New Hits as Antagonists of GPR103 Identified by HTS

Preclinical data indicate that GPR103 receptor and its endogenous neuropeptides QRFP26 and QRFP43 are involved in appetite regulation. A high throughput screening (HTS) for small molecule GPR103 antagonists was performed with the clinical goal to target weight management by modulation of appetite. A high hit rate from the HTS and initial low confirmation with respect to functional versus affinity data challenged us to revise the established screening cascade. To secure high quality data while increasing throughput, the binding assay was optimized on quality to run at single concentration. This strategy enabled evaluation of a larger fraction of chemical clusters and singletons delivering 17 new compound classes for GPR103 antagonism. Representative compounds from three clusters are presented. One of the identified clusters was further investigated, and an initial structure–activity relationship study is reported. The most potent compound identified had a pIC₅₀ of 7.9 with an improved ligand lipophilic efficiency

The acute glucose lowering effect of specific GPR120 activation in mice is mainly driven by glucagon-like peptide 1

<div><p>The mechanism behind the glucose lowering effect occurring after specific activation of GPR120 is not completely understood. In this study, a potent and selective GPR120 agonist was developed and its pharmacological properties were compared with the previously described GPR120 agonist Metabolex-36. Effects of both compounds on signaling pathways and GLP-1 secretion were investigated <i>in vitro</i>. The acute glucose lowering effect was studied in lean wild-type and GPR120 <i>null</i> mice following oral or intravenous glucose tolerance tests. <i>In vitro</i>, in GPR120 overexpressing cells, both agonists signaled through Gα_q, Gα_s and the β-arrestin pathway. However, in mouse islets the signaling pathway was different since the agonists reduced cAMP production. The GPR120 agonists stimulated GLP-1 secretion both <i>in vitro</i> in STC-1 cells and <i>in vivo</i> following oral administration. <i>In vivo</i> GPR120 activation induced significant glucose lowering and increased insulin secretion after intravenous glucose administration in lean mice, while the agonists had no effect in GPR120 <i>null</i> mice. Exendin 9–39, a GLP-1 receptor antagonist, abolished the GPR120 induced effects on glucose and insulin following an intravenous glucose challenge. In conclusion, GLP-1 secretion is an important mechanism behind the acute glucose lowering effect following specific GPR120 activation.</p></div

Metabolex-36 and AZ13581837 increased insulin secretion in IVGTT in lean mice.

<p>Insulin (<b>A</b>) and blood glucose (<b>C</b>) levels following an intravenous glucose challenge after oral administration of Metabolex-36 and AZ13581837 in lean female mice and corresponding AIR (<b>B</b>) and glucose elimination (<b>D</b>). Data represent six (Metabolex-36, n = 33, vehicle n = 34) and two (AZ13581837, n = 14) independent experiments and data are presented as mean ± SEM. Plasma levels of total GLP-1 (<b>E</b>) at time point was determined in separate experiments with n = 10 mice per group. ***p<0.001 and **p<0.01versus vehicle control.</p

Effect of Metabolex-36 and AZ13581837 on oral glucose tolerance in mice.

<p>Effect of Metabolex-36 (<b>A</b>) and AZ13581837 (<b>C</b>) on glucose response after an oral glucose challenge (2g/kg) in male mice and the corresponding unbound circulating concentrations of Metabolex-36 (<b>B</b>) and AZ13581837 (<b>D</b>) during the experiment. AZ13581837 and Metabolex-36 were given in different doses as indicated in the figures with n = 10 mice group and compared to vehicle treated mice (n = 12 mice per group). The EC₅₀ value for each GPR120 agonist assessed on mouse GPR120 using a DMR assay is indicated in figures. Blood glucose levels following oral glucose administration in GPR120 <i>null</i> mice (<b>E</b>) and wild type mice (<b>F</b>) were determined for vehicle (open squares) and Metabolex-36 (filled squares).</p

Exendin 9–39 blocked the Metabolex-36 induced potentiation of insulin secretion in lean mice.

<p>Insulin levels following intravenous glucose challenge (<b>A</b>) and corresponding blood glucose (<b>C</b>), after administration of Metabolex-36, exendin 9–39 or a co-administration of both, with corresponding calculations of AIR (<b>B</b>) and glucose elimination (<b>D</b>). The IVGTT data are from two independent experiments with 6–7 mice per group. Data are presented as mean ± SEM.**p<0.01 and *p<0.05 versus vehicle control.</p

AZ13581837 and Metabolex-36 reduced cAMP production in mouse islets and induced GLP-1 secretion from STC-1 cells.

<p>Effect of 10 μM AZ13581837, 10 μM Metabolex-36, 50 nM Exendin-4 or vehicle control on cAMP production in dispersed islets from wild type (<b>A</b>) and GP120 <i>null</i> mice (<b>B</b>). Data represent mean ± SEM from three independent experiments where islet were isolated from two or four mice of each genotype. cAMP was measured in at least triplicates for both wild type and GPR120 <i>null</i> islet in each experiment. STC-1 cells were stimulated with Metabolex-36, AZ13581837 or vehicle control (0.1% DMSO) for 2 hours and secreted active GLP-1 was measured by ELISA (<b>C</b>). Three independent GLP-1 secretion experiments were run where n = 3 of each control and compound treatment. *p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001 versus vehicle control (two sample, two sided t-test).</p

<i>In vitro</i> pharmacology of AZ13581837 and Metabolex-36.

<p><i>In vitro</i> pharmacology of AZ13581837 and Metabolex-36.</p

Exendin 9–39 blocked the AZ13581837 induced potentiation of insulin secretion in lean mice.

<p>Insulin levels following intravenous glucose challenge (<b>A</b>) and corresponding blood glucose (<b>C</b>), after administration of AZ13581837, exendin 9–39 or a co-administration of both, with corresponding calculations of AIR (<b>B</b>) and glucose elimination (<b>D</b>). The IVGTT data are from two independent experiments with n = 10 mice per group. Data are presented as mean ± SEM.***p<0.001 and *p<0.05 versus vehicle control.</p

Structure of AZ13581837 and Metabolex-36 and specificity of the compounds for human and mouse GPR120 and human or mouse GPR40.

<p><b>A)</b> Chemical structure of AZ13581837 and <b>B)</b> Metabolex-36.<b>C)</b> Effect of AZ13581837 (squares) and Metabolex-36 (circles) on DMR response in CHO-hGPR120 (filled symbols) and CHO (open symbols). <b>D)</b> Activity in CHO-GPR40 cells for AZ13581837 (filled squares), Metabolex-36 (filled circles) and GW9508 (filled triangles). Activity in CHO-hGPR120 cells is shown as reference for AZ13581837 (open squares), Metabolex-36 (open circles) and GW9508 (open triangles). <b>E)</b> Cross species selectivity evaluated in CHO-mGPR120 cells using a DMR assay. Activity of AZ13581837 (squares) and Metabolex-36 (circles) on mouse GPR120 (filled symbols) compared to human GPR120 (open symbols). <b>F)</b> Cross species selectivity for GPR40 evaluated using a calcium mobilization assay. Effect of AZ13581837 (filled squares) and Metabolex-36 (filled circles) on mouse GPR40 with GW9508 (filled triangles) as reference. Activity in CHO-hGPR120 cells is shown as comparison for AZ13581837 (open squares) and Metabolex-36 (open circles). Data are shown as mean ± SEM run in duplicates or more and representative for two or more independent experiments.</p