantly, repolarization parameters (QTc: -6.8%). In dogs with chronic atrioventricular block, LUF7244 prevented dofetilide-induced torsades de pointes arrhythmias in 5/7 animals without normalization of the QTc. Peak LUF7244 plasma levels were 1.75 ± 0.80 during sinus rhythm and 2.34 ± 1.57 μM after chronic atrioventricular block.

Conclusions And Implications: LUF7244 counteracted dofetilide-induced early afterdepolarizations in vitro and torsades de pointes in vivo. Allosteric modulators/activators of K 11.1 channels might neutralize adverse cardiac effects of existing drugs and newly developed compounds that display QTc lengthening.
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http://dx.doi.org/10.1111/bph.14798DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6780032PMC
October 2019

TLR-Induced IL-12 and CCL2 Production by Myeloid Cells Is Dependent on Adenosine A Receptor-Mediated Signaling.

J Immunol 2019 04 25;202(8):2421-2430. Epub 2019 Feb 25.

Alternatives Unit, Biomedical Primate Research Centre, 2288 GJ Rijswijk, the Netherlands;

TLR-induced signaling potently activates cells of the innate immune system and is subject to regulation at different levels. Inflammatory conditions are associated with increased levels of extracellular adenosine, which can modulate TLR-induced production of cytokines through adenosine receptor-mediated signaling. There are four adenosine receptor subtypes that induce different signaling cascades. In this study, we demonstrate a pivotal contribution of adenosine A receptor (AR)-mediated signaling to the TLR4-induced expression of IL-12 in different types of human myeloid APC. In dendritic cells, IL-12 and CCL2 responses as evoked by TLR2, 3, 4, 5, and 8, as well as IL-12 responses evoked by whole pathogens, were all reduced when AR-mediated signaling was blocked. As a result, concomitant production of IFN-γ and IL-17 by T cells was significantly inhibited. We further show that selective inhibition of AR-mediated signaling reduced TLR-induced phosphorylation of the transcription factor STAT1 at tyrosine 701. Next-generation sequencing revealed that AR-mediated signaling controls the expression of metallothioneins, known inhibitors of STAT1 phosphorylation. Together our results reveal a novel regulatory layer of innate immune responses, with a central role for metallothioneins and autocrine/paracrine signaling via ARs.
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http://dx.doi.org/10.4049/jimmunol.1800618DOI Listing
April 2019

A covalent antagonist for the human adenosine A receptor.

Purinergic Signal 2017 06 3;13(2):191-201. Epub 2016 Dec 3.

Division of Medicinal Chemistry, Leiden Academic Centre for Drug Research, Leiden University, P.O. Box 9502, 2300 RA, Leiden, the Netherlands.

The structure of the human A adenosine receptor has been elucidated by X-ray crystallography with a high affinity non-xanthine antagonist, ZM241385, bound to it. This template molecule served as a starting point for the incorporation of reactive moieties that cause the ligand to covalently bind to the receptor. In particular, we incorporated a fluorosulfonyl moiety onto ZM241385, which yielded LUF7445 (4-((3-((7-amino-2-(furan-2-yl)-[1, 2, 4]triazolo[1,5-a][1, 3, 5]triazin-5-yl)amino)propyl)carbamoyl)benzene sulfonyl fluoride). In a radioligand binding assay, LUF7445 acted as a potent antagonist, with an apparent affinity for the hA receptor in the nanomolar range. Its apparent affinity increased with longer incubation time, suggesting an increasing level of covalent binding over time. An in silico A-structure-based docking model was used to study the binding mode of LUF7445. This led us to perform site-directed mutagenesis of the A receptor to probe and validate the target lysine amino acid K153 for covalent binding. Meanwhile, a functional assay combined with wash-out experiments was set up to investigate the efficacy of covalent binding of LUF7445. All these experiments led us to conclude LUF7445 is a valuable molecular tool for further investigating covalent interactions at this receptor. It may also serve as a prototype for a therapeutic approach in which a covalent antagonist may be needed to counteract prolonged and persistent presence of the endogenous ligand adenosine.
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http://dx.doi.org/10.1007/s11302-016-9549-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5432479PMC
June 2017

Scintillation proximity assay (SPA) as a new approach to determine a ligand's kinetic profile. A case in point for the adenosine A1 receptor.

Purinergic Signal 2016 Mar 9;12(1):115-26. Epub 2015 Dec 9.

Division of Medicinal Chemistry, Leiden Academic Centre for Drug Research, Leiden University, P.O. Box 9502, 2300 RA, Leiden, The Netherlands.

