86 results on '"Frimurer TM"'
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2. Multiple recent HCAR2 structures demonstrate a highly dynamic ligand binding and G protein activation mode.
- Author
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Shenol A, Tenente R, Lückmann M, Frimurer TM, and Schwartz TW
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- Humans, Ligands, Binding Sites, Niacin metabolism, Niacin chemistry, Allosteric Regulation, 3-Hydroxybutyric Acid chemistry, 3-Hydroxybutyric Acid metabolism, Molecular Dynamics Simulation, Receptors, G-Protein-Coupled metabolism, Receptors, G-Protein-Coupled chemistry, Receptors, G-Protein-Coupled agonists, Protein Binding, GTP-Binding Proteins metabolism
- Abstract
A surprisingly clear picture of the allosteric mechanism connecting G protein-coupled receptor agonists with G protein binding-and back - is revealed by a puzzle of thirty novel 3D structures of the hydroxycarboxylic acid receptor 2 (HCAR2) in complex with eight different orthosteric and a single allosteric agonist. HCAR2 is a sensor of β-hydroxybutyrate, niacin and certain anti-inflammatory drugs. Surprisingly, agonists with and without on-target side effects bound very similarly and in a completely occluded orthosteric binding site. Thus, despite the many structures we are still left with a pertinent need to understand the molecular dynamics of this and similar systems., (© 2024. The Author(s).)
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- 2024
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3. Molecular dynamics-based identification of binding pathways and two distinct high-affinity sites for succinate in succinate receptor 1/GPR91.
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Shenol A, Lückmann M, Trauelsen M, Lambrughi M, Tiberti M, Papaleo E, Frimurer TM, and Schwartz TW
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- Mice, Rats, Animals, Humans, Molecular Dynamics Simulation, Succinates metabolism, Stress, Physiological, Succinic Acid metabolism, Receptors, G-Protein-Coupled metabolism
- Abstract
SUCNR1 is an auto- and paracrine sensor of the metabolic stress signal succinate. Using unsupervised molecular dynamics (MD) simulations (170.400 ns) and mutagenesis across human, mouse, and rat SUCNR1, we characterize how a five-arginine motif around the extracellular pole of TM-VI determines the initial capture of succinate in the extracellular vestibule (ECV) to either stay or move down to the orthosteric site. Metadynamics demonstrate low-energy succinate binding in both sites, with an energy barrier corresponding to an intermediate stage during which succinate, with an associated water cluster, unlocks the hydrogen-bond-stabilized conformationally constrained extracellular loop (ECL)-2b. Importantly, simultaneous binding of two succinate molecules through either a "sequential" or "bypassing" mode is a frequent endpoint. The mono-carboxylate NF-56-EJ40 antagonist enters SUCNR1 between TM-I and -II and does not unlock ECL-2b. It is proposed that occupancy of both high-affinity sites is required for selective activation of SUCNR1 by high local succinate concentrations., Competing Interests: Declaration of interests M. Trauelsen, T.M.F., and T.W.S. are co-founders of SOLID Therapeutics, and T.W.S. is a co-founder of Embark Biotech., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)
- Published
- 2024
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4. Biosynthesis of natural and halogenated plant monoterpene indole alkaloids in yeast.
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Bradley SA, Lehka BJ, Hansson FG, Adhikari KB, Rago D, Rubaszka P, Haidar AK, Chen L, Hansen LG, Gudich O, Giannakou K, Lengger B, Gill RT, Nakamura Y, de Bernonville TD, Koudounas K, Romero-Suarez D, Ding L, Qiao Y, Frimurer TM, Petersen AA, Besseau S, Kumar S, Gautron N, Melin C, Marc J, Jeanneau R, O'Connor SE, Courdavault V, Keasling JD, Zhang J, and Jensen MK
- Subjects
- Monoterpenes metabolism, Indole Alkaloids metabolism, Plants metabolism, Pharmaceutical Preparations metabolism, Plant Proteins metabolism, Saccharomyces cerevisiae genetics, Saccharomyces cerevisiae metabolism, Catharanthus
- Abstract
Monoterpenoid indole alkaloids (MIAs) represent a large class of plant natural products with marketed pharmaceutical activities against a wide range of indications, including cancer, malaria and hypertension. Halogenated MIAs have shown improved pharmaceutical properties; however, synthesis of new-to-nature halogenated MIAs remains a challenge. Here we demonstrate a platform for de novo biosynthesis of two MIAs, serpentine and alstonine, in baker's yeast Saccharomyces cerevisiae and deploy it to systematically explore the biocatalytic potential of refactored MIA pathways for the production of halogenated MIAs. From this, we demonstrate conversion of individual haloindole derivatives to a total of 19 different new-to-nature haloserpentine and haloalstonine analogs. Furthermore, by process optimization and heterologous expression of a modified halogenase in the microbial MIA platform, we document de novo halogenation and biosynthesis of chloroalstonine. Together, this study highlights a microbial platform for enzymatic exploration and production of complex natural and new-to-nature MIAs with therapeutic potential., (© 2023. The Author(s).)
- Published
- 2023
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5. Deciphering specificity and cross-reactivity in tachykinin NK1 and NK2 receptors.
- Author
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Madsen JJ, Petersen JE, Christensen DP, Hansen JB, Schwartz TW, Frimurer TM, and Olsen OH
- Subjects
- Animals, Humans, Cell Line, Chlorocebus aethiops, Ligands, Neurokinin A metabolism, Neurokinin-1 Receptor Antagonists, Substance P, Receptors, Neurokinin-2 metabolism, Receptors, Neurokinin-1 agonists, Receptors, Neurokinin-1 metabolism, Tachykinins metabolism
- Abstract
The tachykinin receptors neurokinin 1 (NK1R) and neurokinin 2 (NK2R) are G protein-coupled receptors that bind preferentially to the natural peptide ligands substance P and neurokinin A, respectively, and have been targets for drug development. Despite sharing a common C-terminal sequence of Phe-X-Gly-Leu-Met-NH2 that helps direct biological function, the peptide ligands exhibit some degree of cross-reactivity toward each other's non-natural receptor. Here, we investigate the detailed structure-activity relationships of the ligand-bound receptor complexes that underlie both potent activation by the natural ligand and cross-reactivity. We find that the specificity and cross-reactivity of the peptide ligands can be explained by the interactions between the amino acids preceding the FxGLM consensus motif of the bound peptide ligand and two regions of the receptor: the β-hairpin of the extracellular loop 2 (ECL2) and a N-terminal segment leading into transmembrane helix 1. Positively charged sidechains of the ECL2 (R177 of NK1R and K180 of NK2R) are seen to play a vital role in the interaction. The N-terminal positions 1 to 3 of the peptide ligand are entirely dispensable. Mutated and chimeric receptor and ligand constructs neatly swap around ligand specificity as expected, validating the structure-activity hypotheses presented. These findings will help in developing improved agonists or antagonists for NK1R and NK2R., Competing Interests: Conflict of interest D. P. C. and J. B. H. are employees of Embark Biotech ApS. All other authors declare no competing interests., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)
- Published
- 2023
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6. Optimization of First-in-Class Dual-Acting FFAR1/FFAR4 Allosteric Modulators with Novel Mode of Action.
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Lückmann M, Shenol A, Nissen TAD, Petersen JE, Kouvchinov D, Schwartz TW, and Frimurer TM
- Abstract
The free fatty acid receptors FFAR1 and FFAR4 are considered promising therapeutic targets for management of metabolic and inflammatory diseases. However, there is a need for entirely novel chemical scaffolds, since many of the highly similar lipophilic chemotypes in development have been abandoned by the pharmaceutical industry, due to toxic effects on hepatocytes and β-cells. Our group has recently reported the discovery of a 1,3,5-triazine-2-amine-based compound that acts as an allosteric agonist on FFAR1. Here, we present the synthesis and investigation of the structure-activity relationship of an extensive set of analogues of which many display dual-acting agonist properties for both FFAR1 and FFAR4. In several rounds of optimization, we discovered multiple analogues with single-digit nanomolar potency on FFAR1. Pending additional optimization for metabolic stability, the compounds in this study present novel ways of providing beneficial glycemic control while avoiding the notorious toxicity challenges associated with previously identified chemotypes., Competing Interests: The authors declare no competing financial interest., (© 2022 The Authors. Published by American Chemical Society.)
- Published
- 2022
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7. Mechanistic basis of GPCR activation explored by ensemble refinement of crystallographic structures.
- Author
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Madsen JJ, Ye L, Frimurer TM, and Olsen OH
- Subjects
- Molecular Conformation, Protein Binding, Hydrogen Bonding, Crystallography, X-Ray, Ligands, Receptors, Adrenergic, beta-2 chemistry, Receptors, Adrenergic, beta-2 metabolism, Sodium
- Abstract
G protein-coupled receptors (GPCRs) are important drug targets characterized by a canonical seven transmembrane (TM) helix architecture. Recent advances in X-ray crystallography and cryo-EM have resulted in a wealth of GPCR structures that have been used in drug design and formed the basis for mechanistic activation hypotheses. Here, ensemble refinement (ER) of crystallographic structures is applied to explore the impact of binding of agonists and antagonist/inverse agonists to selected structures of cannabinoid receptor 1 (CB1R), β
2 adrenergic receptor (β2 AR), and A2A adenosine receptor (A2A AR). To assess the conformational flexibility and its role in GPCR activation, hydrogen bond (H-bond) networks are analyzed by calculating and comparing H-bond propensities. Mapping pairwise propensity differences between agonist- and inverse agonist/antagonist-bound structures for CB1R and β2 AR shows that agonist binding destabilizes H-bonds in the intracellular parts of TM 5-7, forming the G protein binding cavity, while H-bonds of the extracellular segment of TMs surrounding the orthosteric site are conversely stabilized. Certain class A GPCRs, for example, A2A AR, bind an allosteric sodium ion that negatively modulates agonist binding. The impact of sodium-excluding mutants (D522.50 N, S913.39 A) of A2A AR on agonist binding is examined by applying ER analysis to structures of wildtype and the two mutants in complex with a full agonist. While S913.39 A exhibits normal activity, D522.50 N quenches the downstream signaling. The mainchain H-bond pattern of the latter is stabilized in the intracellular part of TM 7 containing the NPxxY motif, indicating that an induced rigidity of the mutation prevents conformational selection of G proteins resulting in receptor inactivation., (© 2022 The Authors. Protein Science published by Wiley Periodicals LLC on behalf of The Protein Society.)- Published
- 2022
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8. Serotonin G Protein-Coupled Receptor-Based Biosensing Modalities in Yeast.
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Lengger B, Hoch-Schneider EE, Jensen CN, Jakočiu Nas T, Petersen AA, Frimurer TM, Jensen ED, and Jensen MK
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- Humans, Signal Transduction, Biosensing Techniques methods, Receptors, G-Protein-Coupled genetics, Receptors, G-Protein-Coupled metabolism, Saccharomyces cerevisiae genetics, Saccharomyces cerevisiae metabolism, Serotonin analysis
- Abstract
Serotonin is a key neurotransmitter involved in numerous physiological processes and serves as an important precursor for manufacturing bioactive indoleamines and alkaloids used in the treatment of human pathologies. In humans, serotonin sensing and signaling can occur by 12 G protein-coupled receptors (GPCRs) coupled to Gα proteins. In yeast, human serotonin GPCRs coupled to Gα proteins have previously been shown to function as whole-cell biosensors of serotonin. However, systematic characterization of serotonin biosensing modalities between variant serotonin GPCRs and application thereof for high-resolution serotonin quantification is still awaiting. To systematically assess GPCR signaling in response to serotonin, we characterized reporter gene expression at two different pHs of a 144-sized library encoding all 12 human serotonin GPCRs in combination with 12 different Gα proteins engineered in yeast. From this screen, we observed changes in the biosensor sensitivities of >4 orders of magnitude. Furthermore, adopting optimal biosensing designs and pH conditions enabled high-resolution high-performance liquid chromatography-validated sensing of serotonin produced in yeast. Lastly, we used the yeast platform to characterize 19 serotonin GPCR polymorphisms found in human populations. While major differences in signaling were observed among the individual polymorphisms when studied in yeast, a cross-comparison of selected variants in mammalian cells showed both similar and disparate results. Taken together, our study highlights serotonin biosensing modalities of relevance to both biotechnological and potential human health applications.
