Publications by authors named "Laura H Heitman"

98 Publications

Crystal Structure and Subsequent Ligand Design of a Nonriboside Partial Agonist Bound to the Adenosine A Receptor.

J Med Chem 2021 Apr 25;64(7):3827-3842. Epub 2021 Mar 25.

Division of Drug Discovery and Safety, Leiden Academic Centre for Drug Research, Leiden University, Einsteinweg 55, 2333 CC Leiden, The Netherlands.

In this study, we determined the crystal structure of an engineered human adenosine A receptor bound to a partial agonist and compared it to structures cocrystallized with either a full agonist or an antagonist/inverse agonist. The interaction between the partial agonist, belonging to a class of dicyanopyridines, and amino acids in the ligand binding pocket inspired us to develop a small library of derivatives and assess their affinity in radioligand binding studies and potency and intrinsic activity in a functional, label-free, intact cell assay. It appeared that some of the derivatives retained the partial agonist profile, whereas other ligands turned into inverse agonists. We rationalized this remarkable behavior with additional computational docking studies.
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http://dx.doi.org/10.1021/acs.jmedchem.0c01856DOI Listing
April 2021

Design and Characterization of an Intracellular Covalent Ligand for CC Chemokine Receptor 2.

J Med Chem 2021 Mar 18;64(5):2608-2621. Epub 2021 Feb 18.

Division of Drug Discovery and Safety, Leiden Academic Centre for Drug Research, Einsteinweg 55, 2333 CC, Leiden, The Netherlands.

Covalently acting inhibitors constitute a large and growing fraction of approved small-molecule therapeutics as well as useful tools for a variety of and applications. Here, we aimed to develop a covalent antagonist of CC chemokine receptor 2 (CCR2), a class A GPCR that has been pursued as a therapeutic target in inflammation and immuno-oncology. Based on a known intracellularly binding CCR2 antagonist, several covalent derivatives were synthesized and characterized by radioligand binding and functional assays. These studies revealed compound as an intracellular covalent ligand for CCR2. modeling followed by site-directed mutagenesis confirmed that forms a covalent bond with one of three proximal cysteine residues, which can be engaged interchangeably. To our knowledge, compound represents the first covalent ligand reported for CCR2. Due to its unique properties, it may represent a promising tool for ongoing and future studies of CCR2 pharmacology.
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http://dx.doi.org/10.1021/acs.jmedchem.0c01137DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7958898PMC
March 2021

A study of the dopamine transporter using the TRACT assay, a novel in vitro tool for solute carrier drug discovery.

Sci Rep 2021 Jan 14;11(1):1312. Epub 2021 Jan 14.

Division of Drug Discovery and Safety, LACDR, Leiden University, P.O. Box 9502, 2300RA, Leiden, The Netherlands.

Members of the solute carrier (SLC) transporter protein family are increasingly recognized as therapeutic drug targets. The majority of drug screening assays for SLCs are based on the uptake of radiolabeled or fluorescent substrates. Thus, these approaches often have limitations that compromise on throughput or the physiological environment of the SLC. In this study, we report a novel application of an impedance-based biosensor, xCELLigence, to investigate dopamine transporter (DAT) activity via substrate-induced activation of G protein-coupled receptors (GPCRs). The resulting assay, which is coined the 'transporter activity through receptor activation' (TRACT) assay, is based on the hypothesis that DAT-mediated removal of extracellular dopamine directly affects the ability of dopamine to activate cognate membrane-bound GPCRs. In two human cell lines with heterologous DAT expression, dopamine-induced GPCR signaling was attenuated. Pharmacological inhibition or the absence of DAT restored the apparent potency of dopamine for GPCR activation. The inhibitory potencies for DAT inhibitors GBR12909 (pIC = 6.2, 6.6) and cocaine (pIC = 6.3) were in line with values from reported orthogonal transport assays. Conclusively, this study demonstrates the novel use of label-free whole-cell biosensors to investigate DAT activity using GPCR activation as a readout. This holds promise for other SLCs that share their substrate with a GPCR.
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http://dx.doi.org/10.1038/s41598-020-79218-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7809260PMC
January 2021

G protein-coupled receptors expressed and studied in yeast. The adenosine receptor as a prime example.

Biochem Pharmacol 2020 Dec 16:114370. Epub 2020 Dec 16.

Drug Discovery and Safety, Leiden Academic Centre for Drug Research, Einsteinweg 55, 2333 CC Leiden, The Netherlands.

G protein-coupled receptors (GPCRs) are the largest class of membrane proteins with around 800 members in the human genome/proteome. Extracellular signals such as hormones and neurotransmitters regulate various biological processes via GPCRs, with GPCRs being the bodily target of 30-40% of current drugs on the market. Complete identification and understanding of GPCR functionality will provide opportunities for novel drug discovery. Yeast expresses three different endogenous GPCRs regulating pheromone and sugar sensing, with the pheromone pathway offering perspectives for the characterization of heterologous GPCR signaling. Moreover, yeast offers a ''null" background for studies on mammalian GPCRs, including GPCR activation and signaling, ligand identification, and characterization of disease-related mutations. This review focuses on modifications of the yeast pheromone signaling pathway for functional GPCR studies, and on opportunities and usage of the yeast system as a platform for human GPCR studies. Finally, this review discusses in some further detail studies of adenosine receptors heterologously expressed in yeast, and what Geoff Burnstock thought of this approach.
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http://dx.doi.org/10.1016/j.bcp.2020.114370DOI Listing
December 2020

Molecular probes for the human adenosine receptors.