Scintillation proximity assay (SPA) is a radio-isotopic technology format used to measure a wide range of biological interactions, including drug-target binding affinity studies. The assay is homogeneous in nature, as it relies on a "mix and measure" format. It does not involve a filtration step to separate bound from free ligand as is the case in a traditional receptor-binding assay. For G protein-coupled receptors (GPCRs), it has been shown that optimal binding kinetics, next to a high affinity of a ligand, can result in more desirable pharmacological profiles. However, traditional techniques to assess kinetic parameters tend to be cumbersome and laborious. We thus aimed to evaluate whether SPA can be an alternative platform for real-time receptor-binding kinetic measurements on GPCRs. To do so, we first validated the SPA technology for equilibrium binding studies on a prototypic class A GPCR, the human adenosine A1 receptor (hA1R). Differently to classic kinetic studies, the SPA technology allowed us to study binding kinetic processes almost real time, which is impossible in the filtration assay. To demonstrate the reliability of this technology for kinetic purposes, we performed the so-called competition association experiments. The association and dissociation rate constants (k on and k off) of unlabeled hA1R ligands were reliably and quickly determined and agreed very well with the same parameters from a traditional filtration assay performed simultaneously. In conclusion, SPA is a very promising technique to determine the kinetic profile of the drug-target interaction. Its robustness and potential for high-throughput may render this technology a preferred choice for further kinetic studies.
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http://dx.doi.org/10.1007/s11302-015-9485-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4749533PMC
March 2016

The adhesion G protein-coupled receptor G2 (ADGRG2/GPR64) constitutively activates SRE and NFκB and is involved in cell adhesion and migration.

Cell Signal 2015 Dec 29;27(12):2579-88. Epub 2015 Aug 29.

Novo Nordisk Foundation Center for Basic Metabolic Research, Section for Metabolic Receptology and Enteroendocrinology, Faculty of Health Sciences, University of Copenhagen, Denmark.

Adhesion G protein-coupled receptors (ADGRs) are believed to be activated by auto-proteolytic cleavage of their very large extracellular N-terminal domains normally acting as a negative regulator of the intrinsically constitutively active seven transmembrane domain. ADGRG2 (or GPR64) which originally was described to be expressed in the epididymis and studied for its potential role in male fertility, is highly up-regulated in a number of carcinomas, including breast cancer. Here, we demonstrate that ADGRG2 is a functional receptor, which in transfected HEK293 cells signals with constitutive activity through the adhesion- and migration-related transcription factors serum response element (SRE) and nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB) presumably via coupling to Gα12/13 and Gαq. However, activation of these two pathways appears to occur through distinct molecular activation mechanisms as auto-proteolytic cleavage is essential for SRE activation but not required for NFκB signaling. The overall activation mechanism for ADGRG2 is clearly distinct from the established ADGR activation mechanism as it requires the large extracellular N-terminal domain for proper intracellular signal transduction. Knockdown of ADGRG2 by siRNA in the highly motile breast cancer cell lines Hs578T and MDA-MB-231 resulted in a strong reduction in cell adhesion and subsequent cell migration which was associated with a selective reduction in RelB, an NFκB family member. It is concluded that the adhesion GPCR ADGRG2 is critically involved in the adhesion and migration of certain breast cancer cells through mechanisms including a non-canonical NFkB pathway and that ADGRG2 could be a target for treatment of certain types of cancer.
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http://dx.doi.org/10.1016/j.cellsig.2015.08.015DOI Listing
December 2015

A yeast screening method to decipher the interaction between the adenosine A2B receptor and the C-terminus of different G protein α-subunits.

Purinergic Signal 2014 Sep 26;10(3):441-53. Epub 2014 Jan 26.

Division of Medicinal Chemistry, Leiden Academic Centre for Drug Research, Leiden University, P.O. Box 9502, 2300 RA, Leiden, Netherlands.

The expression of human G protein-coupled receptors (GPCRs) in Saccharomyces cerevisiae containing chimeric yeast/mammalian Gα subunits provides a useful tool for the study of GPCR activation. In this study, we used a one-GPCR-one-G protein yeast screening method in combination with molecular modeling and mutagenesis studies to decipher the interaction between GPCRs and the C-terminus of different α-subunits of G proteins. We chose the human adenosine A2B receptor (hA2BR) as a paradigm, a typical class A GPCR that shows promiscuous behavior in G protein coupling in this yeast system. The wild-type hA2BR and five mutant receptors were expressed in 8 yeast strains with different humanized G proteins, covering the four major classes: Gαi, Gαs, Gαq, and Gα12. Our experiments showed that a tyrosine residue (Y) at the C-terminus of the Gα subunit plays an important role in controlling the activation of GPCRs. Receptor residues R103(3.50) and I107(3.54) are vital too in G protein-coupling and the activation of the hA2BR, whereas L213(IL3) is more important in G protein inactivation. Substitution of S235(6.36) to alanine provided the most divergent G protein-coupling profile. Finally, L236(6.37) substitution decreased receptor activation in all G protein pathways, although to a different extent. In conclusion, our findings shed light on the selectivity of receptor/G protein coupling, which may help in further understanding GPCR signaling.
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http://dx.doi.org/10.1007/s11302-014-9407-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4152457PMC
September 2014

Functional selectivity of adenosine A1 receptor ligands?

Purinergic Signal 2013 Mar 28;9(1):91-100. Epub 2012 Sep 28.

Division of Medicinal Chemistry, Leiden/Amsterdam Center for Drug Research, Leiden University, PO Box 9502, 2300 RA, Leiden, The Netherlands.