- Published
- 2022
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9. Discovery of GPR183 Agonists Based on an Antagonist Scaffold.
- Author
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Kjaer VMS, Ieremias L, Daugvilaite V, Lückmann M, Frimurer TM, Ulven T, Rosenkilde MM, and Våbenø J
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- Humans, Molecular Structure, Structure-Activity Relationship, Drug Discovery, Receptors, G-Protein-Coupled agonists, Receptors, G-Protein-Coupled antagonists & inhibitors
- Abstract
The G protein-coupled receptor GPR183/EBI2, which is activated by oxysterols, is a therapeutic target for inflammatory and metabolic diseases where both antagonists and agonists are of potential interest. Using the piperazine diamide core of the known GPR183 antagonist (E)-3-(4-bromophenyl)-1-(4-(4-methoxybenzoyl)piperazin-1-yl)prop-2-en-1-one (NIBR189) as starting point, we identified and sourced 79 structurally related compounds that were commercially available. In vitro screening of this compound collection using a Ca
2+ mobilization assay resulted in the identification of 10 compounds with agonist properties. To enable establishment of initial structure-activity relationship trends, these were supplemented with five in-house compounds, two of which were also shown to be GPR183 agonists. Taken together, our findings suggest that the agonist activity of this compound series is dictated by the substitution pattern of one of the two distal phenyl rings, which functions as a molecular efficacy-switch., (© 2021 The Authors. ChemMedChem published by Wiley-VCH GmbH.)- Published
- 2021
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10. Investigating GIPR (ant)agonism: A structural analysis of GIP and its receptor.
- Author
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Smit FX, van der Velden WJC, Kizilkaya HS, Nørskov A, Lückmann M, Hansen TN, Sparre-Ulrich AH, Qvotrup K, Frimurer TM, and Rosenkilde MM
- Subjects
- Binding Sites, Gastric Inhibitory Polypeptide metabolism, Humans, Hydrogen Bonding, Models, Molecular, Molecular Dynamics Simulation, Mutation, Protein Conformation, Receptors, Gastrointestinal Hormone antagonists & inhibitors, Receptors, Gastrointestinal Hormone genetics, Structural Homology, Protein, Glucagon-Like Peptide-1 Receptor chemistry, Glucagon-Like Peptide-1 Receptor metabolism, Receptors, Gastrointestinal Hormone chemistry, Receptors, Gastrointestinal Hormone metabolism
- Abstract
The glucose-dependent insulinotropic polypeptide (GIP) is a 42-residue metabolic hormone that is actively being targeted for its regulatory role of glycemia and energy balance. Limited structural data of its receptor has made ligand design tedious. This study investigates the structure and function of the GIP receptor (GIPR), using a homology model based on the GLP-1 receptor. Molecular dynamics combined with in vitro mutational data were used to pinpoint residues involved in ligand binding and/or receptor activation. Significant differences in binding mode were identified for the naturally occurring agonists GIP(1-30)NH
2 and GIP(1-42) compared with high potency antagonists GIP(3-30)NH2 and GIP(5-30)NH2 . Residues R1832.60 , R1902.67 , and R3005.40 are shown to be key for activation of the GIPR, and evidence suggests that a disruption of the K293ECL2 -E362ECL3 salt bridge by GIPR antagonists strongly reduces GIPR activation. Combinatorial use of these findings can benefit rational design of ligands targeting the GIPR., Competing Interests: Declaration of interests This study was financially co-supported by Antag Therapeutics ApS. A.H.S.-U. is the CEO and co-founder of Antag Therapeutics ApS. M.M.R. is co-founder of Antag Therapeutics ApS and of Bainan Biotech ApS. M.M.R. is board member at Bainan Biotech. Patents related to this work: The rights to the two PCT applications above and any patents derived therefrom are licensed to Antag Therapeutics ApS, co-founded by A.H.S.-U. and M.M.R. The rights to the two PCT applications above and any patents derived therefrom are licensed to Bainan Biotech ApS, co-founded by M.M.R., (Copyright © 2021 Elsevier Ltd. All rights reserved.)- Published
- 2021
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11. Selective release of gastrointestinal hormones induced by an orally active GPR39 agonist.
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Grunddal KV, Diep TA, Petersen N, Tough IR, Skov LJ, Liu L, Buijink JA, Mende F, Jin C, Jepsen SL, Sørensen LME, Achiam MP, Strandby RB, Bach A, Hartmann B, Frimurer TM, Hjorth SA, Bouvier M, Cox H, and Holst B
- Subjects
- Animals, Appetite Regulation, Bariatric Surgery, Body Weight, Eating, Enteroendocrine Cells, Gastric Inhibitory Polypeptide pharmacology, Ghrelin metabolism, Glucagon-Like Peptide 1 metabolism, Humans, Male, Mice, Mice, Inbred C57BL, Mice, Knockout, Obesity metabolism, Peptide YY metabolism, Receptors, G-Protein-Coupled genetics, Receptors, Gastrointestinal Hormone, Weight Loss, Gastrointestinal Hormones metabolism, Receptors, G-Protein-Coupled agonists, Receptors, G-Protein-Coupled metabolism
- Abstract
Objectives: Obesity is a complex disease associated with a high risk of comorbidities. Gastric bypass surgery, an invasive procedure with low patient eligibility, is currently the most effective intervention that achieves sustained weight loss. This beneficial effect is attributed to alterations in gut hormone signaling. An attractive alternative is to pharmacologically mimic the effects of bariatric surgery by targeting several gut hormonal axes. The G protein-coupled receptor 39 (GPR39) expressed in the gastrointestinal tract has been shown to mediate ghrelin signaling and control appetite, food intake, and energy homeostasis, but the broader effect on gut hormones is largely unknown. A potent and efficacious GPR39 agonist (Cpd1324) was recently discovered, but the in vivo function was not addressed. Herein we studied the efficacy of the GPR39 agonist, Cpd1324, on metabolism and gut hormone secretion., Methods: Body weight, food intake, and energy expenditure in GPR39 agonist-treated mice and GPR39 KO mice were studied in calorimetric cages. Plasma ghrelin, glucose-dependent insulinotropic polypeptide (GIP), glucagon-like peptide-1 (GLP-1), and peptide YY (PYY) levels were measured. Organoids generated from murine and human small intestine and mouse colon were used to study GLP-1 and PYY release. Upon GPR39 agonist administration, dynamic changes in intracellular GLP-1 content were studied via immunostaining and changes in ion transport across colonic mucosa were monitored in Ussing chambers. The G protein activation underlying GPR39-mediated selective release of gut hormones was studied using bioluminescence resonance energy transfer biosensors., Results: The GPR39 KO mice displayed a significantly increased food intake without corresponding increases in respiratory exchange ratios or energy expenditure. Oral administration of a GPR39 agonist induced an acute decrease in food intake and subsequent weight loss in high-fat diet (HFD)-fed mice without affecting their energy expenditure. The tool compound, Cpd1324, increased GLP-1 secretion in the mice as well as in mouse and human intestinal organoids, but not in GPR39 KO mouse organoids. In contrast, the GPR39 agonist had no effect on PYY or GIP secretion. Transepithelial ion transport was acutely affected by GPR39 agonism in a GLP-1- and calcitonin gene-related peptide (CGRP)-dependent manner. Analysis of Cpd1324 signaling properties showed activation of Gα
q and Gαi/o signaling pathways in L cells, but not Gαs signaling., Conclusions: The GPR39 agonist described in this study can potentially be used by oral administration as a weight-lowering agent due to its stimulatory effect on GLP-1 secretion, which is most likely mediated through a unique activation of Gα subunits. Thus, GPR39 agonism may represent a novel approach to effectively treat obesity through selective modulation of gastrointestinal hormonal axes., Competing Interests: Conflicts of interest M.B. is the chairman of the scientific advisory board of Domain Therapeutics to which some of the biosensors used in this study are licensed for commercial use. The biosensors are freely available for academic research from M.B. The rest of the authors declare no conflicts of interest., (Copyright © 2021. Published by Elsevier GmbH.)- Published
- 2021
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12. Extracellular succinate hyperpolarizes M2 macrophages through SUCNR1/GPR91-mediated Gq signaling.
- Author
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Trauelsen M, Hiron TK, Lin D, Petersen JE, Breton B, Husted AS, Hjorth SA, Inoue A, Frimurer TM, Bouvier M, O'Callaghan CA, and Schwartz TW
- Subjects
- Arrestins metabolism, Female, GTP-Binding Protein alpha Subunits, Gi-Go metabolism, Gene Expression Regulation, Gene Ontology, HEK293 Cells, Humans, Ligands, Macrophages immunology, Male, Models, Biological, Protein Subunits metabolism, Receptors, G-Protein-Coupled agonists, Receptors, G-Protein-Coupled genetics, Transcriptional Activation genetics, Type C Phospholipases metabolism, Extracellular Space chemistry, GTP-Binding Protein alpha Subunits, Gq-G11 metabolism, Macrophages metabolism, Receptors, G-Protein-Coupled metabolism, Signal Transduction, Succinic Acid metabolism
- Abstract
Succinate functions both as a classical TCA cycle metabolite and an extracellular metabolic stress signal sensed by the mainly Gi-coupled succinate receptor SUCNR1. In the present study, we characterize and compare effects and signaling pathways activated by succinate and both classes of non-metabolite SUCNR1 agonists. By use of specific receptor and pathway inhibitors, rescue in G-protein-depleted cells and monitoring of receptor G protein activation by BRET, we identify Gq rather than Gi signaling to be responsible for SUCNR1-mediated effects on basic transcriptional regulation. Importantly, in primary human M2 macrophages, in which SUCNR1 is highly expressed, we demonstrate that physiological concentrations of extracellular succinate act through SUCNR1-activated Gq signaling to efficiently regulate transcription of immune function genes in a manner that hyperpolarizes their M2 versus M1 phenotype. Thus, sensing of stress-induced extracellular succinate by SUCNR1 is an important transcriptional regulator in human M2 macrophages through Gq signaling., Competing Interests: Declaration of interests M.B. is the chair of the scientific advisory board for Domain Therapeutics (DT), and some of the BRET-based biosensors used in the present study were licensed to DT for commercial use. The other authors declare no competing interests., (Copyright © 2021. Published by Elsevier Inc.)
- Published
- 2021
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13. The Zinc-Sensing Receptor GPR39 in Physiology and as a Pharmacological Target.
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Laitakari A, Liu L, Frimurer TM, and Holst B
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- Animals, Disease Susceptibility, Humans, Ion Channel Gating drug effects, Ligands, Organ Specificity, Receptors, G-Protein-Coupled agonists, Receptors, G-Protein-Coupled antagonists & inhibitors, Receptors, G-Protein-Coupled chemistry, Signal Transduction drug effects, Structure-Activity Relationship, Zinc metabolism, Biomarkers, Drug Discovery methods, Receptors, G-Protein-Coupled physiology
- Abstract
The G-protein coupled receptor GPR39 is abundantly expressed in various tissues and can be activated by changes in extracellular Zn
2+ in physiological concentrations. Previously, genetically modified rodent models have been able to shed some light on the physiological functions of GPR39, and more recently the utilization of novel synthetic agonists has led to the unraveling of several new functions in the variety of tissues GPR39 is expressed. Indeed, GPR39 seems to be involved in many important metabolic and endocrine functions, but also to play a part in inflammation, cardiovascular diseases, saliva secretion, bone formation, male fertility, addictive and depression disorders and cancer. These new discoveries offer opportunities for the development of novel therapeutic approaches against many diseases where efficient therapeutics are still lacking. This review focuses on Zn2+ as an endogenous ligand as well as on the novel synthetic agonists of GPR39, placing special emphasis on the recently discovered physiological functions and discusses their pharmacological potential.- Published
- 2021
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14. GLP-1 Val8: A Biased GLP-1R Agonist with Altered Binding Kinetics and Impaired Release of Pancreatic Hormones in Rats.