Purinergic Signal 2021 Mar 12;17(1):85-108. Epub 2020 Dec 12.

Division of Drug Discovery and Safety, Leiden Academic Centre for Drug Research, Leiden University, Einsteinweg 55, 2333 CC, Leiden, The Netherlands.

Adenosine receptors, G protein-coupled receptors (GPCRs) that are activated by the endogenous ligand adenosine, have been considered potential therapeutic targets in several disorders. To date however, only very few adenosine receptor modulators have made it to the market. Increased understanding of these receptors is required to improve the success rate of adenosine receptor drug discovery. To improve our understanding of receptor structure and function, over the past decades, a diverse array of molecular probes has been developed and applied. These probes, including radioactive or fluorescent moieties, have proven invaluable in GPCR research in general. Specifically for adenosine receptors, the development and application of covalent or reversible probes, whether radiolabeled or fluorescent, have been instrumental in the discovery of new chemical entities, the characterization and interrogation of adenosine receptor subtypes, and the study of adenosine receptor behavior in physiological and pathophysiological conditions. This review summarizes these applications, and also serves as an invitation to walk another mile to further improve probe characteristics and develop additional tags that allow the investigation of adenosine receptors and other GPCRs in even finer detail.
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http://dx.doi.org/10.1007/s11302-020-09753-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7954947PMC
March 2021

Inhibition of macrophage migration in zebrafish larvae demonstrates in vivo efficacy of human CCR2 inhibitors.

Dev Comp Immunol 2021 Mar 22;116:103932. Epub 2020 Nov 22.

Animal Sciences and Health, Institute of Biology Leiden, Leiden University, Einsteinweg 55room BS1.02, 2333 CC Leiden, the Netherlands. Electronic address:

The chemokine signaling axes CCR2-CCL2 and CXCR3-CXCL11 participate in the inflammatory response by recruiting leukocytes to damaged tissue or sites of infection and are, therefore, potential pharmacological targets to treat inflammatory disorders. Although multiple CCR2 orthosteric and allosteric inhibitors have been developed, none of these compounds has been approved for clinical use, highlighting the need for a fast, simple and robust preclinical test system to determine the in vivo efficacy of CCR2 inhibitors. Herein we show that human CCL2 and CXCL11 drive macrophage recruitment in zebrafish larvae and that CCR2 inhibitors designed for humans also limit macrophage recruitment in this model organism due to the high conservation of the chemokine system. We demonstrated anti-inflammatory activities of three orthosteric and two allosteric CCR2 inhibitors using macrophage recruitment to injury as a functional read-out of their efficiency, while simultaneously evaluating toxicity. These results provide proof-of-principle for screening CCR2 inhibitors in the zebrafish model.
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http://dx.doi.org/10.1016/j.dci.2020.103932DOI Listing
March 2021

Development of the First Potential Nonpeptidic Positron Emission Tomography Tracer for the Imaging of CCR2 Receptors.

ChemMedChem 2021 Feb 23;16(4):640-645. Epub 2020 Nov 23.

Institut für Pharmazeutische und Medizinische Chemie der Universität Münster, Corrensstraße 48, 48149, Münster, Germany.

Herein we report the design and synthesis of a series of highly selective CCR2 antagonists as F-labeled PET tracers. The derivatives were evaluated extensively for their off-target profile at 48 different targets. The most potent and selective candidate was applied in vivo in a biodistribution study, demonstrating a promising profile for further preclinical development. This compound represents the first potential nonpeptidic PET tracer for the imaging of CCR2 receptors.
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http://dx.doi.org/10.1002/cmdc.202000728DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7983900PMC
February 2021

Design and pharmacological profile of a novel covalent partial agonist for the adenosine A receptor.

Biochem Pharmacol 2020 10 10;180:114144. Epub 2020 Jul 10.

Division of Drug Discovery and Safety, Leiden Academic Centre for Drug Research (LACDR), Leiden University, P.O. Box 9502, 2300RA Leiden, The Netherlands. Electronic address:

Partial agonists for G protein-coupled receptors (GPCRs) provide opportunities for novel pharmacotherapies with enhanced on-target safety compared to full agonists. For the human adenosine A receptor (hAAR) this has led to the discovery of capadenoson, which has been in phase IIa clinical trials for heart failure. Accordingly, the design and profiling of novel hAAR partial agonists has become an important research focus. In this study, we report on LUF7746, a capadenoson derivative bearing an electrophilic fluorosulfonyl moiety, as an irreversibly binding hAAR modulator. Meanwhile, a nonreactive ligand bearing a methylsulfonyl moiety, LUF7747, was designed as a control probe in our study. In a radioligand binding assay, LUF7746's apparent affinity increased to nanomolar range with longer pre-incubation time, suggesting an increasing level of covalent binding over time. Moreover, compared to the reference full agonist CPA, LUF7746 was a partial agonist in a hAAR-mediated G protein activation assay and resistant to blockade with an antagonist/inverse agonist. An in silico structure-based docking study combined with site-directed mutagenesis of the hAAR demonstrated that amino acid Y271 was the primary anchor point for the covalent interaction. Additionally, a label-free whole-cell assay was set up to identify LUF7746's irreversible activation of an A receptor-mediated cell morphological response. These results led us to conclude that LUF7746 is a novel covalent hAAR partial agonist and a valuable chemical probe for further mapping the receptor activation process. It may also serve as a prototype for a therapeutic approach in which a covalent partial agonist may cause less on-target side effects, conferring enhanced safety compared to a full agonist.
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http://dx.doi.org/10.1016/j.bcp.2020.114144DOI Listing
October 2020

Characterization of cancer-related somatic mutations in the adenosine A receptor.