The concept of functional selectivity offers great potential for the development of drugs that selectively activate a specific intracellular signaling pathway. During the last few years, it has become possible to systematically analyse compound libraries on G protein-coupled receptors (GPCRs) for this 'biased' form of signaling. We screened over 800 compounds targeting the class of adenosine A(1) receptors using a β-arrestin-mediated signaling assay in U2OS cells as a G protein-independent readout for GPCR activation. A selection of compounds was further analysed in a G protein-mediated GTPγS assay. Additionally, receptor affinity of these compounds was determined in a radioligand binding assay with the agonist [(3)H]CCPA. Of all compounds tested, only LUF5589 9 might be considered as functionally selective for the G protein-dependent pathway, particularly in view of a likely overestimation of β-arrestin signaling in the U2OS cells. Altogether, our study shows that functionally selective ligands for the adenosine A(1) receptor are rare, if existing at all. A thorough analysis of biased signaling on other GPCRs also reveals that only very few compounds can be considered functionally selective. This might indicate that the concept of functional selectivity is less common than speculated.
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http://dx.doi.org/10.1007/s11302-012-9334-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3568431PMC
March 2013

Biological and pharmacological roles of HCA receptors.

Adv Pharmacol 2011 ;62:219-50

Division of Medicinal Chemistry, Leiden/Amsterdam Center for Drug Research, Leiden University, Leiden, The Netherlands.

The hydroxy-carboxylic acid (HCA) receptors HCA(1), HCA(2), and HCA(3) were previously known as GPR81, GPR109A, and GPR109B, respectively, or as the nicotinic acid receptor family. They form a cluster of G protein-coupled receptors with high sequence homology. Recently, intermediates of energy metabolism, all HCAs, have been reported as endogenous ligands for each of these receptors. The HCA receptors are predominantly expressed on adipocytes and mediate the inhibition of lipolysis by coupling to G(i)-type proteins. HCA(1) is activated by lactate, HCA(2) by the ketone body 3-hydroxy-butyrate, and HCA(3) by hydroxylated β-oxidation intermediates, especially 3-hydroxy-octanoic acid. Both HCA(2) and HCA(3) are part of a negative feedback loop which keeps the release of fat stores in check under starvation conditions, whereas HCA(1) plays a role in the antilipolytic (fat-conserving) effect of insulin. HCA(2) was first discovered as the molecular target of the antidyslipidemic drug nicotinic acid (or niacin). Many synthetic agonists have since been designed for HCA(2) and HCA(3), but the development of a new, improved HCA-targeted drug has not been successful so far, despite a number of clinical studies. Recently, it has been shown that the major side effect of nicotinic acid, skin flushing, is mediated by HCA(2) receptors on keratinocytes, as well as on Langerhans cells in the skin. In this chapter, we summarize the latest developments in the field of HCA receptor research, with emphasis on (patho)physiology, receptor pharmacology, major ligand classes, and the therapeutic potential of HCA ligands.
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http://dx.doi.org/10.1016/B978-0-12-385952-5.00005-1DOI Listing
January 2012

Three "hotspots" important for adenosine A(2B) receptor activation: a mutational analysis of transmembrane domains 4 and 5 and the second extracellular loop.

Purinergic Signal 2012 Mar 5;8(1):23-38. Epub 2011 Aug 5.

Division of Medicinal Chemistry, Leiden/Amsterdam Centre for Drug Research, Leiden University, P.O. Box 9502, 2300, Leiden, The Netherlands.

G protein-coupled receptors (GPCRs) are a major drug target and can be activated by a range of stimuli, from photons to proteins. Despite the progress made in the last decade in molecular and structural biology, their exact activation mechanism is still unknown. Here we describe new insights in specific regions essential in adenosine A(2B) receptor activation (A(2B)R), a typical class A GPCR. We applied unbiased random mutagenesis on the middle part of the human adenosine A(2B)R, consisting of transmembrane domains 4 and 5 (TM4 and TM5) linked by extracellular loop 2 (EL2), and subsequently screened in a medium-throughput manner for gain-of-function and constitutively active mutants. For that purpose, we used a genetically engineered yeast strain (Saccharomyces cerevisiae MMY24) with growth as a read-out parameter. From the random mutagenesis screen, 12 different mutant receptors were identified that form three distinct clusters; at the top of TM4, in a cysteine-rich region in EL2, and at the intracellular side of TM5. All mutant receptors show a vast increase in agonist potency and most also displayed a significant increase in constitutive activity. None of these residues are supposedly involved in ligand binding directly. As a consequence, it appears that disrupting the relatively "silent" configuration of the wild-type receptor in each of the three clusters readily causes spontaneous receptor activity.
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http://dx.doi.org/10.1007/s11302-011-9251-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3286537PMC
March 2012

Functional selectivity of adenosine receptor ligands.

Purinergic Signal 2011 Jun 5;7(2):171-92. Epub 2011 May 5.

Division of Medicinal Chemistry, Leiden/Amsterdam Center for Drug Research, Leiden University, PO Box 9502, 2300 RA, Leiden, The Netherlands.