- Author
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van der Velden WJC, Smit FX, Christiansen CB, Møller TC, Hjortø GM, Larsen O, Schiellerup SP, Bräuner-Osborne H, Holst JJ, Hartmann B, Frimurer TM, and Rosenkilde MM
- Abstract
Biased ligands that selectively confer activity in one pathway over another are pharmacologically important because biased signaling may reduce on-target side effects and improve drug efficacy. Here, we describe an N-terminal modification in the incretin hormone glucagon-like peptide (GLP-1) that alters the signaling capabilities of the GLP-1 receptor (GLP-1R) by making it G protein biased over internalization but was originally designed to confer DPP-4 resistance and thereby prolong the half-life of GLP-1. Despite similar binding affinity, cAMP production, and calcium mobilization, substitution of a single amino acid (Ala8 to Val8) in the N-terminus of GLP-1(7-36)NH
2 (GLP-1 Val8) severely impaired its ability to internalize GLP-1R compared to endogenous GLP-1. In-depth binding kinetics analyses revealed shorter residence time for GLP-1 Val8 as well as a slower observed association rate. Molecular dynamics (MD) displayed weaker and less interactions of GLP-1 Val8 with GLP-1R, as well as distinct conformational changes in the receptor compared to GLP-1. In vitro validation of the MD, by receptor alanine substitutions, confirmed stronger impairments of GLP-1 Val8-mediated signaling compared to GLP-1. In a perfused rat pancreas, acute stimulation with GLP-1 Val8 resulted in a lower insulin and somatostatin secretion compared to GLP-1. Our study illustrates that profound differences in molecular pharmacological properties, which are essential for the therapeutic targeting of the GLP-1 system, can be induced by subtle changes in the N-terminus of GLP-1. This information could facilitate the development of optimized GLP-1R agonists., Competing Interests: The authors declare no competing financial interest., (© 2021 American Chemical Society.)- Published
- 2021
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15. Structural basis for GPCR signaling by small polar versus large lipid metabolites-discovery of non-metabolite ligands.
- Author
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Lückmann M, Trauelsen M, Frimurer TM, and Schwartz TW
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- Humans, Ligands, Receptors, G-Protein-Coupled genetics, Signal Transduction, Receptors, G-Protein-Coupled metabolism
- Abstract
Key metabolites act through specific G protein-coupled receptors (GPCRs) as extracellular signals of fuel availability and metabolic stress. Here, we focus on the succinate receptor SUCNR1/GPR91 and the long chain fatty acid receptor FFAR1/GPR40, for which 3D structural information is available. Like other small polar acidic metabolites, succinate is excreted from the cell by transporter proteins to bind to an extracellular, solvent-exposed pocket in SUCNR1. Non-metabolite pharmacological tool compounds are currently being designed based on the structure of the SUCNR1 binding pocket. In FFAR1, differently signaling lipid mimetics bind in two distinct membrane-exposed sites corresponding to each of the lipid bilayer leaflets. Conceivably endogenous lipid ligands gain access to these sites by way of the membrane and probably occupy both sites under physiological circumstances. Design of polar agonists for a dynamic, solvent-exposed pocket in FFAR1 underlines the possibility of structure-based approaches for development of novel tool compounds even in lipid sensing metabolite GPCRs., Competing Interests: Conflict of interest statement The authors declare the following financial interests/personal relationships, which may be considered as potential competing interests: TWS is co-founder and chief scientific godfather of Embark Biotech and TMF is co-founder of Ankrin therapeutics., (Copyright © 2019. Published by Elsevier Ltd.)
- Published
- 2020
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16. Selective Allosteric Modulation of N-Terminally Cleaved, but Not Full Length CCL3 in CCR1.
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Larsen O, Lückmann M, van der Velden WJC, Oliva-Santiago M, Brvar M, Ulven T, Frimurer TM, Karlshøj S, and Rosenkilde MM
- Abstract
Chemokines undergo post-translational modification such as N-terminal truncations. Here, we describe how N-terminal truncation of full length CCL3
(1-70) affects its activity at CCR1. Truncated CCL3(5-70) has 10-fold higher potency and enhanced efficacy in β-arrestin recruitment, but less than 2-fold increased potencies in G protein signaling determined by calcium release, cAMP and IP3 formation. Small positive ago-allosteric ligands modulate the two CCL3 variants differently as the metal ion chelator bipyridine in complex with zinc (ZnBip) enhances the binding of truncated, but not full length CCL3, while a size-increase of the chelator to a chloro-substituted terpyridine (ZnClTerp), eliminates its allosteric, but not agonistic action. By employing a series of receptor mutants and in silico modeling we describe residues of importance for chemokine and small molecule binding. Notably, the chemokine receptor-conserved Glu2877.39 interacts with the N-terminal amine of truncated CCL3(5-70) and with Zn2+ of ZnBip, thereby bridging their binding sites and enabling the positive allosteric effect. Our study emphasizes that small allosteric molecules may act differently toward chemokine variants and thus selectively modulate interactions of specific chemokine subsets with their cognate receptors., Competing Interests: The authors declare no competing financial interest., (Copyright © 2019 American Chemical Society.)- Published
- 2019
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17. Biased Signaling of CCL21 and CCL19 Does Not Rely on N-Terminal Differences, but Markedly on the Chemokine Core Domains and Extracellular Loop 2 of CCR7.
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Jørgensen AS, Larsen O, Uetz-von Allmen E, Lückmann M, Legler DF, Frimurer TM, Veldkamp CT, Hjortø GM, and Rosenkilde MM
- Subjects
- Amino Acid Sequence, Animals, Binding Sites genetics, CHO Cells, Cell Line, Tumor, Chemokine CCL19 genetics, Chemokine CCL19 metabolism, Chemokine CCL21 genetics, Chemokine CCL21 metabolism, Cricetinae, Cricetulus, Humans, Ligands, Mice, Mutation, Protein Binding, Receptors, CCR7 genetics, Receptors, CCR7 metabolism, Sequence Homology, Amino Acid, Signal Transduction genetics, Chemokine CCL19 immunology, Chemokine CCL21 immunology, Receptors, CCR7 immunology, Signal Transduction immunology
- Abstract
Chemokine receptors play important roles in the immune system and are linked to several human diseases. Targeting chemokine receptors have so far shown very little success owing to, to some extent, the promiscuity of the immune system and the high degree of biased signaling within it. CCR7 and its two endogenous ligands display biased signaling and here we investigate the differences between the two ligands, CCL21 and CCL19, with respect to their biased activation of CCR7. We use bystander bioluminescence resonance energy transfer (BRET) based signaling assays and Transwell migration assays to determine (A) how swapping of domains between the two ligands affect their signaling patterns and (B) how receptor mutagenesis impacts signaling. Using chimeric ligands we find that the chemokine core domains are central for determining signaling outcome as the lack of β-arrestin-2 recruitment displayed by CCL21 is linked to its core domain and not N-terminus. Through a mutagenesis screen, we identify the extracellular domains of CCR7 to be important for both ligands and show that the two chemokines interact differentially with extracellular loop 2 (ECL-2). By using in silico modeling, we propose a link between ECL-2 interaction and CCR7 signal transduction. Our mutagenesis study also suggests a lysine in the top of TM3, K130
3.26 , to be important for G protein signaling, but not β-arrestin-2 recruitment. Taken together, the bias in CCR7 between CCL19 and CCL21 relies on the chemokine core domains, where interactions with ECL-2 seem particularly important. Moreover, TM3 selectively regulates G protein signaling as found for other chemokine receptors., (Copyright © 2019 Jørgensen, Larsen, Uetz-von Allmen, Lückmann, Legler, Frimurer, Veldkamp, Hjortø and Rosenkilde.)- Published
- 2019
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18. Allostery in Coagulation Factor VIIa Revealed by Ensemble Refinement of Crystallographic Structures.
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Sorensen AB, Madsen JJ, Frimurer TM, Overgaard MT, Gandhi PS, Persson E, and Olsen OH
- Subjects
- Allosteric Regulation, Apoenzymes chemistry, Apoenzymes metabolism, Benzamidines pharmacology, Crystallography, X-Ray, Disulfides chemistry, Enzyme Activation drug effects, Models, Molecular, Protein Domains, Protein Folding, Trypsin chemistry, Trypsin metabolism, Trypsinogen metabolism, Factor VIIa chemistry, Factor VIIa metabolism
- Abstract
A critical step in injury-induced initiation of blood coagulation is the formation of the complex between the trypsin-like protease coagulation factor VIIa (FVIIa) and its cofactor tissue factor (TF), which converts FVIIa from an intrinsically poor enzyme to an active protease capable of activating zymogens of downstream coagulation proteases. Unlike its constitutively active ancestor trypsin, FVIIa is allosterically activated (by TF). Here, ensemble refinement of crystallographic structures, which uses multiple copies of the entire structure as a means of representing structural flexibility, is applied to explore the impacts of inhibitor binding to trypsin and FVIIa, as well as cofactor binding to FVIIa. To assess the conformational flexibility and its role in allosteric pathways in these proteases, main-chain hydrogen bond networks are analyzed by calculating the hydrogen-bond propensity. Mapping pairwise propensity differences between relevant structures shows that binding of the inhibitor benzamidine to trypsin has a minor influence on the protease flexibility. For FVIIa, in contrast, the protease domain is "locked" into the catalytically competent trypsin-like configuration upon benzamidine binding as indicated by the stabilization of key structural features: the nonprime binding cleft and the oxyanion hole are stabilized, and the effect propagates from the active site region to the calcium-binding site and to the vicinity of the disulphide bridge connecting with the light chain. TF binding to FVIIa furthermore results in stabilization of the 170 loop, which in turn propagates an allosteric signal from the TF-binding region to the active site. Analyses of disulphide bridge energy and flexibility reflect the striking stability difference between the unregulated enzyme and the allosterically activated form after inhibitor or cofactor binding. The ensemble refinement analyses show directly, for the first time to our knowledge, whole-domain structural footprints of TF-induced allosteric networks present in x-ray crystallographic structures of FVIIa, which previously only have been hypothesized or indirectly inferred., (Copyright © 2019 Biophysical Society. Published by Elsevier Inc. All rights reserved.)
- Published
- 2019
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19. Molecular dynamics-guided discovery of an ago-allosteric modulator for GPR40/FFAR1.
- Author
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Lückmann M, Trauelsen M, Bentsen MA, Nissen TAD, Martins J, Fallah Z, Nygaard MM, Papaleo E, Lindorff-Larsen K, Schwartz TW, and Frimurer TM
- Subjects
- Allosteric Site, Benzofurans antagonists & inhibitors, Binding Sites, Crystallography, X-Ray, Humans, Ligands, Molecular Docking Simulation, Mutation, Protein Binding, Protein Conformation, Receptors, G-Protein-Coupled genetics, Sulfones antagonists & inhibitors, Allosteric Regulation drug effects, Molecular Dynamics Simulation, Receptors, G-Protein-Coupled chemistry, Receptors, G-Protein-Coupled drug effects
- Abstract
The long-chain fatty acid receptor FFAR1/GPR40 binds agonists in both an interhelical site between the extracellular segments of transmembrane helix (TM)-III and TM-IV and a lipid-exposed groove between the intracellular segments of these helices. Molecular dynamics simulations of FFAR1 with agonist removed demonstrated a major rearrangement of the polar and charged anchor point residues for the carboxylic acid moiety of the agonist in the interhelical site, which was associated with closure of a neighboring, solvent-exposed pocket between the extracellular poles of TM-I, TM-II, and TM-VII. A synthetic compound designed to bind in this pocket, and thereby prevent its closure, was identified through structure-based virtual screening and shown to function both as an agonist and as an allosteric modulator of receptor activation. This discovery of an allosteric agonist for a previously unexploited, dynamic pocket in FFAR1 demonstrates both the power of including molecular dynamics in the drug discovery process and that this specific, clinically proven, but difficult, antidiabetes target can be addressed by chemotypes different from existing ligands., Competing Interests: The authors declare no conflict of interest., (Copyright © 2019 the Author(s). Published by PNAS.)
- Published
- 2019
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20. Preassociation between the 5-HT 7 serotonin receptor and G protein G s : molecular determinants and association with low potency activation of adenylyl cyclase.