Eur J Pharmacol 2020 Aug 26;880:173126. Epub 2020 Apr 26.

Drug Discovery and Safety, Leiden Academic Centre for Drug Research, Einsteinweg 55, 2333 CC, Leiden, the Netherlands. Electronic address:

In cancer, G protein-coupled receptors (GPCRs) are involved in tumor progression and metastasis. In this study we particularly examined one GPCR, the adenosine A receptor. This receptor is activated by high concentrations of its endogenous ligand adenosine, which suppresses the immune response to fight tumor progression. A series of adenosine A receptor mutations were retrieved from the Cancer Genome Atlas harboring data from patient samples with different cancer types. The main goal of this work was to investigate the pharmacology of these mutant receptors using a 'single-GPCR-one-G protein' yeast assay technology. Concentration-growth curves were obtained with the full agonist NECA for the wild-type receptor and 15 mutants. Compared to wild-type receptor, the constitutive activity levels in mutant receptors F141L, Y202C and L310P were high, while the potency and efficacy of NECA and BAY 60-6583 on Y202C was lower. A 33- and 26-fold higher constitutive activity on F141L and L310P was reduced to wild-type levels in response to the inverse agonist ZM241385. These constitutively active mutants may thus be tumor promoting. Mutant receptors F259S and Y113F showed a more than one log-unit decrease in potency. A complete loss of activation was observed in mutant receptors C29R, W130C and P249L. All mutations were characterized at the structural level, generating hypotheses of their roles on modulating the receptor conformational equilibrium. Taken together, this study is the first to investigate the nature of adenosine A receptor cancer mutations and may thus provide insights in mutant receptor function in cancer.
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http://dx.doi.org/10.1016/j.ejphar.2020.173126DOI Listing
August 2020

Perspective: Implications of Ligand-Receptor Binding Kinetics for Therapeutic Targeting of G Protein-Coupled Receptors.

ACS Pharmacol Transl Sci 2020 Apr 18;3(2):179-189. Epub 2020 Mar 18.

Laboratory for Molecular Pharmacology, Department of Biomedical Sciences, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen DK 2200, Denmark.

The concept of ligand-receptor binding kinetics has been broadly applied in drug development pipelines focusing on G protein-coupled receptors (GPCRs). The ligand residence time (RT) for a receptor describes how long a ligand-receptor complex exists, and is defined as the reciprocal of the dissociation rate constant (). RT has turned out to be a valuable parameter for GPCR researchers focusing on drug development as a good predictor of efficacy. The positive correlation between RT and efficacy has been established for several drugs targeting class A GPCRs (e.g., the neurokinin-1 receptor (NKR), the β adrenergic receptor (βAR), and the muscarinic 3 receptor (MR)) and for drugs targeting class B1 (e.g., the glucagon-like peptide 1 receptor (GLP-1R)). Recently, the association rate constant () has gained similar attention as another parameter affecting efficacy. In the current perspective, we address the importance of studying ligand-receptor binding kinetics for therapeutic targeting of GPCRs, with an emphasis on how binding kinetics can be altered by subtle molecular changes in the ligands and/or the receptors and how such changes affect treatment outcome. Moreover, we speculate on the impact of binding kinetic parameters for functional selectivity and sustained receptor signaling from endosomal compartments; phenomena that have gained increasing interest in attempts to improve therapeutic targeting of GPCRs.
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http://dx.doi.org/10.1021/acsptsci.0c00012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7155193PMC
April 2020

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

Affinity, binding kinetics and functional characterization of draflazine analogues for human equilibrative nucleoside transporter 1 (SLC29A1).

Biochem Pharmacol 2020 02 10;172:113747. Epub 2019 Dec 10.

Division of Drug Discovery and Safety, Leiden Academic Centre for Drug Research (LACDR), Leiden University, P.O. Box 9502, 2300 RA Leiden, the Netherlands. Electronic address:

