Research ArticlePharmacology

Identification of a small-molecule ligand that activates the neuropeptide receptor GPR171 and increases food intake

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Science Signaling  31 May 2016:
Vol. 9, Issue 430, pp. ra55
DOI: 10.1126/scisignal.aac8035
  • 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 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 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 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 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 the mRNAs 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.

  • Table 1 Binding of b-LEN and MS0015203 to different mutants of GPR171.

    Cells expressing wild-type DYK-tagged mouse GPR171 (wild type) or constructs expressing either Y99A, K169A, H177A, C184A, Y239A, or R243A mutations were incubated with 3 nM [125I]b-LEN in the absence or presence of different concentrations of b-LEN or MS0015203 (Binding) (0 to 10 μM). In a separate set of experiments, cells were incubated with b-LEN or MS0015203 (0 to 10 μM), and the amount of cAMP formed was measured (Signaling). Values are means ± SE of two independent experiments in triplicate. IC50, median inhibitory concentration; EC50, median effective concentration; n.d., not determinable.

    GPR171Binding b-LENBinding MS0015203Signaling b-LENSignaling MS0015203
    IC50 (nM)% Displaced at 10 μMIC50 (nM)% Displaced at 10 μMEC50 (nM)% Inhibition at 10 μMEC50 (nM)% Inhibition at 10 μM
    Wild type8 ± 1100 ± 181 ± 185 ± 13 ± 149 ± 3165 ± 337 ± 2
    Y99A376 ± 1100 ± 5445 ± 341 ± 231 ± 438 ± 21934 ± 511 ± 3
    K169A6 ± 199 ± 218 ± 244 ± 12 ± 144 ± 578 ± 416 ± 6
    H177A2 ± 199 ± 86 ± 224 ± 212 ± 535 ± 2n.d.7 ± 2
    C184A35 ± 199 ± 1657 ± 179 ± 27 ± 245 ± 2353 ± 130 ± 2
    Y239A42 ± 2100 ± 450 ± 327 ± 118 ± 339 ± 31119 ± 511 ± 1
    R243A92 ± 299 ± 9103 ± 220 ± 226 ± 230 ± 6n.d.−7 ± 2

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/9/430/ra55/DC1

    Fig. S1. Binding of b-LEN–derived peptides and ligands of P2Y12 or P2Y14 to GPR171.

    Fig. S2. Sequence alignment between mouse GPR171 and P2Y12.

    Fig. S3. Homology model of GPR171 superimposed with the P2Y12 crystal structure.

    Fig. S4. Surface receptor abundance of mutant GPR171 constructs expressed in CHO cells.

    Fig. S5. Selectivity profile MS0015203.

    Fig. S6. Effect of acute administration of MS0015203 on food intake in mice.

    Fig. S7. Effect of GPR171 knockdown on receptor abundance in hypothalamus.

    Table S1. Structures of compounds representative of each cluster.

    Table S2. Displacement of radiolabeled b-LEN by a compound with chemical similarity to MS0015203.

  • Supplementary Materials for:

    Identification of a small-molecule ligand that activates the neuropeptide receptor GPR171 and increases food intake

    Jonathan H. Wardman, Ivone Gomes, Erin N. Bobeck, Jennifer A. Stockert, Abhijeet Kapoor, Paola Bisignano, Achla Gupta, Mihaly Mezei, Sanjai Kumar, Marta Filizola, Lakshmi A. Devi*

    *Corresponding author. Email: lakshmi.devi{at}mssm.edu

    This PDF file includes:

    • Fig. S1. Binding of b-LEN–derived peptides and ligands of P2Y12 or P2Y14 to GPR171.
    • Fig. S2. Sequence alignment between mouse GPR171 and P2Y12.
    • Fig. S3. Homology model of GPR171 superimposed with the P2Y12 crystal structure.
    • Fig. S4. Surface receptor abundance of mutant GPR171 constructs expressed in CHO cells.
    • Fig. S5. Selectivity profile MS0015203.
    • Fig. S6. Effect of acute administration of MS0015203 on food intake in mice.
    • Fig. S7. Effect of GPR171 knockdown on receptor abundance in hypothalamus.
    • Table S1. Structures of compounds representative of each cluster.
    • Table S2. Displacement of radiolabeled b-LEN by a compound with chemical similarity to MS0015203.

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    Citation: J. H. Wardman, I. Gomes, E. N. Bobeck, J. A. Stockert, A. Kapoor, P. Bisignano, A. Gupta, M. Mezei, S. Kumar, M. Filizola, L. A. Devi, Identification of a small-molecule ligand that activates the neuropeptide receptor GPR171 and increases food intake. Sci. Signal. 9, ra55 (2016).

    © 2016 American Association for the Advancement of Science

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