- Author
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Ulsund AH, Dahl M, Frimurer TM, Manfra O, Schwartz TW, Levy FO, and Andressen KW
- Subjects
- Adenylyl Cyclases metabolism, Binding Sites, GTP-Binding Protein alpha Subunits, Gs chemistry, HEK293 Cells, Humans, Protein Binding, Receptors, Serotonin chemistry, GTP-Binding Protein alpha Subunits, Gs metabolism, Receptors, Serotonin metabolism
- Abstract
According to early models of GPCR signaling, G proteins only interact with activated receptors. However, some GPCRs were shown to assemble with G proteins before receptor activation, in accordance with more recent models. Previously, we found that the 5-HT
7 receptor, as opposed to the 5-HT4 receptor, was preassociated with Gs , but the molecular determinants for this interaction are still elusive. In a series of chimeric 5-HT7 receptors with intracellular segments from 5-HT4 , we determined the receptor-G protein interaction by performing antibody-immobilized fluorescence recovery after photobleaching and fluorescence resonance energy transfer. We identified the intracellular loop 3 and C-tail of the 5-HT7 receptor to be responsible for the preassociation with Gs , and we further delineated the TM5 extension in the intracellular loop 3 and helix 8 in the C-tail as the molecular determinants. These chimeric exchanges converted the 5-HT7 receptor into a collision-coupled receptor that recruited G proteins only upon agonist activation, whereas reciprocal exchanges converted 5-HT4 to a preassociated receptor. The 5-HT7 receptor displayed 2-component agonist-induced Gs signaling with high and low potency. In addition, the same segments were involved in low-potency signaling and preassociation. The correspondence between Gs preassociation and low-potency Gs signaling is a novel aspect of GPCR pharmacology.-Ulsund, A. H., Dahl, M., Frimurer, T. M., Manfra, O., Schwartz, T. W., Levy, F. O., Andressen, K. W. Preassociation between the 5-HT7 serotonin receptor and G protein Gs : molecular determinants and association with low potency activation of adenylyl cyclase.- Published
- 2019
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21. Human substance P receptor binding mode of the antagonist drug aprepitant by NMR and crystallography.
- Author
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Chen S, Lu M, Liu D, Yang L, Yi C, Ma L, Zhang H, Liu Q, Frimurer TM, Wang MW, Schwartz TW, Stevens RC, Wu B, Wüthrich K, and Zhao Q
- Subjects
- Aprepitant chemistry, Humans, Protein Binding, Receptors, Neurokinin-1 chemistry, Aprepitant metabolism, Crystallography methods, Magnetic Resonance Spectroscopy methods, Receptors, Neurokinin-1 metabolism
- Abstract
Neurokinin 1 receptor (NK1R) has key regulating functions in the central and peripheral nervous systems, and NK1R antagonists such as aprepitant have been approved for treating chemotherapy-induced nausea and vomiting. However, the lack of data on NK1R structure and biochemistry has limited further drug development targeting this receptor. Here, we combine NMR spectroscopy and X-ray crystallography to provide dynamic and static characterisation of the binding mode of aprepitant in complexes with human NK1R variants.
19 F-NMR showed a slow off-rate in the binding site, where aprepitant occupies multiple substates that exchange with frequencies in the millisecond range. The environment of the bound ligand is affected by the amino acid in position 2.50, which plays a key role in ligand binding and receptor signaling in class A GPCRs. Crystal structures now reveal how receptor signaling relates to the conformation of the conserved NP7.50 xxY motif in transmembrane helix VII.- Published
- 2019
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22. Translating biased signaling in the ghrelin receptor system into differential in vivo functions.
- Author
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Mende F, Hundahl C, Plouffe B, Skov LJ, Sivertsen B, Madsen AN, Lückmann M, Diep TA, Offermanns S, Frimurer TM, Bouvier M, and Holst B
- Subjects
- Animals, Eating drug effects, GTP-Binding Proteins metabolism, HEK293 Cells, Humans, Ligands, Male, Mice, Piperidines pharmacology, Quinazolinones pharmacology, Rats, Rats, Sprague-Dawley, Signal Transduction drug effects, Signal Transduction physiology, beta-Arrestins metabolism, Ghrelin metabolism, Receptors, Ghrelin metabolism
- Abstract
Biased signaling has been suggested as a means of selectively modulating a limited fraction of the signaling pathways for G-protein-coupled receptor family members. Hence, biased ligands may allow modulation of only the desired physiological functions and not elicit undesired effects associated with pharmacological treatments. The ghrelin receptor is a highly sought antiobesity target, since the gut hormone ghrelin in humans has been shown to increase both food intake and fat accumulation. However, it also modulates mood, behavior, growth hormone secretion, and gastric motility. Thus, blocking all pathways of this receptor may give rise to potential side effects. In the present study, we describe a highly promiscuous signaling capacity for the ghrelin receptor. We tested selected ligands for their ability to regulate the various pathways engaged by the receptor. Among those, a biased ligand, YIL781, was found to activate the Gα
q/11 and Gα12 pathways selectively without affecting the engagement of β-arrestin or other G proteins. YIL781 was further characterized for its in vivo physiological functions. In combination with the use of mice in which Gαq/11 was selectively deleted in the appetite-regulating AgRP neurons, this biased ligand allowed us to demonstrate that selective blockade of Gαq/11 , without antagonism at β-arrestin or other G-protein coupling is sufficient to decrease food intake., Competing Interests: The authors declare no conflict of interest.- Published
- 2018
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23. Inhibiting RHOA Signaling in Mice Increases Glucose Tolerance and Numbers of Enteroendocrine and Other Secretory Cells in the Intestine.
- Author
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Petersen N, Frimurer TM, Terndrup Pedersen M, Egerod KL, Wewer Albrechtsen NJ, Holst JJ, Grapin-Botton A, Jensen KB, and Schwartz TW
- Subjects
- Animals, Biomarkers blood, Blood Glucose metabolism, Cell Lineage, Cell Proliferation drug effects, Diet, High-Fat, Disease Models, Animal, Enteroendocrine Cells metabolism, Glucagon-Like Peptide 1 blood, Glucose Intolerance blood, Glucose Intolerance etiology, Glucose Intolerance physiopathology, Ileum metabolism, Insulin blood, Male, Mice, Inbred C57BL, Mice, Transgenic, Organoids drug effects, Organoids metabolism, Phenotype, Signal Transduction drug effects, Stem Cells metabolism, Time Factors, Tissue Culture Techniques, rho-Associated Kinases antagonists & inhibitors, rho-Associated Kinases metabolism, rhoA GTP-Binding Protein, Amides pharmacology, Blood Glucose drug effects, Cell Differentiation drug effects, Enteroendocrine Cells drug effects, Glucose Intolerance drug therapy, Hypoglycemic Agents pharmacology, Ileum drug effects, Insulin Resistance, Protein Kinase Inhibitors pharmacology, Pyridines pharmacology, Stem Cells drug effects, rho GTP-Binding Proteins metabolism
- Abstract
Background & Aims: Glucagon-like peptide 1 (GLP1) is produced by L cells in the intestine, and agonists of the GLP1 receptor are effective in the treatment of diabetes. Levels of GLP1 increase with numbers of L cells. Therefore, agents that increase numbers of L cell might be developed for treatment of diabetes. Ras homologue family member A (RhoA) signaling through Rho-associated coiled-coil-containing protein kinases 1 and 2 (ROCK1 and ROCK2) controls cell differentiation, but it is not clear whether this pathway regulates enteroendocrine differentiation in the intestinal epithelium. We investigated the effects of Y-27632, an inhibitor of ROCK1 and ROCK2, on L-cell differentiation., Methods: We collected intestinal tissues from GLU-Venus, GPR41-RFP, and Neurog3-RFP mice, in which the endocrine lineage is fluorescently labeled, for in vitro culture and histologic analysis. Small intestine organoids derived from these mice were cultured with Y-27632 and we measured percentages of L cells, expression of intestinal cell-specific markers, and secretion of GLP1 in medium. Mice were fed a normal chow or a high-fat diet and given Y-27632 or saline (control) and blood samples were collected for measurement of GLP1, insulin, and glucose., Results: Incubation of intestinal organoids with Y-27632 increased numbers of L cells and secretion of GLP1. These increases were associated with upregulated expression of genes encoding intestinal hormones, neurogenin 3, neurogenic differentiation factor 1, forkhead box A1 and A2, and additional markers of secretory cells. Mice fed the normal chow diet and given Y-27632 had increased numbers of L cells in intestinal tissues, increased plasma levels of GLP1 and insulin, and lower blood levels of glucose compared with mice fed the normal chow diet and given saline. In mice with insulin resistance induced by the high-fat diet, administration of Y-27632 increased secretion of GLP1 and glucose tolerance compared with administration of saline., Conclusions: In mouse intestinal organoids, an inhibitor of RhoA signaling increased the differentiation of the secretory lineage and the development of enteroendocrine cells. Inhibitors of RhoA signaling or other strategies to increase numbers of L cells might be developed for treatment of patients with type 2 diabetes or for increasing glucose tolerance., (Copyright © 2018 AGA Institute. Published by Elsevier Inc. All rights reserved.)
- Published
- 2018
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24. Ligand-selective small molecule modulators of the constitutively active vGPCR US28.
- Author
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Amărandi RM, Lückmann M, Melynis M, Jakobsen MH, Fallah Z, Spiess K, Hjortø GM, Pui A, Frimurer TM, and Rosenkilde MM
- Subjects
- Cells, Cultured, Dose-Response Relationship, Drug, Drug Discovery, HEK293 Cells, Humans, Ligands, Molecular Structure, Small Molecule Libraries chemical synthesis, Small Molecule Libraries chemistry, Structure-Activity Relationship, Receptors, Chemokine agonists, Small Molecule Libraries pharmacology, Viral Proteins agonists
- Abstract
US28 is a broad-spectrum constitutively active G protein-coupled receptor encoded by the human cytomegalovirus (HCMV). It binds and scavenges multiple CC-chemokines as well as CX
3 CL1 (fractalkine) by constitutive receptor endocytosis to escape immune surveillance. We herein report the design and characterization of a novel library of US28-acting commercially available ligands based on the molecular descriptors of two previously reported US28-acting structures. Among these, we identify compounds capable of selectively recognizing CCL2-and CCL4-, but not CX3 CL1-induced receptor conformations. Moreover, we find a direct correlation between the binding properties of small molecule ligands to CCL-induced conformations at the wild-type receptor and functional activity at the C-terminal truncated US28Δ300. As US28Δ300 is devoid of arrestin-recruitment and endocytosis, this highlights the potential usefulness of this construct in future drug discovery efforts aimed at specific US28 conformations. The new scaffolds identified herein represent valuable starting points for the generation of novel anti-HCMV therapies targeting the virus-encoded chemokine receptor US28 in a conformational-selective manner., (Copyright © 2018 Elsevier Masson SAS. All rights reserved.)- Published
- 2018
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25. Structure-Activity Investigations and Optimisations of Non-metabolite Agonists for the Succinate Receptor 1.
- Author
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Rexen Ulven E, Trauelsen M, Brvar M, Lückmann M, Bielefeldt LØ, Jensen LKI, Schwartz TW, and Frimurer TM
- Subjects
- Animals, Crystallography, X-Ray, Humans, Mice, Receptors, G-Protein-Coupled metabolism, Receptors, Purinergic P2Y1 ultrastructure, Succinic Acid metabolism, Receptors, G-Protein-Coupled agonists, Receptors, Purinergic P2Y1 metabolism, Structure-Activity Relationship
- Abstract
The succinate receptor 1 (SUCNR1) is a receptor for the metabolite succinate, which functions as a metabolic stress signal in the liver, kidney, adipose tissue and the retina. However, potent non-metabolite tool compounds are needed to reveal the physiological role and pharmacological potential of SUCNR1. Recently, we published the discovery of a computationally receptor-structure derived non-metabolite SUCNR1 agonist series with high target selectivity. We here report our structure-activity exploration and optimisation that has resulted in the development of agonists with nanomolar potency and excellent solubility and stability properties in a number of in vitro assays. Ligand-guided receptor models with high discriminative power between binding of active and inactive compounds were developed for design of novel chemotypes.
- Published
- 2018
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26. Structural changes at the myrtenol backbone reverse its positive allosteric potential into inhibitory GABAA receptor modulation.