Adenosine receptors are plasma membrane proteins that transduce an extracellular signal into the interior of the cell. Basically every mammalian cell expresses at least one of the four adenosine receptor subtypes. Recent insight in signal transduction cascades teaches us that the current classification of receptor ligands into agonists, antagonists, and inverse agonists relies very much on the experimental setup that was used. Upon activation of the receptors by the ubiquitous endogenous ligand adenosine they engage classical G protein-mediated pathways, resulting in production of second messengers and activation of kinases. Besides this well-described G protein-mediated signaling pathway, adenosine receptors activate scaffold proteins such as β-arrestins. Using innovative and sensitive experimental tools, it has been possible to detect ligands that preferentially stimulate the β-arrestin pathway over the G protein-mediated signal transduction route, or vice versa. This phenomenon is referred to as functional selectivity or biased signaling and implies that an antagonist for one pathway may be a full agonist for the other signaling route. Functional selectivity makes it necessary to redefine the functional properties of currently used adenosine receptor ligands and opens possibilities for new and more selective ligands. This review focuses on the current knowledge of functionally selective adenosine receptor ligands and on G protein-independent signaling of adenosine receptors through scaffold proteins.
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http://dx.doi.org/10.1007/s11302-011-9232-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3146648PMC
June 2011

Chemogenomics approaches for receptor deorphanization and extensions of the chemogenomics concept to phenotypic space.

Curr Top Med Chem 2011 ;11(15):1964-77

Division of Medicinal Chemistry, Leiden/Amsterdam Center for Drug Research, Einsteinweg 55, 2333 CC Leiden, The Netherlands.

Chemogenomic approaches, which link ligand chemistry to bioactivity against targets (and, by extension, to phenotypes) are becoming more and more important due to the increasing number of bioactivity data available both in proprietary databases as well as in the public domain. In this article we review chemogenomics approaches applied in four different domains: Firstly, due to the relationship between protein targets from which an approximate relation between their respective bioactive ligands can be inferred, we investigate the extent to which chemogenomics approaches can be applied to receptor deorphanization. In this case it was found that by using knowledge about active compounds of related proteins, in 93% of all cases enrichment better than random could be obtained. Secondly, we analyze different cheminformatics analysis methods with respect to their behavior in chemogenomics studies, such as subgraph mining and Bayesian models. Thirdly, we illustrate how chemogenomics, in its particular flavor of 'proteochemometrics', can be applied to extrapolate bioactivity predictions from given data points to related targets. Finally, we extend the concept of 'chemogenomics' approaches, relating ligand chemistry to bioactivity against related targets, into phenotypic space which then falls into the area of 'chemical genomics' and 'chemical genetics'; given that this is very often the desired endpoint of approaches in not only the pharmaceutical industry, but also in academic probe discovery, this is often the endpoint the experimental scientist is most interested in.
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http://dx.doi.org/10.2174/156802611796391230DOI Listing
February 2012

How to catch a membrane protein in action: a review of functional membrane protein immobilization strategies and their applications.

Chem Rev 2011 Feb 10;111(2):640-56. Epub 2010 Sep 10.

Division of Medicinal Chemistry, Leiden Amsterdam Center for Drug Research, Leiden University, The Netherlands.

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http://dx.doi.org/10.1021/cr900088sDOI Listing
February 2011

Allosteric modulation of adenosine receptors.

Biochim Biophys Acta 2011 May 27;1808(5):1309-18. Epub 2010 Jun 27.

Leiden/Amsterdam Center for Drug Research, Division of Medicinal Chemistry, Leiden, The Netherlands.

Allosteric ligands for G protein-coupled receptors (GPCRs) may alter receptor conformations induced by an orthosteric ligand. These modulators can thus fine-tune classical pharmacological responses. In this review we will describe efforts to synthesize and characterize allosteric modulators for one particular GPCR subfamily, the adenosine receptors. There are four subtypes of these receptors: A(1), A(2A), A(2B) and A(3). Allosteric enhancers for the adenosine A(1) receptor may have anti-arrythmic and anti-lipolytic activity. They may also act as analgesics and neuroprotective agents. A(3) allosteric enhancers are thought to be beneficial in ischemic conditions or as antitumor agents. We will summarize recent developments regarding the medicinal chemistry of such compounds. Most data have been and are published about the adenosine A(1) and A(3) receptor, whereas limited or no information is available for the A(2A) and A(2B) receptor, respectively. Receptor mutation studies are also discussed, as they may shed light on the localization of the allosteric binding sites. This article is part of a Special Issue entitled: "Adenosine Receptors".
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http://dx.doi.org/10.1016/j.bbamem.2010.06.013DOI Listing
May 2011

Small molecule antagonists for chemokine CCR3 receptors.

Med Res Rev 2010 Sep;30(5):778-817

Leiden/Amsterdam Center for Drug Research, Division of Medicinal Chemistry, PO Box 9502, 2300RA Leiden, The Netherlands.