In the last decade it has been recapitulated that receptor-ligand binding kinetics is a relevant additional parameter in drug discovery to improve in vivo drug efficacy and safety. The equilibrative nucleoside transporter-1 (ENT1, SLC29A1) is an important drug target, as transporter inhibition is a potential treatment of ischemic heart disease, stroke, and cancer. Currently, two non-selective ENT1 inhibitors (dilazep and dipyridamole) are on the market as vasodilators. However, their binding kinetics are unknown; moreover, novel, more effective and selective inhibitors are still needed. Hence, this study focused on the incorporation of binding kinetics for finding new and improved ENT1 inhibitors. We developed a radioligand competition association assay to determine the binding kinetics of ENT1 inhibitors with four chemical scaffolds (including dilazep and dipyridamole). The kinetic parameters were compared to the affinities obtained from a radioligand displacement assay. Three of the scaffolds presented high affinities with relatively fast dissociation kinetics, yielding short to moderate residence times (RTs) at the protein (1-44 min). While compounds from the fourth scaffold, i.e. draflazine analogues, also had high affinity, they displayed significantly longer RTs, with one analogue (4) having a RT of over 10 h. Finally, a label-free assay was used to evaluate the impact of divergent ENT1 inhibitor binding kinetics in a functional assay. It was shown that the potency of compound 4 increased with longer incubation times, which was not observed for draflazine, supporting the importance of long RT for increased target-occupancy and effect. In conclusion, our research shows that high affinity ENT1 inhibitors show a large variation in residence times at this transport protein. As a consequence, incorporation of binding kinetic parameters adds to the design criteria and may thus result in a different lead compound selection. Taken together, this kinetic approach could inspire future drug discovery in the field of ENT1 and membrane transport proteins in general.
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http://dx.doi.org/10.1016/j.bcp.2019.113747DOI Listing
February 2020

Synthesis and Pharmacological Evaluation of Triazolopyrimidinone Derivatives as Noncompetitive, Intracellular Antagonists for CC Chemokine Receptors 2 and 5.

J Med Chem 2019 12 13;62(24):11035-11053. Epub 2019 Dec 13.

Division of Drug Discovery and Safety, Leiden Academic Centre for Drug Research , Leiden University , P.O. Box 9502, 2300 RA Leiden , The Netherlands.

CC chemokine receptors 2 (CCR2) and 5 (CCR5) are involved in many inflammatory diseases; however, most CCR2 and CCR5 clinical candidates have been unsuccessful. (Pre)clinical evidence suggests that dual CCR2/CCR5 inhibition might be more effective in the treatment of such multifactorial diseases. In this regard, the highly conserved intracellular binding site in chemokine receptors provides a new avenue for the design of multitarget ligands. In this study, we synthesized and evaluated the biological activity of a series of triazolopyrimidinone derivatives in CCR2 and CCR5. Radioligand binding assays first showed that they bind to the intracellular site of CCR2, and in combination with functional assays on CCR5, we explored structure-affinity/activity relationships in both receptors. Although most compounds were CCR2-selective, and inhibited β-arrestin recruitment in CCR5 with high potency. Moreover, these compounds displayed an insurmountable mechanism of inhibition in both receptors, which holds promise for improved efficacy in inflammatory diseases.
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http://dx.doi.org/10.1021/acs.jmedchem.9b00742DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6935887PMC
December 2019

A multiple classifier system identifies novel cannabinoid CB2 receptor ligands.

J Cheminform 2019 Nov 7;11(1):66. Epub 2019 Nov 7.

Drug Discovery and Safety, LACDR, Leiden University, Einsteinweg 55, 2333 CC, Leiden, The Netherlands.

Drugs have become an essential part of our lives due to their ability to improve people's health and quality of life. However, for many diseases, approved drugs are not yet available or existing drugs have undesirable side effects, making the pharmaceutical industry strive to discover new drugs and active compounds. The development of drugs is an expensive process, which typically starts with the detection of candidate molecules (screening) after a protein target has been identified. To this end, the use of high-performance screening techniques has become a critical issue in order to palliate the high costs. Therefore, the popularity of computer-based screening (often called virtual screening or in silico screening) has rapidly increased during the last decade. A wide variety of Machine Learning (ML) techniques has been used in conjunction with chemical structure and physicochemical properties for screening purposes including (i) simple classifiers, (ii) ensemble methods, and more recently (iii) Multiple Classifier Systems (MCS). Here, we apply an MCS for virtual screening (D2-MCS) using circular fingerprints. We applied our technique to a dataset of cannabinoid CB2 ligands obtained from the ChEMBL database. The HTS collection of Enamine (1,834,362 compounds), was virtually screened to identify 48,232 potential active molecules using D2-MCS. Identified molecules were ranked to select 21 promising novel compounds for in vitro evaluation. Experimental validation confirmed six highly active hits (> 50% displacement at 10 µM and subsequent Ki determination) and an additional five medium active hits (> 25% displacement at 10 µM). Hence, D2-MCS provided a hit rate of 29% for highly active compounds and an overall hit rate of 52%.
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http://dx.doi.org/10.1186/s13321-019-0389-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6836644PMC
November 2019

Label-free detection of transporter activity via GPCR signalling in living cells: A case for SLC29A1, the equilibrative nucleoside transporter 1.

Sci Rep 2019 09 24;9(1):13802. Epub 2019 Sep 24.

Division of Drug Discovery and Safety, Leiden Academic Centre for Drug Research (LACDR), Leiden University, P.O. Box 9502, 2300 RA, Leiden, The Netherlands.

