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. 2016 May 31;9(430):ra55.
doi: 10.1126/scisignal.aac8035.

Identification of a Small-Molecule Ligand That Activates the Neuropeptide Receptor GPR171 and Increases Food Intake

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Free PMC article

Identification of a Small-Molecule Ligand That Activates the Neuropeptide Receptor GPR171 and Increases Food Intake

Jonathan H Wardman et al. Sci Signal. .
Free PMC article

Abstract

Several neuropeptide systems in the hypothalamus, including neuropeptide Y and agouti-related protein (AgRP), control food intake. Peptides derived from proSAAS, a precursor implicated in the regulation of body weight, also control food intake. GPR171 is a heterotrimeric guanine nucleotide-binding protein (G protein)-coupled receptor (GPCR) for BigLEN (b-LEN), a peptide derived from proSAAS. To facilitate studies exploring the physiological role of GPR171, we sought to identify small-molecule ligands for this receptor by performing a virtual screen of a compound library for interaction with a homology model of GPR171. We identified MS0015203 as an agonist of GPR171 and demonstrated the selectivity of MS0015203 for GPR171 by testing the binding of this compound to 80 other membrane proteins, including family A GPCRs. Reducing the expression of GPR171 by shRNA (short hairpin RNA)-mediated knockdown blunted the cellular and tissue response to MS0015203. Peripheral injection of MS0015203 into mice increased food intake and body weight, and these responses were significantly attenuated in mice with decreased expression of GPR171 in the hypothalamus. Together, these results suggest that MS0015203 is a useful tool to probe the pharmacological and functional properties of GPR171 and that ligands targeting GPR171 may eventually lead to therapeutics for food-related disorders.

