Design and synthesis of downsized metastin (45-54) analogs with maintenance of high GPR54 agonistic activity

Metastin has been identified as a metastasis suppressor gene product that mediates its function through a G protein coupled receptor, GPR54. To refine insight into the critical pharmacophore for the activation of GPR54, we have conducted alanine and d-amino acid scanning on a biologically active metastin fragment (45-54). Based on these data and structures of peptides previously reported to activate GPR54, a series of shortened metastin (45-54) derivatives were synthesized and tested for the ability to induce GPR54 signaling. These biological experiments were performed in yeast containing human GPR54 that was coupled to the pheromone response pathway and a pheromone responsive lacZ reporter gene. Compounds 32, 33, and 39, which possess an N-terminal basic group and a C-terminal RW-amide motif, were strong agonists, similar to the level of metastin. This may provide an approach to reverse the pro-metastatic effect of metastin deletion in multiple malignant tumors.     

Structure-activity relationship study on small peptidic GPR54 agonists

Metastin (kisspeptin-54) is an endogenous ligand that modulates gonadotropin-releasing hormone (GnRH) secretion through the interaction with a G protein-coupled receptor (GPCR), GPR54. The short-chain C-terminal decapeptide amide, metastin (45-54) (kisspeptin-10), exerts the identical bioactivities to metastin, such as metastasis suppression of cancer cells and inhibition of trophoblast migration and invasion. In order to understand the structural requirement for GPR54 agonistic activity, structure-activity relationship (SAR) study on pentapeptide-based C-terminal metastin analogues was carried out. As a result, H-Amb-Nal(2)-Gly-Leu-Arg-Trp-NH2 34 was identified as a novel GPR54 agonist that possessed the most potent GPR54 agonistic activity reported so far.     

Characterization of the receptor binding residues of kisspeptins by positional scanning using peptide photoaffinity probes

Kisspeptins, endogenous peptide ligands for GPR54, play an important role in GnRH secretion. Since in vivo administration of kisspeptins induces increased plasma LH levels, GPR54 agonists hold promise as therapeutic agents for the treatment of hormonal secretion diseases. To facilitate the design of novel potent GPR54 ligands, residues in kisspeptins that involve in the interaction with GPR54 were investigated by kisspeptin-based photoaffinity probes. Herein, we report the design and synthesis of novel kisspeptin-based photoaffinity probes, and the application to crosslinking experiments for GPR54-expressing cells.     

GPR54 (KISS1R) transactivates EGFR to promote breast cancer cell invasiveness

Kisspeptins (Kp), peptide products of the Kisspeptin-1 (KISS1) gene are endogenous ligands for a G protein-coupled receptor 54 (GPR54). Previous findings have shown that KISS1 acts as a metastasis suppressor in numerous cancers in humans. However, recent studies have demonstrated that an increase in KISS1 and GPR54 expression in human breast tumors correlates with higher tumor grade and metastatic potential. At present, whether or not Kp signaling promotes breast cancer cell invasiveness, required for metastasis and the underlying mechanisms, is unknown. We have found that kisspeptin-10 (Kp-10), the most potent Kp, stimulates the invasion of human breast cancer MDA-MB-231 and Hs578T cells using Matrigel-coated Transwell chamber assays and induces the formation of invasive stellate structures in three-dimensional invasion assays. Furthermore, Kp-10 stimulated an increase in matrix metalloprotease (MMP)-9 activity. We also found that Kp-10 induced the transactivation of epidermal growth factor receptor (EGFR). Knockdown of the GPCR scaffolding protein, β-arrestin 2, inhibited Kp-10-induced EGFR transactivation as well as Kp-10 induced invasion of breast cancer cells via modulation of MMP-9 secretion and activity. Finally, we found that the two receptors associate with each other under basal conditions, and FRET analysis revealed that GPR54 interacts directly with EGFR. The stability of the receptor complex formation was increased upon treatment of cells by Kp-10. Taken together, our findings suggest a novel mechanism by which Kp signaling via GPR54 stimulates breast cancer cell invasiveness.     

Synthesis and structure–activity relationships of 2-acylamino-4,6-diphenylpyridine derivatives as novel antagonists of GPR54

GPR54 is a G protein-coupled receptor (GPCR) which was formerly an orphan receptor. Recent functional study of GPR54 revealed that the receptor has an essential role to modulate sex-hormones including GnRH. Though antagonists of GPR54 are expected to be novel drugs for sex-hormone dependent diseases such as prostate cancer or endometriosis, small molecule GPR54 antagonists have not been reported. We have synthesized a series of 2-acylamino-4,6-diphenylpyridines to identify potent GPR54 antagonists. Detailed structure–activity relationship studies led to compound 9l with an IC₅₀ value of 3.7 nM in a GPR54 binding assay, and apparent antagonistic activity in a cellular functional assay.     

Design,synthesis and SAR of a novel series of heterocyclic phenylpropanoic acids as GPR120 agonists

A novel series of 5-membered heterocycle-containing phenylpropanoic acid derivatives was discovered as potent GPR120 agonists with low clearance, high oral bioavailability and in vivo antidiabetic activity in rodents.     

Discovery of novel pyrrole derivatives as potent agonists for the niacin receptor GPR109A

Novel pyrrole derivatives were discovered as potent agonists of the niacin receptor, GPR109A. During the derivatization, compound 16 was found to be effective both in vitro and in vivo. The compound 16 exhibited a significant reduction of the non-esterified fatty acid in human GPR109A transgenic rats, and the duration of its in vivo efficacy was much longer than niacin.     