Transporters are important therapeutic but yet understudied targets due to lack of available assays. Here we describe a novel label-free, whole-cell method for the functional assessment of Solute Carrier (SLC) inhibitors. As many SLC substrates are also ligands for G protein-coupled receptors (GPCRs), transporter inhibition may affect GPCR signalling due to a change in extracellular concentration of the substrate/ligand, which can be monitored by an impedance-based label-free assay. For this study, a prototypical SLC/GPCR pair was selected, i.e. the equilibrative nucleoside transporter-1 (SLC29A1/ENT1) and an adenosine receptor (AR), for which adenosine is the substrate/ligand. ENT1 inhibition with three reference compounds was monitored sensitively via AR activation on human osteosarcoma cells. Firstly, the inhibitor addition resulted in an increased apparent potency of adenosine. Secondly, all inhibitors concentration-dependently increased the extracellular adenosine concentration, resulting in an indirect quantitative assessment of their potencies. Additionally, AR activation was abolished by AR antagonists, confirming that the monitored impedance was AR-mediated. In summary, we developed a novel assay as an in vitro model system that reliably assessed the potency of SLC29A1 inhibitors via AR signalling. As such, the method may be applied broadly as it has the potential to study a multitude of SLCs via concomitant GPCR signalling.
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http://dx.doi.org/10.1038/s41598-019-48829-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6760145PMC
September 2019

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

A live cell NanoBRET binding assay allows the study of ligand-binding kinetics to the adenosine A receptor.

Purinergic Signal 2019 06 27;15(2):139-153. Epub 2019 Mar 27.

Cell Signalling and Pharmacology Research Group, Division of Physiology, Pharmacology and Neuroscience, School of Life Sciences, University of Nottingham, Nottingham, UK.

There is a growing interest in understanding the binding kinetics of compounds that bind to G protein-coupled receptors prior to progressing a lead compound into clinical trials. The widely expressed adenosine A receptor (AAR) has been implicated in a range of diseases including immune conditions, and compounds that aim to selectively target this receptor are currently under development for arthritis. Kinetic studies at the AAR have been performed using a radiolabelled antagonist, but due to the kinetics of this probe, they have been carried out at 10 °C in membrane preparations. In this study, we have developed a live cell NanoBRET ligand binding assay using fluorescent AAR antagonists to measure kinetic parameters of labelled and unlabelled compounds at the AAR at physiological temperatures. The kinetic profiles of four fluorescent antagonists were determined in kinetic association assays, and it was found that XAC-ser-tyr-X-BY630 had the longest residence time (RT = 288 ± 62 min) at the AAR. The association and dissociation rate constants of three antagonists PSB-11, compound 5, and LUF7565 were also determined using two fluorescent ligands (XAC-ser-tyr-X-BY630 or AV039, RT = 6.8 ± 0.8 min) as the labelled probe and compared to those obtained using a radiolabelled antagonist ([H]PSB-11, RT = 44.6 ± 3.9 min). There was close agreement in the kinetic parameters measured with AV039 and [H]PSB-11 but significant differences to those obtained using XAC-S-ser-S-tyr-X-BY630. These data indicate that selecting a probe with the appropriate kinetics is important to accurately determine the kinetics of unlabelled ligands with markedly different kinetic profiles.
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http://dx.doi.org/10.1007/s11302-019-09650-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6635573PMC
June 2019

Long residence time adenosine A receptor agonists produce sustained wash-resistant antilipolytic effect in rat adipocytes.

Biochem Pharmacol 2019 06 21;164:45-52. Epub 2019 Mar 21.

Jiangsu Key Laboratory of New Drug Research and Clinical Pharmacy, Xuzhou Medical University, 209 Tongshan Road, Xuzhou 221004, Jiangsu, China. Electronic address:

Elevated circulating free fatty acid (FFA) level is closely linked to the pathogenesis of insulin resistance and type 2 diabetes mellitus. Activation of the adenosine A receptor (AR) inhibits lipolysis in adipocytes and hence reduces the concentration of FFA, which represents a potential target for the development of antilipolytic agents. We aimed to assess the binding affinity as well as target binding kinetics of AR agonists and further delineate a possible relationship with their antilipolytic effect in adipocytes. Radioligand binding assays were performed to determine the affinity and kinetics of three representative AR agonists, namely CPA, LUF6944 and LUF6941, on the rat AR. Functional responses to these agonists were examined in both a recombinant cell system and physiologically relevant rat adipocytes. The three AR agonists displayed similar affinity while divergent target binding kinetics on the rat AR. Irrespective of equilibrium binding affinity, temporal analysis of receptor signaling demonstrated persistent functional responses for the long residence time agonist, despite removal of excess agonist, in both a recombinant cell system and in rat adipocytes. By contrast, such effect was less pronounced or even lost for agonists with medium or short receptor residence time, respectively. Our results indicate that ligand receptor binding kinetics rather than their affinity or potency play an essential role in regulating cellular responses. The long residence time AR agonist produces a sustained wash-resistant antilipolytic effect in rat adipocytes and thus may represent a potential antilipolytic alternative for further investigation.
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http://dx.doi.org/10.1016/j.bcp.2019.03.032DOI Listing
June 2019

Development of Covalent Ligands for G Protein-Coupled Receptors: A Case for the Human Adenosine A Receptor.

J Med Chem 2019 04 28;62(7):3539-3552. Epub 2019 Mar 28.

Division of Drug Discovery and Safety, Leiden Academic Centre for Drug Research , Leiden University , Einsteinweg 55 , 2333 CC Leiden , The Netherlands.