- Author
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Milanos S, Kuenzel K, Gilbert DF, Janzen D, Sasi M, Buettner A, Frimurer TM, and Villmann C
- Subjects
- Allosteric Regulation drug effects, Bicyclic Monoterpenes, HEK293 Cells, Humans, Molecular Structure, GABA-A Receptor Antagonists chemistry, GABA-A Receptor Antagonists pharmacology, Monoterpenes chemistry, Monoterpenes pharmacology, Receptors, GABA-A metabolism
- Abstract
GABAA receptors are ligand-gated anion channels that form pentameric arrangements of various subunits. Positive allosteric modulators of GABAA receptors have been reported as being isolated either from plants or synthesized analogs of known GABAA receptor targeting drugs. Recently, we identified monoterpenes, e.g. myrtenol as a positive allosteric modulator at α1β2 GABAA receptors. Here, along with pharmacophore-based virtual screening studies, we demonstrate that scaffold modifications of myrtenol resulted in the loss of modulatory activity. Two independent approaches, fluorescence-based compound analysis and electrophysiological recordings in whole-cell configurations were used for analysis of transfected cells. C-atoms 1 and 2 of the myrtenol backbone were identified as crucial to preserve positive allosteric potential. A modification at C-atom 2 and lack of the hydroxyl group at C-atom 1 exhibited significantly reduced GABAergic currents at α1β2, α1β2γ, α2β3, α2β3γ and α4β3δ receptors. This effect was independent of the γ2 subunit. A sub-screen with side chain length and volume differences at the C-atom 1 identified two compounds that inhibited GABAergic responses but without receptor subtype specificity. Our combined approach of pharmacophore-based virtual screening and functional readouts reveals that side chain modifications of the bridged six-membered ring structure of myrtenol are crucial for its modulatory potential at GABAA receptors.
- Published
- 2018
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27. The HETE Is on FFAR1 and Pancreatic Islet Cells.
- Author
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Trauelsen M, Lückmann M, Frimurer TM, and Schwartz TW
- Subjects
- Hydroxyeicosatetraenoic Acids, Insulin Secretion, Islets of Langerhans, Receptors, G-Protein-Coupled, Glucose, Insulin
- Abstract
It is known but generally unappreciated that the fatty acid receptor FFAR1 (GPR40) is responsible for a major part of glucose-induced insulin secretion. This puzzling fact is now explained by Tunaru et al. (2018), who demonstrate that glucose-induced 20-hydroxyeicosatetraenoic acid (20-HETE) amplifies insulin secretion through autocrine activation of FFAR1., (Copyright © 2018 Elsevier Inc. All rights reserved.)
- Published
- 2018
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28. Receptor structure-based discovery of non-metabolite agonists for the succinate receptor GPR91.
- Author
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Trauelsen M, Rexen Ulven E, Hjorth SA, Brvar M, Monaco C, Frimurer TM, and Schwartz TW
- Subjects
- Cells, Cultured, Drug Discovery methods, HEK293 Cells, Humans, Protein Binding, Quantitative Structure-Activity Relationship, Receptors, G-Protein-Coupled chemistry, Receptors, G-Protein-Coupled metabolism, Small Molecule Libraries chemistry, Molecular Docking Simulation, Receptors, G-Protein-Coupled agonists, Small Molecule Libraries pharmacology
- Abstract
Objective: Besides functioning as an intracellular metabolite, succinate acts as a stress-induced extracellular signal through activation of GPR91 (SUCNR1) for which we lack suitable pharmacological tools., Methods and Results: Here we first determined that the cis conformation of the succinate backbone is preferred and that certain backbone modifications are allowed for GPR91 activation. Through receptor modeling over the X-ray structure of the closely related P2Y1 receptor, we discovered that the binding pocket is partly occupied by a segment of an extracellular loop and that succinate therefore binds in a very different mode than generally believed. Importantly, an empty side-pocket is identified next to the succinate binding site. All this information formed the basis for a substructure-based search query, which, combined with molecular docking, was used in virtual screening of the ZINC database to pick two serial mini-libraries of a total of only 245 compounds from which sub-micromolar, selective GPR91 agonists of unique structures were identified. The best compounds were backbone-modified succinate analogs in which an amide-linked hydrophobic moiety docked into the side-pocket next to succinate as shown by both loss- and gain-of-function mutagenesis. These compounds displayed GPR91-dependent activity in altering cytokine expression in human M2 macrophages similar to succinate, and importantly were devoid of any effect on the major intracellular target, succinate dehydrogenase., Conclusions: These novel, synthetic non-metabolite GPR91 agonists will be valuable both as pharmacological tools to delineate the GPR91-mediated functions of succinate and as leads for the development of GPR91-targeted drugs to potentially treat low grade metabolic inflammation and diabetic complications such as retinopathy and nephropathy., (Copyright © 2017 The Authors. Published by Elsevier GmbH.. All rights reserved.)
- Published
- 2017
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29. Structural biology: Full monty of family B GPCRs.
- Author
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Schwartz TW and Frimurer TM
- Subjects
- Glucagon-Like Peptide-1 Receptor, Molecular Biology, Protein Binding, Peptides, Receptors, G-Protein-Coupled
- Published
- 2017
- Full Text
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30. Biased agonism and allosteric modulation of G protein-coupled receptor 183 - a 7TM receptor also known as Epstein-Barr virus-induced gene 2.
- Author
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Daugvilaite V, Madsen CM, Lückmann M, Echeverria CC, Sailer AW, Frimurer TM, Rosenkilde MM, and Benned-Jensen T
- Subjects
- Allosteric Regulation drug effects, Animals, Binding, Competitive drug effects, CHO Cells, Cells, Cultured, Cricetulus, Dose-Response Relationship, Drug, HEK293 Cells, Humans, Models, Molecular, Mutagenesis, Site-Directed, Receptors, G-Protein-Coupled genetics, Receptors, G-Protein-Coupled metabolism, Signal Transduction drug effects, Sodium pharmacology, Structure-Activity Relationship, Hydroxycholesterols pharmacology, Receptors, G-Protein-Coupled agonists
- Abstract
Background and Purpose: The GPCR Epstein-Barr virus-induced gene 2 (EBI2, also known as GPR183) is activated by oxysterols and plays a pivotal role in the regulation of B cell migration during immune responses. While the molecular basis of agonist binding has been addressed in several studies, the concept of biased agonism of the EBI2 receptor has not been explored., Experimental Approach: We investigated the effects of the EBI2 endogenous agonist 7α,25-dihydroxycholesterol (7α,25-OHC) on G protein-dependent and -independent pathways as well as sodium ion allosterism using site-directed mutagenesis and functional studies. Moreover, we generated a homology model of the EBI2 receptor to investigate the structural basis of the allosteric modulation by sodium., Key Results: Residue N114, located in the middle of transmembrane-III at position III:11/3.35, was found to function as an efficacy switch. Thus, substituting N114 with an alanine (N114A) completely abolished heterotrimeric G protein subunit Gi α activation by 7α,25-OHC even though the specific binding of [
3 H]-7α,25-OHC increased. In contrast, the N114A mutant was still able to recruit β-arrestin and even had an enhanced potency (18.7-fold) compared with EBI2 wild type. Sodium had a negative allosteric effect on oxysterol binding that was mediated via N114, verifying the key role of N114. This was further supported by molecular modelling of the ion binding site based on a EBI2 receptor homology model., Conclusions and Implications: Collectively, our data point to N114 as a key residue for EBI2 signalling controlling the balance between G protein-dependent and -independent pathways and facilitating sodium binding., (© 2017 The British Pharmacological Society.)- Published
- 2017
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31. Structure-based discovery of novel US28 small molecule ligands with different modes of action.
- Author
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Lückmann M, Amarandi RM, Papargyri N, Jakobsen MH, Christiansen E, Jensen LJ, Pui A, Schwartz TW, Rosenkilde MM, and Frimurer TM
- Subjects
- Animals, COS Cells drug effects, Calcium metabolism, Drug Design, Drug Evaluation, Preclinical methods, High-Throughput Screening Assays methods, Humans, Ligands, Models, Molecular, Piperidines chemistry, Piperidines metabolism, Receptors, Chemokine agonists, Receptors, Chemokine metabolism, Small Molecule Libraries chemistry, Structure-Activity Relationship, Viral Proteins agonists, Viral Proteins metabolism, Receptors, Chemokine chemistry, Small Molecule Libraries pharmacology, Viral Proteins chemistry
- Abstract
The human cytomegalovirus-encoded G protein-coupled receptor US28 is a constitutively active receptor, which can recognize various chemokines. Despite the recent determination of its 2.9 Å crystal structure, potent and US28-specific tool compounds are still scarce. Here, we used structural information from a refined US28:VUF2274 complex for virtual screening of >12 million commercially available small molecule compounds. Using a combined receptor- and ligand-based approach, we tested 98 of the top 0.1% ranked compounds, revealing novel chemotypes as compared to the ~1.45 million known ligands in the ChEMBL database. Two compounds were confirmed as agonist and inverse agonist, respectively, in both IP accumulation and Ca
2+ mobilization assays. The screening setup presented in this work is computationally inexpensive and therefore particularly useful in an academic setting as it enables simultaneous testing in binding as well as in different functional assays and/or species without actual chemical synthesis., (© 2016 John Wiley & Sons A/S.)- Published
- 2017
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32. Model-Based Discovery of Synthetic Agonists for the Zn 2+ -Sensing G-Protein-Coupled Receptor 39 (GPR39) Reveals Novel Biological Functions.
- Author
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Frimurer TM, Mende F, Graae AS, Engelstoft MS, Egerod KL, Nygaard R, Gerlach LO, Hansen JB, Schwartz TW, and Holst B
- Subjects
- Allosteric Regulation, Drug Discovery, Gastric Mucosa metabolism, Humans, Insulin metabolism, Insulin Secretion, Molecular Structure, Receptors, G-Protein-Coupled metabolism, Structure-Activity Relationship, Receptors, G-Protein-Coupled agonists, Zinc metabolism
- Abstract
The G-protein-coupled receptor 39 (GPR39) is a G-protein-coupled receptor activated by Zn
2+ . We used a homology model-based approach to identify small-molecule pharmacological tool compounds for the receptor. The method focused on a putative binding site in GPR39 for synthetic ligands and knowledge of ligand binding to other receptors with similar binding pockets to select iterative series of minilibraries. These libraries were cherry-picked from all commercially available synthetic compounds. A total of only 520 compounds were tested in vitro, making this method broadly applicable for tool compound development. The compounds of the initial library were inactive when tested alone, but lead compounds were identified using Zn2+ as an allosteric enhancer. Highly selective, highly potent Zn2+ -independent GPR39 agonists were found in subsequent minilibraries. These agonists identified GPR39 as a novel regulator of gastric somatostatin secretion.- Published
- 2017
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33. Molecular Mechanism of Action for Allosteric Modulators and Agonists in CC-chemokine Receptor 5 (CCR5).
- Author
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Karlshøj S, Amarandi RM, Larsen O, Daugvilaite V, Steen A, Brvar M, Pui A, Frimurer TM, Ulven T, and Rosenkilde MM
- Subjects
- Animals, Binding Sites, COS Cells, Chlorocebus aethiops, Crystallography, X-Ray, Humans, Models, Molecular, Protein Binding, Protein Conformation, Pyridines chemistry, Allosteric Regulation drug effects, Chelating Agents pharmacology, Chemokine CCL3 metabolism, Pyridines pharmacology, Receptors, CCR5 chemistry, Receptors, CCR5 metabolism
- Abstract
The small molecule metal ion chelators bipyridine and terpyridine complexed with Zn
2+ (ZnBip and ZnTerp) act as CCR5 agonists and strong positive allosteric modulators of CCL3 binding to CCR5, weak modulators of CCL4 binding, and competitors for CCL5 binding. Here we describe their binding site using computational modeling, binding, and functional studies on WT and mutated CCR5. The metal ion Zn2+ is anchored to the chemokine receptor-conserved Glu-283VII:06/7.39 Both chelators interact with aromatic residues in the transmembrane receptor domain. The additional pyridine ring of ZnTerp binds deeply in the major binding pocket and, in contrast to ZnBip, interacts directly with the Trp-248VI:13/6.48 microswitch, contributing to its 8-fold higher potency. The impact of Trp-248 was further confirmed by ZnClTerp, a chloro-substituted version of ZnTerp that showed no inherent agonism but maintained positive allosteric modulation of CCL3 binding. Despite a similar overall binding mode of all three metal ion chelator complexes, the pyridine ring of ZnClTerp blocks the conformational switch of Trp-248 required for receptor activation, thereby explaining its lack of activity. Importantly, ZnClTerp becomes agonist to the same extent as ZnTerp upon Ala mutation of Ile-116III:16/3.40 , a residue that constrains the Trp-248 microswitch in its inactive conformation. Binding studies with125 I-CCL3 revealed an allosteric interface between the chemokine and the small molecule binding site, including residues Tyr-37I:07/1.39 , Trp-86II:20/2.60 , and Phe-109III:09/3.33 The small molecules and CCL3 approach this interface from opposite directions, with some residues being mutually exploited. This study provides new insight into the molecular mechanism of CCR5 activation and paves the way for future allosteric drugs for chemokine receptors., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)- Published
- 2016
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34. Role of Conserved Disulfide Bridges and Aromatic Residues in Extracellular Loop 2 of Chemokine Receptor CCR8 for Chemokine and Small Molecule Binding.