Figures

Fig. 1
Fig. 1. Identification of a GPR171 agonist and its predicted binding mode
(A) The indicated ligands (10 µM) predicted to bind to GPR171 were tested by measuring stimulation of Ca+2 signal in CHO cells expressing GPR171 and Gα15/i3. Ca2+ fluorescence was measured with Fluo-4 NW, ATP served as a positive control (activating endogenous purinergic receptors), b-LEN served as a GPR171-selective positive control, and l-LEN (LENSSPQAPA) served as a GPR171-negative control. Left graph shows normalized data (means ± SE; n = 6) with buffer conditions considered 100%. ***P < 0.001, one-way analysis of variance (ANOVA), Bonferroni’s test; F16,34 = 27.73. Right graph shows representative time course data of cells exposed to l-LEN, b-LEN, or MS0015203. Relative fluorescence is normalized to the baseline conditions (100%) before the indicated ligand was added. (B) Two-dimensional (2D) structure of MS0015203 and three-dimensional (3D) representation of the MS0015203-GPR171 complex showing amino acid residues involved in direct ligand-receptor interactions in the binding pocket. H-bond interactions are indicated with dotted lines. GPR171 residues are numbered and indicated with Ballesteros-Weinstein numbering (superscript). (C) Displacement of [125I]Tyr–b-LEN binding (3 nM) with MS0015203 (0 to 10 µM) using CHO cells expressing either mouse GPR171 (wild type) or the indicated mutant. Binding in the absence of MS0015203 was taken as 100%.
Fig. 2
Fig. 2. Characterization of molecular pharmacological properties of MS0015203
(A) Ability of MS0015203 (0 to 10 µM) to displace [125I]Tyr–b-LEN (3 nM) binding from rat hypothalamic membranes. Binding in the absence of cold ligands was taken as 100%. (B) Effect of MS0015203 (0 to 1 µM) on [35S]GTPγS binding in rat hypothalamic membranes. (C) Effect of MS0015203 (0 to 10 µM) on forskolin-stimulated adenylyl cyclase activity in rat hypothalamic membranes. (D) Effect of MS0015203 (0 to 10 µM) on cAMP concentrations in wild-type Neuro2A cells (N2A) and Neuro2A cells in which GPR171 is knocked down (N2A + KDV). Data in all panels represent means ± SE (n = 3 to 6). **P < 0.01; ***P < 0.0001, b-LEN versus MS0015203 (Student’s t test).
Fig. 3
Fig. 3. Effect of systemic administration of MS0015203 on c-Fos activity in PVN neurons
(A) Mice were injected twice intraperitoneally at 30 and 60 min with either vehicle [6% dimethyl sulfide (DMSO); top panel] or MS0015203 (3 mg/kg, intraperitoneally; bottom panel) before perfusion, and c-Fos activity was measured. Representative images are shown. An area like the one outlined in white (87.5 mm2) was used for analysis and is shown in the enlarged image. Arrows indicate c-Fos activation in GPR171-positive cells. DAPI, 4′,6-diamidino-2-phenylindole. (B) Images (×10) were quantified for total cells, GPR171-positive cells, or c-Fos–positive cells using ImageJ particle analysis. Two equal-sized areas from either side of the ventricle were quantified for each image. (C) Percentage of cells quantified in (B) shown as percentage of total number of cells that are positive for GPR171 or c-Fos after vehicle or MS0015203 injection. (D) Cells were randomly selected that contained GPR171 (40 per image) or lacked GPR171 (40 per image). Percent of each of these cells that contain c-Fos was quantified. n = 3 to 4 mice per group; two to four sections per mouse. Scale bars, 50 µm. *P < 0.05; **P < 0.01; ****P < 0.0001, vehicle versus MS0015203, two-way ANOVA with Bonferroni’s post hoc test.
Fig. 4
Fig. 4. Effect of acute administration of MS0015203 on food intake
(A) Cumulative food intake in fasted mice administered MS0015203 (3 mg/kg, intraperitoneally; n = 6) or vehicle (6% DMSO; n = 5). *P < 0.05; **P < 0.01, vehicle versus MS0015203 [two-way ANOVA with Bonferroni’s post hoc comparisons; Interaction: F3,27 = 2.261, P = 0.1041; Time: F3,27 = 197.9, P < 0.0001; Drug: F1,9 = 6.946, P < 0.0271; Subjects (matching): F9,27 = 8.511, P < 0.0001]. Food intake on an hour-by-hour basis is shown in fig. S6. (B) Effect of administration of MS0015203 (3 mg/kg, intraperitoneally) or vehicle (6% DMSO) to mice that were administered control virus (CV) or GPR171-knockdown virus (KDV) and that were fed a high-fat diet. **P < 0.01, one-way ANOVA (Bonferroni’s multiple comparison test). (C) Effect of administration of MS0015203 (2.5 mg/kg, intracerebroventricularly) or vehicle (6% DMSO) to mice that were administered control virus (CV) or GPR171-knockdown virus (KDV) and were fed regular chow. *P < 0.05; **P < 0.01, vehicle versus MS0015203 (two-way ANOVA with Bonferroni’s post hoc comparisons; Interaction: F9,48 = 0.3331, P = 0.9595; Time: F3,48 = 10.07, P <0.0001; Drug: F3,48 = 17.52, P < 0.0001). (D) Comparison of expression of GPR171 mRNA in hypothalami of mice administered control virus or GPR171-knockdown virus [n = 7 mice (control virus) or 9 mice (GPR171-knockdown virus)]. ***P < 0.001, control virus versus GPR171-knockdown virus, Student’s t test.
Fig. 5
Fig. 5. Effect of chronic administration of MS0015203 on body weight
(A) Effect of administration of MS0015203 (3 mg/kg, intraperitoneally; n = 5 per group) or vehicle (6% DMSO) every third day on body weight of mice fed a high-fat diet. *P < 0.05, vehicle versus MS0015203 (two-way ANOVA with Bonferroni’s post hoc comparisons; Interaction: F10,88 = 1.219, P = 0.2901; Time: F10,88 = 14.87, P < 0.0001; Drug: F1,88 = 45.79, P < 0.0001). (B) Effect of administration of MS0015203 (3 mg/kg, intraperitoneally) or vehicle (6% DMSO) every third day to mice that were administered control virus (n = 7) or GPR171-knockdown virus (n = 9) and that were fed a high-fat diet. **P < 0.01, control virus versus GPR171-knockdown virus (two-way ANOVA with Bonferroni’s post hoc comparisons; Interaction: F7,80X = 0.1145, P = X; Time: F7,80 = 22.69, P < 0.001; Treatment: F1,80 = 46.15, P < 0.0001). (C and D) Effect of chronic administration of MS0015203 on them RNAs encoding proSAAS, NPY, AgRP, orexin, GPR171, neuropeptide Y1 receptor (Y1R), neuropeptide Y5 receptor (Y5R), melanocortin 4 receptor (MC4R), hypocretin 1 receptor (HCRT1R), and hypocretin 2 receptor (HCRT2R) in the ventral hypothalamus relative to vehicle-injected controls (n = 5 per group). *P < 0.05; **P < 0.01; ***P < 0.001, vehicle versus MS0015203, Student’s t test. (E and F) Effect of chronic administration of MS0015203 on the mRNAs encoding the indicated neuropeptides or receptors in the dorsal hypothalamus relative to vehicle-injected controls (n = 5 per group). *P < 0.05; **P < 0.01; ***P < 0.001, vehicle versus MS0015203, Student’s t test.

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