The development of covalent ligands for G protein-coupled receptors (GPCRs) is not a trivial process. Here, we report a streamlined workflow thereto from synthesis to validation, exemplified by the discovery of a covalent antagonist for the human adenosine A receptor (hAAR). Based on the 1 H,3 H-pyrido[2,1- f]purine-2,4-dione scaffold, a series of ligands bearing a fluorosulfonyl warhead and a varying linker was synthesized. This series was subjected to an affinity screen, revealing compound 17b as the most potent antagonist. In addition, a nonreactive methylsulfonyl derivative 19 was developed as a reversible control compound. A series of assays, comprising time-dependent affinity determination, washout experiments, and [S]GTPγS binding assays, then validated 17b as the covalent antagonist. A combined in silico hAAR-homology model and site-directed mutagenesis study was performed to demonstrate that amino acid residue Y265 was the unique anchor point of the covalent interaction. This workflow might be applied to other GPCRs to guide the discovery of covalent ligands.
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http://dx.doi.org/10.1021/acs.jmedchem.8b02026DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6466477PMC
April 2019

Label-free impedance-based whole cell assay to study GPCR pharmacology.

Methods Cell Biol 2019 11;149:179-194. Epub 2018 Sep 11.

Division of Drug Discovery and Safety, Leiden Academic Centre for Drug Research (LACDR), Leiden University, Leiden, The Netherlands. Electronic address:

The superfamily of G protein-coupled receptors (GPCRs) represents the largest group of cell surface receptors in the human body. It is estimated that around 40% of the drugs currently on the market target GPCRs. As only a very small number of GPCRs is targeted by these marketed drugs, the potential of GPCRs as novel drug targets remains enormous. As opposed to conventional in vitro assays, label-free cellular assays using a biosensor provide new opportunities for studying GPCRs. Integrated receptor-mediated responses are measured in real-time rather than a single downstream signaling pathway, without the need for the use of any label (e.g., fluorescent or radioactive). Here, we describe a protocol to study GPCR pharmacology using the label-free whole cell impedance-based biosensor system xCELLigence. This assay allows quantification of compound-induced GPCR-mediated responses in real-time. Finally, we have also discussed the analysis and interpretation of the results obtained with this assay using the mGlu receptor as a model system.
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http://dx.doi.org/10.1016/bs.mcb.2018.08.003DOI Listing
April 2019

Pyrrolone Derivatives as Intracellular Allosteric Modulators for Chemokine Receptors: Selective and Dual-Targeting Inhibitors of CC Chemokine Receptors 1 and 2.

J Med Chem 2018 10 11;61(20):9146-9161. Epub 2018 Oct 11.

Division of Drug Discovery and Safety , Leiden Academic Centre for Drug Research, Leiden University , P.O. Box 9502, 2300 RA Leiden , The Netherlands.

The recent crystal structures of CC chemokine receptors 2 and 9 (CCR2 and CCR9) have provided structural evidence for an allosteric, intracellular binding site. The high conservation of residues involved in this site suggests its presence in most chemokine receptors, including the close homologue CCR1. By using [H]CCR2-RA-[ R], a high-affinity, CCR2 intracellular ligand, we report an intracellular binding site in CCR1, where this radioligand also binds with high affinity. In addition, we report the synthesis and biological characterization of a series of pyrrolone derivatives for CCR1 and CCR2, which allowed us to identify several high-affinity intracellular ligands, including selective and potential multitarget antagonists. Evaluation of selected compounds in a functional [S]GTPγS assay revealed that they act as inverse agonists in CCR1, providing a new manner of pharmacological modulation. Thus, this intracellular binding site enables the design of selective and multitarget inhibitors as a novel therapeutic approach.
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http://dx.doi.org/10.1021/acs.jmedchem.8b00605DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6328288PMC
October 2018

Bioisosteric replacement of central 1,2,4-oxadiazole ring of high affinity CB ligands by regioisomeric 1,3,4-oxadiazole ring.

Medchemcomm 2017 Aug 19;8(8):1697-1705. Epub 2017 Jul 19.

Institut für Pharmazeutische und Medizinische Chemie der Universität Münster , Corrensstraße 48 , D-48149 Münster , Germany . Email: ; ; Tel: +49 251 8333311.

It has been reported that bioisosteric replacement of an 1,2,4-oxadiazole ring by an 1,3,4-oxadiazole ring leads to higher polarity, reduced metabolic degradation by human liver microsomes and reduced interaction with hERG channels. In a seven to eight step synthesis 1,3,4-oxadiazles were synthesized as bioisosteric analogs of high-affinity but rather lipophilic CB ligands containing an 1,2,4-oxadiazole ring. The 1,3,4-oxadiazole derivatives and show 10- and 50-fold reduced CB affinity compared to the 1,2,4-oxadiazole derivatives and , respectively. However, the 1,3,4-oxadiazole has high CB affinity ( = 25 nM) and high selectivity over the CB receptor.
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http://dx.doi.org/10.1039/c7md00296cDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6072079PMC
August 2017

An Affinity-Based Probe for the Human Adenosine A Receptor.

J Med Chem 2018 09 21;61(17):7892-7901. Epub 2018 Aug 21.

Discovery Sciences, IMED Biotech Unit , AstraZeneca , Gothenburg , Sweden.

