Discovery of a Potent and Selective GPR120 Agonist

GPR120 is a receptor of unsaturated long-chain fatty acids reported to mediate GLP-1 secretion, insulin sensitization, anti-inflammatory, and anti-obesity effects and is therefore emerging as a new potential target for treatment of type 2 diabetes and metabolic diseases. Further investigation is however hindered by the lack of suitable receptor modulators. Screening of FFA1 ligands provided a lead with moderate activity on GPR120 and moderate selectivity over FFA1. Optimization led to the discovery of the first potent and selective GPR120 agonist

Design, Synthesis, and Evaluation of a Diazirine Photoaffinity Probe for Ligand-Based Receptor Capture Targeting G Protein-Coupled Receptors.

Chemoproteomic approaches to identify ligand-receptor interactions have gained popularity. However, identifying transmembrane receptors remains challenging. A new trifunctional probe to aid the nonbiased identification of such receptors was developed and synthesized using a convenient seven-step synthesis. This probe contained three functional groups: 1) an N-hydroxysuccinimide ester for ligand-coupling through free amines, 2) a diazirine moiety to capture the receptor of interest upon irradiation with UV light, and 3) a biotin group which allowed affinity purification of the final adduct using streptavidin. The interaction between the G protein-coupled tachykinin neurokinin 1 (NK1) receptor, expressed in an inducible manner, and the peptidic ligand substance P was used as a test system. Liquid chromatography-mass spectrometry analysis confirmed successful coupling of the probe to substance P, while inositol monophosphate accumulation assays demonstrated that coupling of the probe did not interfere substantially with the substance P-NK1 receptor interaction. Confocal microscopy and western blotting provided evidence of the formation of a covalent bond between the probe and the NK1 receptor upon UV activation. As proof of concept, the probe was used in full ligand-based receptor-capture experiments to identify the substance P-binding receptor via liquid chromatography-tandem mass spectrometry, resulting in the successful identification of only the NK1 receptor. This provides proof of concept toward general utilization of this probe to define interactions between ligands and previously unidentified plasma-membrane receptors

capaR is tubule-specific and localized to principal cells; manipulation of capaR expression levels modulates [Ca²⁺]_i and fluid transport rates.

<p>(<b>A</b>) Mean mRNA expression data ± SEM were collated from Affymetrix tissue-specific array datasets described in flyatlas.org <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029897#pone.0029897-Chintapalli1" target="_blank">[14]</a> for adult and larval tissues as indicated. Blue shading (dark-adult; light-larvae) indicates epithelial tissues; whereas green shading (dark-adult; light-larvae) indicates fat body or tissues containing fat body <i>eg.</i>, adult head and carcass. ‘mRNA signal’ indicates how abundant <i>capaR</i> mRNA is; and for each tissue, <i>capaR</i> mRNA was detectably expressed in 4 out of 4 arrays (flyatlas.org). In order to assess the expression pattern of capaR <i>in vivo</i>, the capaR promoter-driven GAL4 line, capaR-GAL4, was generated and crossed with UAS-GFP, and fluorescence examined by GFP histochemistry in tissues from progeny of the cross (top left panel). For orientation, tubule regions are indicated by M (main segment); I (initial segment); L (lower tubule). Expression of capaR-driven GFP occurs in the principal cells in the tubule main segment, exclusion of a stellate cell (arrowed, top right panel). (<b>B–D</b>) <i>Drosophila</i> capa receptor is expressed in principal cells of the Malpighian tubule. (<b>B</b>) Tubules were processed with pre-immune serum and only low-level non-specific staining of intracellular vesicles was observed, confirming the specificity of the antibody. (<b>C</b>) Immunocytochemistry using anti-capaR rabbit polyclonal antibody and anti-Rabbit IgG-Texas Red conjugate reveal basolateral membrane localization of capaR in tubule principal cells. (<b>D</b>) Merge of z-stacks from (<b>B</b>) picture reveals exclusion of a stellate cell (arrowed). In panels A, B–D, nuclei are labelled blue with DAPI, scale bar represents 30 µM. (<b>E</b>) Manipulation of capaR affects cytosolic [Ca²⁺]_i levels in intact tubules. Tubules were dissected from c42>UAS-apoaequorin flies (c42aeq), c42aeq>UAS-capaR RNAi flies and c42aeq>UAS-capaR. Resting cytoslic [Ca²⁺]_i levels were measured, after which tubules were stimulated with 10⁻⁷ M capa-1 to obtain stimulated cytosolic [Ca²⁺]_i readings. Primary and secondary pooled data for cytosolic [Ca²⁺]_i levels are shown as nM [Ca²⁺]_i ± SEM, <i>N</i> = 6, where * P<0.05, Student's <i>t</i>-test. (<b>F</b>) Fluid transport by <i>Drosophila</i> c42-GAL4>capaR RNAi renal tubules is significantly decreased (as determined using a Student's <i>t</i>-test (*P<0.05)) compared to the parental GAL4 line when the tubule is stimulated by application of capa-1 (10⁻⁷ M). Secretion rates are expressed as nl/min ± SEM (<i>N</i> = 6).</p

Capa, Neuromedin and Hugin receptor <i>(CG8795)</i>-associated calcium signatures.