The chemokine receptor CCR3 is believed to play a role in the development of allergic diseases such as asthma, atopic dermatitis, and allergic rhinitis. Despite the conflicting results that have been reported regarding the importance of eosinophils and CCR3 in allergic inflammation, inhibition of this receptor with small molecule antagonists is thought to provide a valuable approach for the treatment of these diseases. This review describes the structure-activity relationships (SAR) of small molecule CCR3 antagonists as reported in the scientific and patent literature. Various chemical classes of small molecule CCR3 antagonists have been described so far, including (bi)piperidine and piperazine derivatives, N-arylalkylpiperidine urea derivatives and (N-ureidoalkyl)benzylpiperidines, phenylalanine derivatives, morpholinyl derivatives, pyrrolidinohydroquinazolines, arylsulfonamides, amino-alkyl amides, imidazole- and pyrimidine-based antagonists, and bicyclic diamines. The (N-ureidoalkyl)benzylpiperidines are the best studied class in view of their generally high affinity and antagonizing potential. For many of these antagonists subnanomolar IC(50) values were reported for binding to CCR3 along with the ability to effectively inhibit intracellular calcium mobilization and eosinophil chemotaxis induced by CCR3 agonist ligands in vitro.
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http://dx.doi.org/10.1002/med.20181DOI Listing
September 2010

The endocannabinoid 2-arachidonylglycerol is a negative allosteric modulator of the human A3 adenosine receptor.

Biochem Pharmacol 2010 Jan 7;79(1):48-56. Epub 2009 Aug 7.

Division of Medicinal Chemistry, Leiden/Amsterdam Centre for Drug Research, Leiden University, P.O. Box 9502, 2300 RA Leiden, The Netherlands.

Studies of endogenous cannabinoid agonists, such as 2-arachidonylglycerol (2-AG), have revealed their potential to exert modulatory actions on other receptor systems in addition to their ability to activate cannabinoid receptors. This study investigated the effect of cannabinoid ligands on the human adenosine A(3) (hA(3)R) receptor. The endocannabinoid 2-AG was able to inhibit agonist ([125I]N(6)-(4-amino-3-iodobenzyl) adenosine-5'-(N-methyluronamide)--[125I] AB MECA) binding at the hA(3)R. This inhibition occurred over a narrow range of ligand concentration and was characterized by high Hill coefficients suggesting a non-competitive interaction. Furthermore, in the presence of 2-AG, the rate of [125I] AB MECA dissociation was increased, consistent with an action as a negative allosteric modulator of the hA(3)R. Moreover, by measuring intracellular cAMP levels, we demonstrate that 2-AG decreases both the potency of an agonist at the hA(3)R and the basal signalling of this receptor. Since the hA(3)R has been shown to be expressed in astrocytes and microglia, these findings may be particularly relevant in certain pathological states such as cerebral ischemia where levels of 2-AG and anandamide are raised.
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http://dx.doi.org/10.1016/j.bcp.2009.07.024DOI Listing
January 2010

Differential expression of adenosine A3 receptors controls adenosine A2A receptor-mediated inhibition of TLR responses in microglia.

J Immunol 2009 Jun;182(12):7603-12

Alternatives Unit, Biomedical Primate Research Centre, Rijswijk, The Netherlands.

Microglia activation is a prominent feature in many neuroinflammatory disorders. Unrestrained activation can generate a chronic inflammatory environment that might lead to neurodegeneration and autoimmunity. Extracellular adenosine modulates cellular activation through adenosine receptor (ADORA)-mediated signaling. There are four ADORA subtypes that can either increase (A(2A) and A(2B) receptors) or decrease (A(1) and A(3) receptors) intracellular cyclic AMP levels. The expression pattern of the subtypes thus orchestrates the cellular response to extracellular adenosine. We have investigated the expression of ADORA subtypes in unstimulated and TLR-activated primary rhesus monkey microglia. Activation induced an up-regulation of A(2A) and a down-regulation of A(3) receptor (A(3)R) levels. The altered ADORA-expression pattern sensitized microglia to A(2A) receptor (A(2A)R)-mediated inhibition of subsequent TLR-induced cytokine responses. By using combinations of subtype-specific agonists and antagonists, we revealed that in unstimulated microglia, A(2A)R-mediated inhibitory signaling was effectively counteracted by A(3)R-mediated signaling. In activated microglia, the decrease in A(3)R-mediated signaling sensitized them to A(2A)R-mediated inhibitory signaling. We report a differential, activation state-specific expression of ADORA in microglia and uncover a role for A(3)R as dynamically regulated suppressors of A(2A)R-mediated inhibition of TLR-induced responses. This would suggest exploration of combinations of A(2A)R agonists and A(3)R antagonists to dampen microglial activation during chronic neuroinflammatory conditions.
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http://dx.doi.org/10.4049/jimmunol.0803383DOI Listing
June 2009

Allosteric modulation of adenosine receptors.

Purinergic Signal 2009 Mar 10;5(1):51-61. Epub 2008 Jul 10.

Division of Medicinal Chemistry, Leiden/Amsterdam Center for Drug Research, P.O. Box 9502, 2300, RA, Leiden, The Netherlands.