Using activity-based protein profiling (ABPP), functional proteins can be interrogated in their native environment. Despite their pharmaceutical relevance, G protein-coupled receptors (GPCRs) have been difficult to address through ABPP. In the current study, we took the prototypical human adenosine A receptor (hAR) as the starting point for the construction of a chemical toolbox allowing two-step affinity-based labeling of GPCRs. First, we equipped an irreversibly binding hAR ligand with a terminal alkyne to serve as probe. We showed that our probe irreversibly and concentration-dependently labeled purified hAR. Click-ligation with a sulfonated cyanine-3 fluorophore allowed us to visualize the receptor on SDS-PAGE. We further demonstrated that labeling of the purified hAR by our probe could be inhibited by selective antagonists. Lastly, we showed successful labeling of the receptor in cell membranes overexpressing hAR, making our probe a promising affinity-based tool compound that sets the stage for the further development of probes for GPCRs.
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http://dx.doi.org/10.1021/acs.jmedchem.8b00860DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6150691PMC
September 2018

Impact of allosteric modulation: Exploring the binding kinetics of glutamate and other orthosteric ligands of the metabotropic glutamate receptor 2.

Biochem Pharmacol 2018 09 17;155:356-365. Epub 2018 Jul 17.

Division of Drug Discovery and Safety, Leiden Academic Centre for Drug Research (LACDR), Leiden University, P.O. Box 9502, 2300RA Leiden, The Netherlands. Electronic address:

While many orthosteric ligands have been developed for the mGlu receptor, little is known about their target binding kinetics and how these relate to those of the endogenous agonist glutamate. Here, the kinetic rate constants, i.e. k and k, of glutamate were determined for the first time followed by those of the synthetic agonist LY354740 and antagonist LY341495. To increase the understanding of the binding mechanism and impact of allosteric modulation thereon, kinetic experiments were repeated in the presence of allosteric modulators. Functional assays were performed to further study the interplay between the orthosteric and allosteric binding sites, including an impedance-based morphology assay. We found that dissociation rate constants of orthosteric mGlu ligands were all within a small 6-fold range, whereas association rate constants were ranging over more than three orders of magnitude and correlated to both affinity and potency. The latter showed that target engagement of orthosteric mGlu ligands is k-driven in vitro. Moreover, only the off-rates of the two agonists were decreased by a positive allosteric modulator (PAM), thereby increasing their affinity. Interestingly, a PAM increased the duration of a glutamate-induced cellular response. A negative allosteric modulator (NAM) increased both on- and off-rate of glutamate without changing its affinity, while it did not affect these parameters for LY354740, indicating probe-dependency. In conclusion, we found that affinity- or potency-based orthosteric ligand optimization primarily results in ligands with high k values. Moreover, positive allosteric modulators alter the binding kinetics of orthosteric agonists mainly by decreasing k, which we were able to correlate to a lengthened cellular response. Together, this study shows the importance of studying binding kinetics in early drug discovery, as this may provide important insights towards improved efficacy in vivo.
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http://dx.doi.org/10.1016/j.bcp.2018.07.014DOI Listing
September 2018

Development of a Cannabinoid-Based Photoaffinity Probe to Determine the Δ-Tetrahydrocannabinol Protein Interaction Landscape in Neuroblastoma Cells.

Cannabis Cannabinoid Res 2018 1;3(1):136-151. Epub 2018 Jul 1.

Department of Molecular Physiology, Leiden Institute of Chemistry, Leiden University, Leiden, The Netherlands.

Δ-Tetrahydrocannabinol (THC), the principle psychoactive ingredient in , is widely used for its therapeutic effects in a large variety of diseases, but it also has numerous neurological side effects. The cannabinoid receptors (CBRs) are responsible to a large extent for these, but not all biological responses are mediated via the CBRs. The identification of additional target proteins of THC to enable a better understanding of the (adverse) physiological effects of THC. In this study, a chemical proteomics approach using a two-step photoaffinity probe is applied to identify potential proteins that may interact with THC. Photoaffinity probe , containing a diazirine as a photocrosslinker, and a terminal alkyne as a ligation handle, was synthesized in 14 steps. It demonstrated high affinity for both CBRs. Subsequently, two-step photoaffinity labeling in neuroblastoma cells led to identification of four potential novel protein targets of THC. The identification of these putative protein hits is a first step towards a better understanding of the protein interaction profile of THC, which could ultimately lead to the development of novel therapeutics based on THC.
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http://dx.doi.org/10.1089/can.2018.0003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6038054PMC
July 2018

Intracellular Receptor Modulation: Novel Approach to Target GPCRs.

Trends Pharmacol Sci 2018 06 10;39(6):547-559. Epub 2018 Apr 10.

Division of Drug Discovery and Safety, Leiden Academic Centre for Drug Research, Leiden University, Einsteinweg 55, 2333 CC, Leiden, The Netherlands. Electronic address:

Recent crystal structures of multiple G protein-coupled receptors (GPCRs) have revealed a highly conserved intracellular pocket that can be used to modulate these receptors from the inside. This novel intracellular site partially overlaps with the G protein and β-arrestin binding site, providing a new manner of pharmacological intervention. Here we provide an update of the architecture and function of the intracellular region of GPCRs, until now portrayed as the signaling domain. We review the available evidence on the presence of intracellular binding sites among chemokine receptors and other class A GPCRs, as well as different strategies to target it, including small molecules, pepducins, and nanobodies. Finally, the potential advantages of intracellular (allosteric) ligands over orthosteric ligands are also discussed.
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http://dx.doi.org/10.1016/j.tips.2018.03.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7048003PMC
June 2018

Constitutive activity of the metabotropic glutamate receptor 2 explored with a whole-cell label-free biosensor.