<p>(<b>A</b>) Typical cytoplasmic Ca²⁺ response in S2 cells expressing capaR and apoaequorin challenged with Drm-capa-1, Drm-PK-1, hugg, Drm-PK-2 and NMU-25 at a concentration of 10⁻⁷ M. (<b>B</b>) Typical cytoplasmic Ca²⁺ response in S2 cells expressing NMUR 2 and apoaequorin challenged with Drm-capa-1, Drm-PK-1, hugg, Drm-PK-2 and NMU-25 at a concentration of 10⁻⁷ M. (<b>C</b>) Typical cytoplasmic Ca²⁺ response in S2 cells expressing <i>CG8795</i>and apoaequorin challenged with Drm-capa-1, Drm-PK-1, hugg, Drm-PK-2 and NMU-25 at a concentration of 10⁻⁷ M. (<b>D</b>) Human NMU-25 dose-response curve in S2 cells and intact tubule. NMU-25 peptide stimulation of NMUR 2- or capaR- and apoaequorin-co-transfected S2 cells; and of tubule principal cells expressing apoaequorin transgene. Cells or tubules were challenged with increasing concentrations of agonist, and [Ca²⁺]_i was measured. Values were expressed as maximal (nM) - background (nM) (mean ± S.E.M., <i>N</i> = 3).</p

Immunocytochemical localization of the capa-expressing neurons with a vertebrate NMU antibody.

<p>Larval central nervous system was doubly labeled using an antibody against capa precursor (<b>A</b>, red), an antibody against porcine neuromedinU-8 (<b>B</b>, green). The immunoreactive cells bodies and neurohemal organs, the retrocerebral complex for the 2 cell bodies in the subesophageal neuromere, and the abdominal median transverse nerves for the 3 pairs of abdominal neuroendocrine cells all co-localize (<b>C</b>, yellow, merge). Scale bar 100 µm.</p

Functional role of NMU <i>in vivo</i>.

<p>(<b>A</b>) Fluid transport by <i>Drosophila</i> wild-type tubule is significantly increased after application of the peptides Drm-capa-1 and NMU-25 at 10⁻⁷ M but not with Drm-PK-1, hugg, Drm-PK-2. (<b>B</b>) [Ca²⁺]_i levels in NMU-25-stimulated (10⁻⁷ M) transgenic tubules expressing UAS-NMUR 2 and apoaequorin transgenes driven by c42-GAL4 (grey) compared with a typical control response (black, c42aeq). The calcium trace in blue represent a typical biphasic capa-1 (10⁻⁷ M) response in tubule principal cells. Pooled cytosolic [Ca²⁺] data from separate experiments are shown where data are nM [Ca²⁺] ± SEM, <i>N</i> = 6, where <i>P</i><0.05. (<b>C</b>) NMU-25 (10⁻⁷ M) stimulates increased fluid transport in transgenic tubules, in which UAS-NMUR 2 was driven by actin-GAL4. (<b>D</b>) capa-1 (10⁻⁷ M) stimulates increased fluid transport in transgenic tubules, in which UAS-NMUR 2 was driven by actin-GAL4. Data are expressed as mean fluid transport rate (nl/min) ± SEM, N = 6–10. The level of significance in A, C and D was determined using a Student's <i>t</i>-test (* P<0.05) and in C and D, statistical analysis was confined to the comparison between the parental and progeny response.</p

Desensitization and internalisation of capa-1-stimulated capaR.