Allosteric modulators for adenosine receptors may have potential therapeutic advantage over orthosteric ligands. Allosteric enhancers at the adenosine A(1) receptor have been linked to antiarrhythmic and antilipolytic activity. They may also have therapeutic potential as analgesics and neuroprotective agents. A(3) allosteric enhancers are postulated to be useful against ischemic conditions or as antitumor agents. In this review, we address recent developments regarding the medicinal chemistry of such compounds. Most efforts have been and are directed toward adenosine A(1) and A(3) receptors, whereas limited or no information is available for A(2A) and A(2B) receptors. We also discuss some findings, mostly receptor mutation studies, regarding localization of the allosteric binding sites on the receptors.
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http://dx.doi.org/10.1007/s11302-008-9105-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2721769PMC
March 2009

Synthesis and evaluation of homodimeric GnRHR antagonists having a rigid bis-propargylated benzene core.

Bioorg Med Chem 2008 Apr 2;16(7):3744-58. Epub 2008 Feb 2.

Department of Bio-organic Synthesis, Leiden Institute of Chemistry, Gorlaeus Laboratories, Leiden University, PO Box 9502, 2300 RA Leiden, The Netherlands.

The fact that GPCRs might function in a dimeric fashion is currently well accepted. For GnRHR, a GPCR that regulates gonadotropin release, there is evidence that the receptor also functions as a dimer. We here describe the design and synthesis of a set of dimeric GnRHR antagonists in order to understand the interaction of dimeric ligands to the receptor and to address the question whether GnRHR dimerization is a prerequisite for signalling. Biological evaluation of the compounds shows no discrimination between monomeric and dimeric-ligands in respect to binding affinities, however, the dimeric ligands appear to have different functional properties.
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http://dx.doi.org/10.1016/j.bmc.2008.01.054DOI Listing
April 2008

GPCR NaVa database: natural variants in human G protein-coupled receptors.

Hum Mutat 2008 Jan;29(1):39-44

Division of Medicinal Chemistry, Leiden-Amsterdam Center for Drug Research, Leiden University, Leiden, The Netherlands.

The superfamily of human G protein-coupled receptors (GPCRs) is large and regulates a plethora of important physiological processes by transducing extracellular signals over cell membranes. A diversity of natural variants occurs in these receptors, including rare mutations and common polymorphisms. These variants differ in their impact on DNA, ranging from single nucleotide polymorphisms (SNPs) to copy number variants, and in their impact on protein function. Natural variants furthermore vary in their effects on human phenotypes from neutral to disease-associated. As mutation data are highly dispersed over numerous sources, a single resource for variants would aid investigators of GPCRs. The GPCR NaVa database therefore integrates data on natural variants in human GPCRs from online databases, the scientific literature, and patents. Where available, variants contain information on their location in the DNA (and protein sequence), the involved nucleotides (and amino acids), the average frequency of each allele, reported disease associations, and references to public databases and the scientific literature. The GPCR NaVa database aims to facilitate studies into pharmacogenetics, genotype-phenotype, and structure-function relationships of GPCRs. The GPCR NaVa database is interlinked with the family-specific GPCRDB resource and is accessible as a stand-alone database through a user-friendly website at http://nava.liacs.nl (last accessed 28 August 2007).
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http://dx.doi.org/10.1002/humu.20638DOI Listing
January 2008

Synthesis and evaluation of homo-bivalent GnRHR ligands.

Bioorg Med Chem 2007 Jul 6;15(14):4841-56. Epub 2007 May 6.

Department of Bio-organic Synthesis, Leiden Institute of Chemistry, Gorlaeus Laboratories, Leiden University, PO Box 9502, 2300 RA Leiden, The Netherlands.

G protein coupled receptors (GPCRs) are important drug targets in pharmaceutical research. Traditionally, most research efforts have been devoted towards the design of small molecule agonists and antagonists. An interesting, yet poorly investigated class of GPCR modulators comprise the bivalent ligands, in which two receptor pharmacophores are incorporated. Here, we set out to develop a general strategy for the synthesis of bivalent compounds that are projected to bind to the human gonadotropin-releasing hormone receptor (GnRHR). Our results on the dimerisation of a known GnRHR antagonist, with as key step the Huisgen 1,3-cycloaddition, and their ability to bind to and antagonize GnRH-induced GnRHR stimulation, are presented here.
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http://dx.doi.org/10.1016/j.bmc.2007.04.065DOI Listing
July 2007

Characteristic amino acid combinations in olfactory G protein-coupled receptors.

Proteins 2007 Apr;67(1):154-66

Division of Medicinal Chemistry, Leiden/Amsterdam Center for Drug Research, Leiden University, Leiden, The Netherlands.