Biochem Pharmacol 2018 06 30;152:201-210. Epub 2018 Mar 30.

Division of Drug Discovery and Safety, Leiden Academic Centre for Drug Research (LACDR), Leiden University, P.O. Box 9502, 2300RA Leiden, The Netherlands. Electronic address:

Label-free cellular assays using a biosensor provide new opportunities for studying G protein-coupled receptor (GPCR) signaling. As opposed to conventional in vitro assays, integrated receptor-mediated cellular responses are determined in real-time rather than a single downstream signaling pathway. In this study, we examined the potential of a label-free whole cell impedance-based biosensor system (i.e. xCELLigence) to study the pharmacology of one GPCR in particular, the mGlu receptor. This receptor is a target for the treatment of several psychiatric diseases such as schizophrenia and depression. After optimization of assay conditions to prevent interference of endogenous glutamate in the culture medium, detailed pharmacological assessments were performed. Concentration-response curves showed a concentration-dependent increase in impedance for agonists and positive allosteric modulators, whereas receptor inhibition by an antagonist or negative allosteric modulator resulted in a concentration-dependent decrease in cellular impedance. Interestingly, constitutive receptor activity was observed that was decreased by LY341495, which therefore behaved as an inverse agonist here, a property that was heretofore unappreciated. This was confirmed by concentration-dependent modulation of LY341495 potency and efficacy by a allosteric modulators. In summary, the use of the xCELLigence system to study mGlu receptor pharmacology was validated. This is the first class C GPCR to be characterized extensively by such method, opening new avenues to study receptor pharmacology including inverse agonism and demonstrating its value for future drug discovery efforts of mGlu receptors as well as other GPCRs.
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http://dx.doi.org/10.1016/j.bcp.2018.03.026DOI Listing
June 2018

Structure-kinetic relationship studies of cannabinoid CB receptor agonists reveal substituent-specific lipophilic effects on residence time.

Biochem Pharmacol 2018 06 21;152:129-142. Epub 2018 Mar 21.

Division of Drug Discovery and Safety, Leiden Academic Centre for Drug Research, University of Leiden, Einsteinweg 55, 2333 AR, The Netherlands. Electronic address:

A decade ago, the drug-target residence time model has been (re-)introduced, which describes the importance of binding kinetics of ligands on their protein targets. Since then, it has been applied successfully for multiple protein targets, including GPCRs, for the development of lead compounds with slow dissociation kinetics (i.e. long target residence time) to increase in vivo efficacy or with short residence time to prevent on-target associated side effects. To date, this model has not been applied in the design and pharmacological evaluation of novel selective ligands for the cannabinoid CB receptor (CBR), a GPCR with therapeutic potential in the treatment of tissue injury and inflammatory diseases. Here, we have investigated the relationships between physicochemical properties, binding kinetics and functional activity in two different signal transduction pathways, G protein activation and β-arrestin recruitment. We synthesized 24 analogues of 3-cyclopropyl-1-(4-(6-((1,1-dioxidothiomorpholino)methyl)-5-fluoropyridin-2-yl)benzyl)imidazoleidine-2,4-dione (LEI101), our previously reported in vivo active and CBR-selective agonist, with varying basicity and lipophilicity. We identified a positive correlation between target residence time and functional potency due to an increase in lipophilicity on the alkyl substituents, which was not the case for the amine substituents. Basicity of the agonists did not show a relationship with affinity, residence time or functional activity. Our findings provide important insights about the effects of physicochemical properties of the specific substituents of this scaffold on the binding kinetics of agonists and their CBR pharmacology. This work therefore shows how CBR agonists can be designed to have optimal kinetic profiles, which could aid the lead optimization process in drug discovery for the study or treatment of inflammatory diseases.
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http://dx.doi.org/10.1016/j.bcp.2018.03.018DOI Listing
June 2018

Covalent Allosteric Probe for the Metabotropic Glutamate Receptor 2: Design, Synthesis, and Pharmacological Characterization.

J Med Chem 2019 01 7;62(1):223-233. Epub 2018 Mar 7.

Division of Drug Discovery and Safety, Leiden Academic Centre for Drug Research (LACDR) , Leiden University , P.O. Box 9502, 2300RA Leiden , The Netherlands.

Covalent labeling of G protein-coupled receptors (GPCRs) by small molecules is a powerful approach to understand binding modes, mechanism of action, pharmacology, and even facilitate structure elucidation. We report the first covalent positive allosteric modulator (PAM) for a class C GPCR, the mGlu receptor. Three putatively covalent mGlu PAMs were designed and synthesized. Pharmacological characterization identified 2 to bind the receptor covalently. Computational modeling combined with receptor mutagenesis revealed T791 as the likely position of covalent interaction. We show how this covalent ligand can be used to characterize the PAM binding mode and that it is a valuable tool compound in studying receptor function and binding kinetics. Our findings advance the understanding of the mGlu PAM interaction and suggest that 2 is a valuable probe for further structural and chemical biology approaches.
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http://dx.doi.org/10.1021/acs.jmedchem.8b00051DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6331142PMC
January 2019