<p>S2 cells were transfected with eYFP-tagged capaR, left un-treated or treated with capa-1, and viewed by confocal microscopy after immunocytochemistry with anti-GFP antibody. (<b>A</b>) Control. (<b>B</b>) capa-1 stimulated, 15 min. (<b>C</b>) sample was incubated for 15 minutes with capa-1, washed three times with culture medium followed by 30 minutes incubation in culture medium to allow resensitization. Nuclei are labelled blue with DAPI, scale bar represents 10 µM. (<b>D</b>) S2 cells expressing capaR were left untreated (0), or treated for 5, 10, 20 or 30 minutes (indicated) with 10⁻⁷ M capa-1 to induce receptor internalization. An additional sample was incubated for 30 minutes with capa-1, washed three times with culture medium without capa-1 followed by 30 minutes incubation in culture medium to allow resensitization (Res.). A sample of untransfected cells serves as a negative control. Samples were subjected to cell surface biotinylation to label plasma membrane proteins. We found that the protein concentration of biotinylated samples are generally lower than that of the total lysates; therefore, the equivalent of 5000 cells were loaded for the total lysate, and an equivalent of 15,000 cells were loaded for the biotinylated samples. Total lysates and biotinylated samples were subjected to western blot analysis. Immunoblot using anti-capaR antibody identified a band of the predicted size of 52 kDa which confirms the specificity of the antibody and an additional non specific 75 kDa protein absent in the cell-surface (biotinylated) fraction. (<b>E</b>) Samples from the cell surface biotinylation experiment were semi-quantified and corrected for total receptor expression. Relative cell surface expression is shown as a percentage of the non-treated S2 cells expressing capaR (t = 0). Bars indicated with an asterisk were significantly (P<0.05 as determined by one-way ANOVA) reduced compared to t = 0. (<b>F</b>) Calcium measurements in S2 cells transfected with expression constructs for aequorin and the capa receptor. S2 cells were challenged with 10⁻⁷ M capa-1, pre-treated with capa-1 for 15 min (Desensitization (Des.)), followed by ligand removal after which S2 cells were challenged at 15 min or 30 min (Resensitization (Res.)) with 10⁻⁷ M capa-1 and cytosolic [Ca²⁺]_i levels measured. Bars indicated with an asterisk were significantly (P<0.05 as determined by Student's <i>t</i>-test) reduced compared to control. (<b>G</b>) Analysis of capaR-β-arrestin-2 interactions. S2 cells were co-transfected with capa receptor tagged with <i>Renilla</i> luciferase and β-arrestin-2 tagged with eYFP. Bioluminescence Resonance Energy Transfer (BRET) signals were monitored after treatment of the cells for 15 min with varying concentrations of capa-1. Data are expressed as mBRET units ± SEM, <i>N</i> = 3.</p

Free Fatty Acid Receptor 1 (FFA1/GPR40) Agonists: Mesylpropoxy Appendage Lowers Lipophilicity and Improves ADME Properties

FFA1 (GPR40) is a new target for treatment of type 2 diabetes. We recently identified the potent FFA1 agonist TUG-469 (<b>5</b>). Inspired by the structurally related TAK-875, we explored the effects of a mesylpropoxy appendage on <b>5</b>. The appendage significantly lowers lipophilicity and improves metabolic stability while preserving potency, resulting in discovery of the potent FFA1 agonist <b>13</b>

Requirements and ontology for a G protein-coupled receptor oligomerization knowledge base-1

<p><b>Copyright information:</b></p><p>Taken from "Requirements and ontology for a G protein-coupled receptor oligomerization knowledge base"</p><p>http://www.biomedcentral.com/1471-2105/8/177</p><p>BMC Bioinformatics 2007;8():177-177.</p><p>Published online 30 May 2007</p><p>PMCID:PMC1904246.</p><p></p>olecularStructure concept. Each MolecularStructure is created with an instance of MethodType, and may be analyzed by many computational methods (instances of Analysis). MethodType has two subclasses: IdentificationMethod, which is used to identify the oligomer, and CreationMethod, which is used to create the MolecularStructure. IdentificationMethod and CreationMethod can have many sub-concepts that describe the precise type of method. In this figure we show only a few examples of such concepts

Requirements and ontology for a G protein-coupled receptor oligomerization knowledge base-2

<p><b>Copyright information:</b></p><p>Taken from "Requirements and ontology for a G protein-coupled receptor oligomerization knowledge base"</p><p>http://www.biomedcentral.com/1471-2105/8/177</p><p>BMC Bioinformatics 2007;8():177-177.</p><p>Published online 30 May 2007</p><p>PMCID:PMC1904246.</p><p></p> protomers oligomerize. There are three types of phenotypic change that are described by the ontology: changes in internalization, changes in signaling, and differences in the ligand binding of the oligomer as compared to any of the constituent protomers. The effect that ligand(s) binding to one or more of the protomers in an oligomer may have on binding of ligands to other protomers, or on the change in signaling, is described by the CrossTalk concept. The Internalization concept is used to describe changes that different ligands have on the trafficking of the Oligomer to the cell membrane. Any information that is available about the mechanism of activation of the Oligomer is stored in the MechanismOfActivation concept. The PhysiologicalRelevance concept stores information about the Oligomer