- Author
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Barington L, Rummel PC, Lückmann M, Pihl H, Larsen O, Daugvilaite V, Johnsen AH, Frimurer TM, Karlshøj S, and Rosenkilde MM
- Subjects
- Animals, COS Cells, Chemokine CCL1 metabolism, Chemokines, CC metabolism, Chlorocebus aethiops, Disulfides metabolism, Humans, Inositol 1,4,5-Trisphosphate metabolism, Protein Binding, Protein Domains, Protein Structure, Secondary, Receptors, CCR8 genetics, Receptors, CCR8 metabolism, Viral Proteins metabolism, Chemokine CCL1 chemistry, Chemokines, CC chemistry, Disulfides chemistry, Inositol 1,4,5-Trisphosphate chemistry, Receptors, CCR8 chemistry, Viral Proteins chemistry
- Abstract
Chemokine receptors play important roles in the immune system and are linked to several human diseases. The initial contact of chemokines with their receptors depends on highly specified extracellular receptor features. Here we investigate the importance of conserved extracellular disulfide bridges and aromatic residues in extracellular loop 2 (ECL-2) for ligand binding and activation in the chemokine receptor CCR8. We used inositol 1,4,5-trisphosphate accumulation and radioligand binding experiments to determine the impact of receptor mutagenesis on both chemokine and small molecule agonist and antagonist binding and action in CCR8. We find that the seven-transmembrane (TM) receptor conserved disulfide bridge (7TM bridge) linking transmembrane helix III (TMIII) and ECL-2 is crucial for chemokine and small molecule action, whereas the chemokine receptor conserved disulfide bridge between the N terminus and TMVII is needed only for chemokines. Furthermore, we find that two distinct aromatic residues in ECL-2, Tyr(184) (Cys + 1) and Tyr(187) (Cys + 4), are crucial for binding of the CC chemokines CCL1 (agonist) and MC148 (antagonist), respectively, but not for small molecule binding. Finally, using in silico modeling, we predict an aromatic cluster of interaction partners for Tyr(187) in TMIV (Phe(171)) and TMV (Trp(194)). We show in vitro that these residues are crucial for the binding and action of MC148, thus supporting their participation in an aromatic cluster with Tyr(187) This aromatic cluster appears to be present in a large number of CC chemokine receptors and thereby could play a more general role to be exploited in future drug development targeting these receptors., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)
- Published
- 2016
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35. In Silico Investigation of the Neurotensin Receptor 1 Binding Site: Overlapping Binding Modes for Small Molecule Antagonists and the Endogenous Peptide Agonist.
- Author
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Lückmann M, Holst B, Schwartz TW, and Frimurer TM
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- Binding Sites, Humans, Ligands, Models, Molecular, Molecular Structure, Neurotensin chemistry, Neurotensin pharmacology, Peptides chemistry, Peptides pharmacology, Protein Binding, Protein Domains, Pyrazoles chemistry, Pyrazoles pharmacology, Quinolines chemistry, Quinolines pharmacology, Receptors, Neurotensin chemistry, Signal Transduction drug effects, Computational Biology methods, Computer Simulation, Receptors, Neurotensin agonists, Receptors, Neurotensin antagonists & inhibitors
- Abstract
The neurotensin receptor 1 (NTSR1) belongs to the family of 7TM, G protein-coupled receptors, and is activated by the 13-amino-acid peptide neurotensin (NTS) that has been shown to play important roles in neurological disorders and the promotion of cancer cells. Recently, a high-resolution x-ray crystal structure of NTSR1 in complex with NTS8-13 has been determined, providing novel insights into peptide ligand recognition by 7TM receptors. SR48692, a potent and selective small molecule antagonist has previously been used extensively as a tool compound to study NTSR1 receptor signaling properties. To investigate the binding mode of SR48692 and other small molecule compounds to NTSR1, we applied an Automated Ligand-guided Backbone Ensemble Receptor Optimization protocol (ALiBERO), taking receptor flexibility and ligand knowledge into account. Structurally overlapping binding poses for SR48692 and NTS8-13 were observed, despite their distinct chemical nature and inverse pharmacological profiles. The optimized models showed significantly improved ligand recognition in a large-scale virtual screening assessment compared to the crystal structure. Our models provide new insights into small molecule ligand binding to NTSR1 and could facilitate the structure-based design of non-peptide ligands for the evaluation of the pharmacological potential of NTSR1 in neurological disorders and cancer., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)
- Published
- 2016
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36. Mutation-Guided Unbiased Modeling of the Fat Sensor GPR119 for High-Yield Agonist Screening.
- Author
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Norn C, Hauge M, Engelstoft MS, Kim SH, Lehmann J, Jones RM, Schwartz TW, and Frimurer TM
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- Amino Acid Sequence, Drug Discovery methods, Humans, Molecular Docking Simulation, Molecular Sequence Data, Oxadiazoles pharmacology, Protein Binding, Pyrimidines pharmacology, Receptors, G-Protein-Coupled chemistry, Receptors, G-Protein-Coupled genetics, Algorithms, High-Throughput Screening Assays methods, Mutation, Receptors, G-Protein-Coupled agonists
- Abstract
Recent benchmark studies have demonstrated the difficulties in obtaining accurate predictions of ligand binding conformations to comparative models of G-protein-coupled receptors. We have developed a data-driven optimization protocol, which integrates mutational data and structural information from multiple X-ray receptor structures in combination with a fully flexible ligand docking protocol to determine the binding conformation of AR231453, a small-molecule agonist, in the GPR119 receptor. Resulting models converge to one conformation that explains the majority of data from mutation studies and is consistent with the structure-activity relationship for a large number of AR231453 analogs. Another key property of the refined models is their success in separating active ligands from decoys in a large-scale virtual screening. These results demonstrate that mutation-guided receptor modeling can provide predictions of practical value for describing receptor-ligand interactions and drug discovery., (Copyright © 2015 Elsevier Ltd. All rights reserved.)
- Published
- 2015
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37. Biased Gs versus Gq proteins and β-arrestin signaling in the NK1 receptor determined by interactions in the water hydrogen bond network.
- Author
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Valentin-Hansen L, Frimurer TM, Mokrosinski J, Holliday ND, and Schwartz TW
- Subjects
- Alanine chemistry, Allosteric Site, Animals, COS Cells, Chlorocebus aethiops, Crystallography, X-Ray, DNA Mutational Analysis, Humans, Hydrogen Bonding, Molecular Dynamics Simulation, Monte Carlo Method, Protein Conformation, Receptors, Ghrelin metabolism, Signal Transduction, Sodium chemistry, Transfection, Water chemistry, beta-Arrestins, Arrestins metabolism, GTP-Binding Protein alpha Subunits, Gq-G11 metabolism, GTP-Binding Protein alpha Subunits, Gs metabolism, Receptors, G-Protein-Coupled metabolism, Receptors, Neurokinin-1 metabolism
- Abstract
X-ray structures, molecular dynamics simulations, and mutational analysis have previously indicated that an extended water hydrogen bond network between trans-membranes I-III, VI, and VII constitutes an allosteric interface essential for stabilizing different active and inactive helical constellations during the seven-trans-membrane receptor activation. The neurokinin-1 receptor signals efficiently through Gq, Gs, and β-arrestin when stimulated by substance P, but it lacks any sign of constitutive activity. In the water hydrogen bond network the neurokinin-1 has a unique Glu residue instead of the highly conserved AspII:10 (2.50). Here, we find that this GluII:10 occupies the space of a putative allosteric modulating Na(+) ion and makes direct inter-helical interactions in particular with SerIII:15 (3.39) and AsnVII:16 (7.49) of the NPXXY motif. Mutational changes in the interface between GluII:10 and AsnVII:16 created receptors that selectively signaled through the following: 1) Gq only; 2) β-arrestin only; and 3) Gq and β-arrestin but not through Gs. Interestingly, increased constitutive Gs but not Gq signaling was observed by Ala substitution of four out of the six core polar residues of the network, in particular SerIII:15. Three residues were essential for all three signaling pathways, i.e. the water-gating micro-switch residues TrpVI:13 (6.48) of the CWXP motif and TyrVII:20 (7.53) of the NPXXY motif plus the totally conserved AsnI:18 (1.50) stabilizing the kink in trans-membrane VII. It is concluded that the interface between position II:10 (2.50), III:15 (3.39), and VII:16 (7.49) in the center of the water hydrogen bond network constitutes a focal point for fine-tuning seven trans-membrane receptor conformations activating different signal transduction pathways., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)
- Published
- 2015
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38. Optimisation of in silico derived 2-aminobenzimidazole hits as unprecedented selective kappa opioid receptor agonists.
- Author
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Sasmal PK, Krishna CV, Adabala SS, Roshaiah M, Rawoof KA, Thadi E, Sukumar KP, Cheera S, Abbineni C, Rao KV, Prasanthi A, Nijhawan K, Jaleel M, Iyer LR, Chaitanya TK, Tiwari NK, Krishna NL, Potluri V, Khanna I, Frimurer TM, Lückmann M, Rist Ø, Elster L, and Högberg T
- Subjects
- Amino Acid Sequence, Computer Simulation, Humans, Molecular Sequence Data, Receptors, Opioid, kappa chemistry, Sequence Homology, Amino Acid, Structure-Activity Relationship, Benzimidazoles pharmacology, Receptors, Opioid, kappa agonists
- Abstract
Kappa opioid receptor (KOR) is an important mediator of pain signaling and it is targeted for the treatment of various pains. Pharmacophore based mining of databases led to the identification of 2-aminobenzimidazole derivative as KOR agonists with selectivity over the other opioid receptors DOR and MOR. A short SAR exploration with the objective of identifying more polar and hence less brain penetrant agonists is described herewith. Modeling studies of the recently published structures of KOR, DOR and MOR are used to explain the receptor selectivity. The synthesis, biological evaluation and SAR of novel benzimidazole derivatives as KOR agonists are described. The in vivo proof of principle for anti-nociceptive effect with a lead compound from this series is exemplified., (Copyright © 2014 Elsevier Ltd. All rights reserved.)
- Published
- 2015
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39. Structural basis for constitutive activity and agonist-induced activation of the enteroendocrine fat sensor GPR119.
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Engelstoft MS, Norn C, Hauge M, Holliday ND, Elster L, Lehmann J, Jones RM, Frimurer TM, and Schwartz TW
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- Animals, COS Cells, Chlorocebus aethiops, Endocannabinoids, Insulin Secretion, Molecular Docking Simulation, Protein Structure, Tertiary, Receptors, G-Protein-Coupled metabolism, Transfection, Enteroendocrine Cells metabolism, Insulin metabolism, Oleic Acids pharmacology, Oxadiazoles pharmacology, Pyrimidines pharmacology, Receptors, G-Protein-Coupled agonists
- Abstract
Background and Purpose: GPR119 is a Gαs-coupled 7TM receptor activated by endogenous lipids such as oleoylethanolamide (OEA) and by the dietary triglyceride metabolite 2-monoacylglycerol. GPR119 stimulates enteroendocrine hormone and insulin secretion. But despite massive drug discovery efforts in the field, very little is known about the basic molecular pharmacology of GPR119., Experimental Approach: GPR119 receptor signalling was studied in transfected cells. Mutational mapping (30 mutations in 23 positions) was performed on residues required for ligand-independent and agonist-induced GPR119 activation (AR231453 and OEA). Novel Rosetta-based receptor modelling was applied, using a composite template approach with segments from different X-ray structures and fully flexible ligand docking., Key Results: The increased signalling induced by increasing the cell surface expression of GPR119 in the absence of agonist and the inhibitory effect of two synthetic inverse agonists demonstrated that GRP119 signals with a high degree of constitutive activity through the Gαs pathway. The mutational maps for AR231453 and OEA were very similar and, surprisingly, also similar to the mutational map for residues affecting the constitutive signalling - albeit with key differences. Surprisingly, almost all residues in extracellular loop-2b were important for the constitutive activity. The molecular modelling and docking demonstrated that AR231453 binds in a 'vertical' pocket in between mutational hits reaching from the centre of the receptor out to extracellular loop-2b., Conclusions and Implications: The high constitutive activity of GPR119 should be taken into account in future drug discovery efforts, which can now be guided by the detailed knowledge of the physiochemical properties of the extended ligand-binding pocket., (© 2014 The British Pharmacological Society.)