The human olfactory subgenome has recently been fully characterized with over 1000 genes. Although as many as two thirds of them are expected to be pseudogenes, it still leaves us with about half of all human G protein-coupled receptors being olfactory. It is therefore of great interest to characterize olfactory receptors with high precision. Usually it is done through sequence motifs that are not fully conserved, making an exact characterization difficult. In this paper, we propose a rule-based characterization of olfactory receptors derived from a multiple sequence alignment of human GPCRs. We show that just seven alignment sites are sufficient to characterize 99% of human olfactory GPCRs with one feature, a tyrosine at site 7.41, being of particular importance. We also show dependencies between sites near the extracellular and intracellular region of a membrane-embedded receptor, indicating that olfactory receptors are characterized by a combination of important residues in these two areas, whereas nonolfactory receptors tend to have residues of lower importance at the same sites.
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http://dx.doi.org/10.1002/prot.21112DOI Listing
April 2007

ZM241385, DPCPX, MRS1706 are inverse agonists with different relative intrinsic efficacies on constitutively active mutants of the human adenosine A2B receptor.

J Pharmacol Exp Ther 2007 Feb 31;320(2):637-45. Epub 2006 Oct 31.

Division of Medicinal Chemistry, Leiden/Amsterdam Center for Drug Research, Leiden University, 2300 RA Leiden, The Netherlands.

The human adenosine A(2B) receptor belongs to class A G protein-coupled receptors (GPCRs). In our previous work, constitutively active mutant (CAM) human adenosine A(2B) receptors were identified from a random mutation bank. In the current study, three known A(2B) receptor antagonists, 4-{2-[7-amino-2-(2-furyl)[1,2,4]triazolo-[2,3-a][1,3,5]triazin-5-yl-amino]ethyl}phenol (ZM241385), 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), and N-(4-acetylphenyl)-2-[4-(2,3,6,7-tetrahydro-2,6-dioxo-1,3-dipropyl-1H-purin-8-yl)phenoxy]acetamide (MRS1706) were tested on wild-type and nine CAM A(2B) receptors with different levels of constitutive activity in a yeast growth assay. All three compounds turned out to be inverse agonists for the adenosine A(2B) receptor because they were able to fully reverse the basal activity of four low-level constitutively active A(2B) receptor mutants and to partially reverse the basal activity of three medium-level constitutively active A(2B) receptor mutants. We also discovered two highly constitutively active mutants whose basal activity could not be reversed by any of the three compounds. A two-state receptor model was used to explain the experimental observations; fitting these yielded the following relative intrinsic efficacies for the three inverse agonists ZM241385, DPCPX, and MRS1706: 0.14 +/- 0.03, 0.35 +/- 0.03, and 0.31 +/- 0.02, respectively. Moreover, varying L, the ratio of active versus inactive receptors in this model, from 0.11 for mutant F84L to 999 for two highly constitutively active mutants yielded simulated dose-response curves that mimicked the experimental curves. This study is the first description of inverse agonists for the human adenosine A(2B) receptor. Moreover, the use of receptor mutants with varying levels of constitutive activity enabled us to determine the relative intrinsic efficacy of these inverse agonists.
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http://dx.doi.org/10.1124/jpet.106.111203DOI Listing
February 2007

TreeSOM: Cluster analysis in the self-organizing map.

Neural Netw 2006 Jul-Aug;19(6-7):935-49. Epub 2006 Jun 15.

Division of Medicinal Chemistry, Leiden/Amsterdam Center for Drug Research, Leiden University, Einsteinweg 55, 2333CC Leiden, The Netherlands.

Clustering problems arise in various domains of science and engineering. A large number of methods have been developed to date. The Kohonen self-organizing map (SOM) is a popular tool that maps a high-dimensional space onto a small number of dimensions by placing similar elements close together, forming clusters. Cluster analysis is often left to the user. In this paper we present the method TreeSOM and a set of tools to perform unsupervised SOM cluster analysis, determine cluster confidence and visualize the result as a tree facilitating comparison with existing hierarchical classifiers. We also introduce a distance measure for cluster trees that allows one to select a SOM with the most confident clusters.
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http://dx.doi.org/10.1016/j.neunet.2006.05.003DOI Listing
October 2006

Mining a chemical database for fragment co-occurrence: discovery of "chemical clichés".

J Chem Inf Model 2006 Mar-Apr;46(2):553-62

Division of Medicinal Chemistry, Leiden/Amsterdam Center for Drug Research, Leiden University, Einsteinweg 55, 2300 RA Leiden, The Netherlands.

Nowadays millions of different compounds are known, their structures stored in electronic databases. Analysis of these data could yield valuable insights into the laws of chemistry and the habits of chemists. We have therefore explored the public database of the National Cancer Institute (>250,000 compounds) by pattern searching. We split the molecules of this database into fragments to find out which fragments exist, how frequent they are, and whether the occurrence of one fragment in a molecule is related to the occurrence of another, nonoverlapping fragment. It turns out that some fragments and combinations of fragments are so frequent that they can be called "chemical clichés". We believe that the fragment data can give insight into the chemical space explored so far by synthesis. The lists of fragments and their (co-)occurrences can help create novel chemical compounds by (i) systematically listing the most popular and therefore most easily used substituents and ring systems for synthesizing new compounds, (ii) being an easily accessible repository for rarer fragments suitable for lead compound optimization, and (iii) pointing out some of the yet unexplored parts of chemical space.
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http://dx.doi.org/10.1021/ci050370cDOI Listing
September 2006

The molecule evoluator. An interactive evolutionary algorithm for the design of drug-like molecules.