- Published
- 2014
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40. GPR40 (FFAR1) - Combined Gs and Gq signaling in vitro is associated with robust incretin secretagogue action ex vivo and in vivo.
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Hauge M, Vestmar MA, Husted AS, Ekberg JP, Wright MJ, Di Salvo J, Weinglass AB, Engelstoft MS, Madsen AN, Lückmann M, Miller MW, Trujillo ME, Frimurer TM, Holst B, Howard AD, and Schwartz TW
- Abstract
Objectives: GPR40 (FFAR1), a clinically proven anti-diabetes target, is a Gq-coupled receptor for long chain fatty acids (LCFA) stimulating insulin secretion directly and mediating a major part of the dietary triglyceride-induced secretion of the incretins GLP-1 and GIP. In phase-II studies the GPR40 agonist TAK-875 decreased blood glucose but surprisingly without stimulating incretins., Methods and Results: Here we find that GPR40 can signal through not only Gq and IP3 but also Gs and cAMP when stimulated with certain agonists such as AM-1638 and AM-5262 in contrast to the endogenous LCFA ligands and agonists such as TAK-875 and AM-837, which only signal through Gq. In competition binding against [3H]AM-1638 and [3H]L358 the Gq + Gs and the Gq-only agonists either competed for or showed positive cooperativity by increasing the binding of the two different radio-ligands, in opposite ways. Nevertheless, both the Gq-only and the Gq + Gs agonists all docked surprisingly well into the binding site for TAK-875 in the X-ray structure of GPR40. In murine intestinal primary cell-cultures the endogenous LCFAs and the Gq-only agonists stimulated GLP-1 secretion with rather poor efficacy as compared with the high efficacy Gq + Gs GPR40 agonists and a prototype GPR119 agonist. Similarly, in fasting both male and female mice the Gq + Gs agonists showed significantly higher efficacy than the Gq-only agonists in respect of increasing plasma GLP-1 and plasma GIP in a GPR40-dependent manner., Conclusions: It is concluded that stimulation of GPR40 by endogenous LCFAs or by Gq-only synthetic agonists result in a rather limited incretin response, whereas Gq + Gs GPR40 agonists stimulate incretin secretion robustly.
- Published
- 2014
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41. Gating function of isoleucine-116 in TM-3 (position III:16/3.40) for the activity state of the CC-chemokine receptor 5 (CCR5).
- Author
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Steen A, Sparre-Ulrich AH, Thiele S, Guo D, Frimurer TM, and Rosenkilde MM
- Subjects
- Amino Acid Sequence, Animals, COS Cells, Chlorocebus aethiops, Enzyme-Linked Immunosorbent Assay, Humans, Molecular Sequence Data, Receptors, CCR5 chemistry, Sequence Homology, Amino Acid, Ion Channel Gating, Isoleucine physiology, Receptors, CCR5 physiology
- Abstract
Background and Purpose: A conserved amino acid within a protein family indicates a significance of the residue. In the centre of transmembrane helix (TM)-5, position V:13/5.47, an aromatic amino acid is conserved among class A 7TM receptors. However, in 37% of chemokine receptors - a subgroup of 7TM receptors - it is a leucine indicating an altered function. Here, we describe the significance of this position and its possible interaction with TM-3 for CCR5 activity., Experimental Approach: The effects of [L203F]-CCR5 in TM-5 (position V:13/5.47), [I116A]-CCR5 in TM-3 (III:16/3.40) and [L203F;G286F]-CCR5 (V:13/5.47;VII:09/7.42) were determined in G-protein- and β-arrestin-coupled signalling. Computational modelling monitored changes in amino acid conformation., Key Results: [L203F]-CCR5 increased the basal level of G-protein coupling (20-70% of Emax ) and β-arrestin recruitment (50% of Emax ) with a threefold increase in agonist potency. In silico, [I116A]-CCR5 switched χ1-angle in [L203F]-CCR5. Furthermore, [I116A]-CCR5 was constitutively active to a similar degree as [L203F]-CCR5. Tyr(244) in TM-6 (VI:09/6.44) moved towards TM-5 in silico, consistent with its previously shown function for CCR5 activation. On [L203F;G286F]-CCR5 the antagonist aplaviroc was converted to a superagonist., Conclusions and Implications: The results imply that an aromatic amino acid in the centre of TM-5 controls the level of receptor activity. Furthermore, Ile(116) acts as a gate for the movement of Tyr(244) towards TM-5 in the active state, a mechanism proposed previously for the β2 -adrenoceptor. The results provide an understanding of chemokine receptor function and thereby information for the development of biased and non-biased antagonists and inverse agonists., (© 2013 The British Pharmacological Society.)
- Published
- 2014
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42. Structure-activity relationships and identification of optmized CC-chemokine receptor CCR1, 5, and 8 metal-ion chelators.
- Author
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Chalikiopoulos A, Thiele S, Malmgaard-Clausen M, Rydberg P, Isberg V, Ulven T, Frimurer TM, Rosenkilde MM, and Gloriam DE
- Subjects
- 2,2'-Dipyridyl chemistry, Animals, CCR5 Receptor Antagonists, COS Cells, Chlorocebus aethiops, Gene Expression, Halogenation, Humans, Hydrophobic and Hydrophilic Interactions, Ligands, Receptors, CCR1 agonists, Receptors, CCR1 antagonists & inhibitors, Receptors, CCR1 genetics, Receptors, CCR5 agonists, Receptors, CCR5 genetics, Receptors, CCR8 agonists, Receptors, CCR8 antagonists & inhibitors, Receptors, CCR8 genetics, Structure-Activity Relationship, 2,2'-Dipyridyl analogs & derivatives, Chelating Agents chemistry, Phenanthrolines chemistry, Pyridines chemistry, Receptors, CCR1 chemistry, Receptors, CCR5 chemistry, Receptors, CCR8 chemistry, Zinc chemistry
- Abstract
Chemokine receptors are involved in trafficking of leukocytes and represent targets for autoimmune conditions, inflammatory diseases, viral infections, and cancer. We recently published CCR1, CCR8, and CCR5 agonists and positive modulators based on a three metal-ion chelator series: 2,2'-bipyridine, 1,10-phenanthroline, and 2,2';6',2″-terpyridine. Here, we have performed an in-depth structure-activity relationship study and tested eight new optimized analogs. Using density functional theory calculations we demonstrate that the chelator zinc affinities depend on how electron-donating and -withdrawing substituents modulate the partial charges of chelating nitrogens. The zinc affinity was found to constitute the major factor for receptor potency, although the activity of some chelators deviate suggesting favorable or unfavorable interactions. Hydrophobic and halogen substituents are generally better accommodated in the receptors than polar groups. The new analog brominated terpyridine (29) resulted in the highest chelator potencies observed so far CCR1 (EC50: 0.49 μM) and CCR8 (EC50: 0.28 μM). Furthermore, we identified the first selective CCR5 agonist chelator, meta dithiomethylated bipyridine (23). The structure-activity relationships contribute to small-molecule drug development, and the novel chelators constitute valuable tools for studies of structural mechanisms for chemokine receptor activation.
- Published
- 2013
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43. Assessment and challenges of ligand docking into comparative models of G-protein coupled receptors.
- Author
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Nguyen ED, Norn C, Frimurer TM, and Meiler J
- Subjects
- Amino Acid Sequence, Binding Sites, Databases, Protein, Humans, Ligands, Molecular Sequence Data, Monte Carlo Method, Protein Binding, Protein Structure, Secondary, Structural Homology, Protein, Thermodynamics, Molecular Docking Simulation, Receptors, G-Protein-Coupled chemistry, Software
- Abstract
The rapidly increasing number of high-resolution X-ray structures of G-protein coupled receptors (GPCRs) creates a unique opportunity to employ comparative modeling and docking to provide valuable insight into the function and ligand binding determinants of novel receptors, to assist in virtual screening and to design and optimize drug candidates. However, low sequence identity between receptors, conformational flexibility, and chemical diversity of ligands present an enormous challenge to molecular modeling approaches. It is our hypothesis that rapid Monte-Carlo sampling of protein backbone and side-chain conformational space with Rosetta can be leveraged to meet this challenge. This study performs unbiased comparative modeling and docking methodologies using 14 distinct high-resolution GPCRs and proposes knowledge-based filtering methods for improvement of sampling performance and identification of correct ligand-receptor interactions. On average, top ranked receptor models built on template structures over 50% sequence identity are within 2.9 Å of the experimental structure, with an average root mean square deviation (RMSD) of 2.2 Å for the transmembrane region and 5 Å for the second extracellular loop. Furthermore, these models are consistently correlated with low Rosetta energy score. To predict their binding modes, ligand conformers of the 14 ligands co-crystalized with the GPCRs were docked against the top ranked comparative models. In contrast to the comparative models themselves, however, it remains difficult to unambiguously identify correct binding modes by score alone. On average, sampling performance was improved by 10(3) fold over random using knowledge-based and energy-based filters. In assessing the applicability of experimental constraints, we found that sampling performance is increased by one order of magnitude for every 10 residues known to contact the ligand. Additionally, in the case of DOR, knowledge of a single specific ligand-protein contact improved sampling efficiency 7 fold. These findings offer specific guidelines which may lead to increased success in determining receptor-ligand complexes.
- Published
- 2013
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44. Biased and constitutive signaling in the CC-chemokine receptor CCR5 by manipulating the interface between transmembrane helices 6 and 7.
- Author
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Steen A, Thiele S, Guo D, Hansen LS, Frimurer TM, and Rosenkilde MM
- Subjects
- Amino Acid Motifs, Animals, COS Cells, Chlorocebus aethiops, Humans, Protein Structure, Tertiary, Receptors, CCR5 genetics, Amino Acid Substitution, Mutation, Missense, Receptors, CCR5 metabolism, Signal Transduction
- Abstract
The equilibrium state of CCR5 is manipulated here toward either activation or inactivation by introduction of single amino acid substitutions in the transmembrane domains (TMs) 6 and 7. Insertion of a steric hindrance mutation in the center of TM7 (G286F in position VII:09/7.42) resulted in biased signaling. Thus, β-arrestin recruitment was eliminated, whereas constitutive activity was observed in Gαi-mediated signaling. Furthermore, the CCR5 antagonist aplaviroc was converted to a full agonist (a so-called efficacy switch). Computational modeling revealed that the position of the 7TM receptor-conserved Trp in TM6 (Trp-248 in position VI:13/6.48, part of the CWXP motif) was influenced by the G286F mutation, causing Trp-248 to change orientation away from TM7. The essential role of Trp-248 in CCR5 activation was supported by complete inactivity of W248A-CCR5 despite maintaining chemokine binding. Furthermore, replacing Trp-248 with a smaller aromatic amino acid (Tyr/Phe) impaired the β-arrestin recruitment, yet with maintained G protein activity (biased signaling); also, here aplaviroc switched to a full agonist. Thus, the altered positioning of Trp-248, induced by G286F, led to a constraint of G protein active, but β-arrestin inactive and thus biased, CCR5 conformation. These results provide important information on the molecular interplay and impact of TM6 and TM7 for CCR5 activity, which may be extrapolated to other chemokine receptors and possibly to other 7TM receptors.
- Published
- 2013
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45. PheVI:09 (Phe6.44) as a sliding microswitch in seven-transmembrane (7TM) G protein-coupled receptor activation.
- Author
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Valentin-Hansen L, Holst B, Frimurer TM, and Schwartz TW
- Subjects
- Amino Acid Substitution, Animals, COS Cells, Chlorocebus aethiops, Humans, Hydrophobic and Hydrophilic Interactions, Mutation, Missense, Protein Structure, Secondary, Receptors, Adrenergic, beta-2 genetics, Receptors, Adrenergic, beta-2 metabolism, Receptors, G-Protein-Coupled genetics, Receptors, G-Protein-Coupled metabolism, Receptors, Neurokinin-1 genetics, Receptors, Neurokinin-1 metabolism, Receptors, Adrenergic, beta-2 chemistry, Receptors, G-Protein-Coupled chemistry, Receptors, Neurokinin-1 chemistry
- Abstract
In seven-transmembrane (7TM), G protein-coupled receptors, highly conserved residues function as microswitches, which alternate between different conformations and interaction partners in an extended allosteric interface between the transmembrane segments performing the large scale conformational changes upon receptor activation. Computational analysis using x-ray structures of the β(2)-adrenergic receptor demonstrated that PheVI:09 (6.44), which in the inactive state is locked between the backbone and two hydrophobic residues in transmembrane (TM)-III, upon activation slides ∼2 Å toward TM-V into a tight pocket generated by five hydrophobic residues protruding from TM-III and TM-V. Of these, the residue in position III:16 (3.40) (often an Ile or Val) appears to function as a barrier or gate for the transition between inactive and active conformation. Mutational analysis showed that PheVI:09 is essential for the constitutive and/or agonist-induced signaling of the ghrelin receptor, GPR119, the β(2)-adrenergic receptor, and the neurokinin-1 receptor. Substitution of the residues constituting the hydrophobic pocket between TM-III and TM-V in the ghrelin receptor in four of five positions impaired receptor signaling. In GPR39, representing the 12% of 7TM receptors lacking an aromatic residue at position VI:09, unchanged agonist-induced signaling was observed upon Ala substitution of LeuVI:09 despite reduced cell surface expression of the mutant receptor. It is concluded that PheVI:09 constitutes an aromatic microswitch that stabilizes the active, outward tilted conformation of TM-VI relative to TM-III by sliding into a tight hydrophobic pocket between TM-III and TM-V and that the hydrophobic residue in position III:16 constitutes a gate for this transition.
- Published
- 2012
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46. Melanin concentrating hormone receptor 1 (MCHR1) antagonists-Still a viable approach for obesity treatment?
- Author
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Högberg T, Frimurer TM, and Sasmal PK
- Subjects
- Anti-Obesity Agents chemistry, Anti-Obesity Agents therapeutic use, Biphenyl Compounds chemistry, Biphenyl Compounds therapeutic use, Crystallography, X-Ray, Drug Design, Ether-A-Go-Go Potassium Channels antagonists & inhibitors, Humans, Models, Molecular, Molecular Structure, Naphthalenes chemistry, Naphthalenes therapeutic use, Piperidines chemistry, Piperidines therapeutic use, Pyrimidines chemistry, Pyrimidines therapeutic use, Structure-Activity Relationship, Anti-Obesity Agents pharmacology, Biphenyl Compounds pharmacology, Naphthalenes pharmacology, Obesity drug therapy, Piperidines pharmacology, Pyrimidines pharmacology, Receptors, Somatostatin antagonists & inhibitors
- Abstract
Obesity is a global epidemic associated with multiple severe diseases. Several pharmacotherapies have been investigated including the melanin concentrating hormone (MCH) and its receptor 1. The development of MCHR1 antagonists are described with a specific perspective on different chemotypes investigated in efforts to overcome hERG liabilities while having orally active, potent and selective compounds with sufficient brain penetration. A chemometric comparison of ∼2000 diverse MCHR1 and ∼1000 diverse hERG ligands underline the structural similarities. A binding pocket analysis of a MCHR1 model and recent X-ray structures of GPCRs invoked in selectivity issues indicate a way to support future drug design., (Copyright © 2012 Elsevier Ltd. All rights reserved.)
- Published
- 2012
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47. Modulation of constitutive activity and signaling bias of the ghrelin receptor by conformational constraint in the second extracellular loop.
- Author
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Mokrosiński J, Frimurer TM, Sivertsen B, Schwartz TW, and Holst B
- Subjects
- Alanine chemistry, Amino Acid Sequence, Animals, Arrestins metabolism, COS Cells, Chlorocebus aethiops, DNA Mutational Analysis, HEK293 Cells, Humans, Models, Biological, Models, Molecular, Molecular Sequence Data, Protein Conformation, Protein Structure, Secondary, Protein Structure, Tertiary, Receptors, G-Protein-Coupled chemistry, Receptors, Ghrelin chemistry, Signal Transduction, beta-Arrestins, Receptors, Ghrelin metabolism
- Abstract
Based on a rare, natural Glu for Ala-204(C+6) variant located six residues after the conserved Cys residue in extracellular loop 2b (ECL2b) associated with selective elimination of the high constitutive signaling of the ghrelin receptor, this loop was subjected to a detailed structure functional analysis. Introduction of Glu in different positions demonstrated that although the constitutive signaling was partly reduced when introduced in position 205(C+7) it was only totally eliminated in position 204(C+6). No charge-charge interaction partner could be identified for the Glu(C+6) variant despite mutational analysis of a number of potential partners in the extracellular loops and outer parts of the transmembrane segments. Systematic probing of position 204(C+6) with amino acid residues of different physicochemical properties indicated that a positively charged Lys surprisingly provided phenotypes similar to those of the negatively charged Glu residue. Computational chemistry analysis indicated that the propensity for the C-terminal segment of extracellular loop 2b to form an extended α-helix was increased from 15% in the wild type to 89 and 82% by introduction in position 204(C+6) of a Glu or a Lys residue, respectively. Moreover, the constitutive activity of the receptor was inhibited by Zn(2+) binding in an engineered metal ion site, stabilizing an α-helical conformation of this loop segment. It is concluded that the high constitutive activity of the ghrelin receptor is dependent upon flexibility in the C-terminal segment of extracellular loop 2 and that mutations or ligand binding that constrains this segment and thereby conceivably the movements of transmembrane domain V relative to transmembrane domain III inhibits the high constitutive signaling.
- Published
- 2012
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48. Modulation in selectivity and allosteric properties of small-molecule ligands for CC-chemokine receptors.
- Author
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Thiele S, Malmgaard-Clausen M, Engel-Andreasen J, Steen A, Rummel PC, Nielsen MC, Gloriam DE, Frimurer TM, Ulven T, and Rosenkilde MM
- Subjects
- Allosteric Regulation drug effects, Animals, Binding Sites, COS Cells, Chelating Agents chemistry, Chlorocebus aethiops, Copper chemistry, Glutamic Acid, Humans, Ligands, Models, Molecular, Organometallic Compounds chemical synthesis, Organometallic Compounds metabolism, Protein Structure, Tertiary, Pyridines chemistry, Receptors, CCR agonists, Receptors, CCR chemistry, Substrate Specificity, Zinc chemistry, Organometallic Compounds chemistry, Organometallic Compounds pharmacology, Receptors, CCR metabolism
- Abstract
Among 18 human chemokine receptors, CCR1, CCR4, CCR5, and CCR8 were activated by metal ion Zn(II) or Cu(II) in complex with 2,2'-bipyridine or 1,10-phenanthroline with similar potencies (EC(50) from 3.9 to 172 μM). Besides being agonists, they acted as selective allosteric enhancers of CCL3. These actions were dependent on a conserved glutamic acid at TM-7 (VII:06/7.39). A screening of 20 chelator analogues in complex with Zn(II) identified compounds with increased potencies, with 7 reaching highest potency at CCR1 (EC(50) of 0.85 μM), 20 at CCR8 (0.39 μM), and 8 at CCR5 (1.0 μM). Altered selectivity for CCR1 and CCR8 over CCR5 (11, 12) and a receptor-dependent separation of allosteric from intrinsic properties were achieved (20). The pocket similarities of CCR1 and CCR8, contrary to CCR5 as proposed by the ligand screen, were elaborated by computational modeling. These studies facilitate exploration of chemokine receptors as possible targets for therapeutic intervention.
- Published
- 2012
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49. The arginine of the DRY motif in transmembrane segment III functions as a balancing micro-switch in the activation of the β2-adrenergic receptor.
- Author
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Valentin-Hansen L, Groenen M, Nygaard R, Frimurer TM, Holliday ND, and Schwartz TW
- Subjects
- Alanine chemistry, Amino Acid Motifs, Animals, Arrestin chemistry, CHO Cells, COS Cells, Cell Membrane metabolism, Chlorocebus aethiops, Cricetinae, GTP-Binding Proteins metabolism, Gene Silencing, Models, Molecular, Molecular Dynamics Simulation, Mutagenesis, Site-Directed, Protein Binding, Receptors, Adrenergic, beta-2 metabolism, Structure-Activity Relationship, Arginine chemistry, Receptors, Adrenergic, beta-2 chemistry
- Abstract
Recent high resolution x-ray structures of the β2-adrenergic receptor confirmed a close salt-bridge interaction between the suspected micro-switch residue ArgIII:26 (Arg3.50) and the neighboring AspIII:25 (Asp3.49). However, neither the expected "ionic lock" interactions between ArgIII:26 and GluVI:-06 (Glu6.30) in the inactive conformation nor the interaction with TyrV:24 (Tyr5.58) in the active conformation were observed in the x-ray structures. Here we find through molecular dynamics simulations, after removal of the stabilizing T4 lysozyme, that the expected salt bridge between ArgIII:26 and GluVI:-06 does form relatively easily in the inactive receptor conformation. Moreover, mutational analysis of GluVI:-06 in TM-VI and the neighboring AspIII:25 in TM-III demonstrated that these two residues do function as locks for the inactive receptor conformation as we observed increased G(s) signaling, arrestin mobilization, and internalization upon alanine substitutions. Conversely, TyrV:24 appears to play a role in stabilizing the active receptor conformation as loss of function of G(s) signaling, arrestin mobilization, and receptor internalization was observed upon alanine substitution of TyrV:24. The loss of function of the TyrV:24 mutant could partly be rescued by alanine substitution of either AspIII:25 or GluVI:-06 in the double mutants. Surprisingly, removal of the side chain of the ArgIII:26 micro-switch itself had no effect on G(s) signaling and internalization and only reduced arrestin mobilization slightly. It is suggested that ArgIII:26 is equally important for stabilizing the inactive and the active conformation through interaction with key residues in TM-III, -V, and -VI, but that the ArgIII:26 micro-switch residue itself apparently is not essential for the actual G protein activation.
- Published
- 2012
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50. An aromatic region to induce a switch between agonism and inverse agonism at the ghrelin receptor.
- Author
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Els S, Schild E, Petersen PS, Kilian TM, Mokrosinski J, Frimurer TM, Chollet C, Schwartz TW, Holst B, and Beck-Sickinger AG
- Subjects
- Animals, COS Cells, Chlorocebus aethiops, Feeding Behavior drug effects, Humans, Models, Molecular, Mutagenesis, Oligopeptides chemistry, Oligopeptides pharmacology, Rats, Receptors, Ghrelin agonists, Receptors, Ghrelin antagonists & inhibitors, Receptors, Ghrelin drug effects
- Abstract
The ghrelin receptor displays a high constitutive activity suggested to be involved in the regulation of appetite and food intake. Here, we have created peptides with small changes in the core binding motif -wFw- of the hexapeptide KwFwLL-NH(2) that can swap the peptide behavior from inverse agonism to agonism, indicating the importance of this sequence. Introduction of β-(3-benzothienyl)-d-alanine (d-Bth), 3,3-diphenyl-d-alanine (d-Dip) and 1-naphthyl-d-alanine (d-1-Nal) at position 2 resulted in highly potent and efficient inverse agonists, whereas the substitution of d-tryptophane at position 4 with 1-naphthyl-d-alanine (d-1-Nal) and 2-naphthyl-d-alanine (d-2-Nal) induces agonism in functional assays. Competitive binding studies showed a high affinity of the inverse agonist K-(d-1-Nal)-FwLL-NH(2) at the ghrelin receptor. Moreover, mutagenesis studies of the receptor revealed key positions for the switch between inverse agonist and agonist response. Hence, only minor changes in the peptide sequence can decide between agonism and inverse agonism and have a major impact on the biological activity.
- Published
- 2012
- Full Text
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