J Chem Inf Model 2006 Mar-Apr;46(2):545-52

Leiden/Amsterdam Center for Drug Research, Division of Medicinal Chemistry, P.O. Box 9502, 2300RA Leiden, The Netherlands.

We developed a software tool to design drug-like molecules, the "Molecule Evoluator", which we introduce and describe here. An atom-based evolutionary approach was used allowing both several types of mutation and crossover to occur. The novelty, we claim, is the unprecedented interactive evolution, in which the user acts as a fitness function. This brings a human being's creativity, implicit knowledge, and imagination into the design process, next to the more standard chemical rules. Proof-of-concept was demonstrated in a number of ways, both computationally and in the lab. Thus, we synthesized a number of compounds designed with the aid of the Molecule Evoluator. One of these is described here, a new chemical entity with activity on alpha-adrenergic receptors.
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http://dx.doi.org/10.1021/ci050369dDOI Listing
September 2006

Recent developments in constitutive receptor activity and inverse agonism, and their potential for GPCR drug discovery.

Trends Pharmacol Sci 2006 Feb 6;27(2):92-6. Epub 2006 Jan 6.

Department of Pharmacological and Pharmaceutical Sciences, University of Houston, 4800 Calhoun, Houston, TX 77204-5037, USA.

The concept of constitutively active G-protein-coupled receptors is now firmly rooted in receptor pharmacology. Many independent research groups have contributed to its acceptance since its introduction by Costa and Herz in 1989. This concept necessitated a revised ligand classification, and a new category of inverse agonists was introduced alongside existing agonist and antagonist ligands. Initially, it was hoped that new therapeutic modalities would become available. However, the drug industry has not adopted inverse agonism as a design criterion and instead accepted that some compounds emerge as (neutral) antagonists in compound screening, whereas other compounds possess inverse agonistic activity. In this article, we summarize aspects of the impact of constitutive activity on the drug-discovery process: for example, its use in orphan receptor assays, its link with pharmacogenetics and genomics, and its relevance for currently marketed drugs.
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http://dx.doi.org/10.1016/j.tips.2005.12.007DOI Listing
February 2006

Allosteric modulators affect the internalization of human adenosine A1 receptors.

Eur J Pharmacol 2005 Oct 7;522(1-3):1-8. Epub 2005 Oct 7.

Department of Medicinal Chemistry, Leiden/Amsterdam Center for Drug Research, Leiden University, The Netherlands.

To study the effect of allosteric modulators on the internalization of human adenosine A(1) receptors, the receptor was equipped with a C-terminal yellow fluorescent protein tag. The introduction of this tag did not affect the radioligand binding properties of the receptor. CHO cells stably expressing this receptor were subjected during 16 h to varying concentrations of the agonist N(6)-cyclopentyladenosine (CPA) in the absence or presence of 10 microM of the allosteric enhancer PD 81,723 ((2-amino-4,5-dimethyl-3-thienyl)-[3-(trifluoromethyl)phenyl]methanone) or the allosteric inhibitor SCH-202676 (N-(2,3-diphenyl-1,2,4-thiadiazol-5(2H)-ylidene)methanamine). CPA itself was able to internalize 25% and 40% of the receptors at a concentration of 400 nM or 4 muM, respectively. Addition of either PD 81,723 or SCH-202676 alone had no effect on internalization. However, with PD 81,723 a slight amount of internalization was obtained already at 40 nM of CPA and at 400 nM CPA 59% of the receptors internalized. SCH-202676 on the other hand effectively prevented CPA-induced internalization of the receptor.
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http://dx.doi.org/10.1016/j.ejphar.2005.08.052DOI Listing
October 2005

A "locked-on," constitutively active mutant of the adenosine A1 receptor.

Eur J Pharmacol 2005 Mar;510(1-2):1-8

Division of Medicinal Chemistry, Leiden/Amsterdam Center for Drug Research, Leiden University, P.O. Box 9502, 2300 RA Leiden, The Netherlands.

We studied the wild-type human adenosine A1 receptor and three mutant receptors, in which the glycine at position 14 had been changed into an alanine, a leucine, or a threonine residue. All receptors were characterized in radioligand binding experiments, the wild-type and the Gly14Thr mutant receptor in greater detail. Both receptors were allosterically modulated by sodium ions and PD81,723 (2-amino-4,5-dimethyl-3-thienyl-[3(trifluoromethyl)-phenyl]methanone), although in a different way. All mutant receptors appeared to be spontaneously or "constitutively" active in a [35S]GTPgammaS binding assay, the first demonstration of the existence of such CAM (constitutively active mutant) receptors for the adenosine A1 receptor. The Gly14Thr mutant receptor was also constitutively active in another functional assay, i.e., the inhibition of forskolin-induced cAMP production in intact cells. Importantly, this mutant displayed a peculiar "locked-on" phenotype, i.e., neither agonist nor inverse agonist was capable of modulating the basal activity in both the GTPgammaS and the cAMP assay, unlike the wild-type and the two other mutant receptors.
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http://dx.doi.org/10.1016/j.ejphar.2005.01.007DOI Listing
March 2005