5-Hydroxy-eicosapentaenoic acid is an endogenous GPR119 agonist and enhances glucose-dependent insulin secretion

GPR119 is one of the G-protein-coupled receptors expressed in pancreatic β-cells and intestinal endocrine cells. Since agonists to GPR119 stimulate glucose-dependent insulin secretion, GPR119 agonists are anticipated to promote anti-diabetic effects and control of glucose homeostasis. Here, we reported that an omega-3 unsaturated fatty acid metabolite, 5-hydroxy-eicosapentaenoic acid (5-HEPE), was a potent agonist for GPR119 and enhanced glucose-dependent insulin secretion. 5-HEPE stimulated cAMP accumulation in mouse MIN6 insulinoma cells and human HuTu80 intestinal adenocarcinoma cells. These effects were blunted by GPR119-specific siRNA. Recombinant GPR119 also responded to 5-HEPE as well as authentic agonists. Several previous reports have indicated the beneficial biological effects of omega-3 unsaturated fatty acids, and epidemiological studies have suggested that these fatty acids plays a protective role against diabetes. However, the molecular pharmacology and receptor identifications of omega-3 unsaturated fatty acids and their metabolites have not yet been well investigated. It is hoped that our findings will encourage novel investigations into the molecular relationships between omega-3 fatty acids and diabetes.     

Design,synthesis and biological evaluation of novel 7-azaspiro[3.5]nonane derivatives as GPR119 agonists

The design and synthesis of a novel class of 7-azaspiro[3.5]nonane GPR119 agonists are described. In this series, optimization of the right piperidine N-capping group (R²) and the left aryl group (R³) led to the identification of compound 54g as a potent GPR119 agonist. Compound 54g showed a desirable PK profile in Sprague-Dawley (SD) rats and a favorable glucose lowering effect in diabetic rats.     

Discovery, optimisation and in vivo evaluation of novel GPR119 agonists

GPR119 is increasingly seen as an attractive target for the treatment of type II diabetes and other elements of the metabolic syndrome. During a programme aimed at developing agonists of the GPR119 receptor, we identified compounds that were potent with reduced hERG liabilities, that had good pharmacokinetic properties and that displayed excellent glucose-lowering effects in vivo. However, further profiling in a GPR119 knock-out (KO) mouse model revealed that the biological effects were not exclusively due to GPR119 agonism, highlighting the value of transgenic animals in drug discovery programs.     

Cyclic dibenzoylhydrazines reproducing the conformation of ecdysone agonists, RH-5849

We have investigated the biologically active conformation of the non-steroidal ecdysone agonist, 1-tert-butyl-1,2-dibenzoylhydrazine (RH-5849) by means of design, synthesis and conformational analysis of cyclic derivatives of RH-5849. Among the synthesized compounds, a 6-membered cyclic hydrazine bearing two benzoyl groups (5) exists in three conformational states in solution, and the major unsymmetrical conformer of 5 is similar to that of RH-5849 on the basis of 1H NMR and X-ray analyses. The 3,3-dimethyl derivative of 5 (10) exists as a single unsymmetrical conformer. Although there is conformational similarity of the cyclic derivatives with RH-5849, these compounds did not show any hormonal or insecticidal activity. The hydrogen bonding character of the amide N-H group of the dibenzoylhydrazine seems to play a critical role in the appearance of the biological activity.     

2-Acylamino-4,6-diphenylpyridine derivatives as novel GPR54 antagonists with good brain exposure and in vivo efficacy for plasma LH level in male rats

GPR54 is a G protein-coupled receptor (GPCR) which was formerly an orphan receptor. Recent functional study of GPR54 revealed that the receptor plays an essential role to modulate sex-hormones including GnRH. Thus, antagonists of GPR54 are expected to be novel drugs for sex-hormone dependent diseases such as prostate cancer or endometriosis. We recently reported 2-acylamino-4,6-diphenylpyridines as the first small molecule GPR54 antagonists with high potency. However, the representative compound 1 showed low brain exposure, where GPR54 acts as a modulator of gonadotropins by binding with its endogenous ligand, metastin. In order to discover compounds that have not only potent GPR54 antagonistic activity but also good brain permeability, we focused on converting the primary amine on the side chain to a secondary or tertiary amine, and finally we identified 15a containing a piperazine group. This compound exhibited high affinity to human and rat GPR54, apparent antagonistic activity, and high brain exposure. In addition, intravenous administration of 15a to castrated male rat suppressed plasma LH level, which indicates the possibility of a small molecule GPR54 antagonist as a novel drug for sex-hormone dependent diseases.     

Novel Zn2+ Modulated GPR39 Receptor Agonists Do Not Drive Acute Insulin Secretion in Rodents

Type 2 diabetes (T2D) occurs when there is insufficient insulin release to control blood glucose, due to insulin resistance and impaired β-cell function. The GPR39 receptor is expressed in metabolic tissues including pancreatic β-cells and has been proposed as a T2D target. Specifically, GPR39 agonists might improve β-cell function leading to more adequate and sustained insulin release and glucose control. The present study aimed to test the hypothesis that GPR39 agonism would improve glucose stimulated insulin secretion in vivo. A high throughput screen, followed by a medicinal chemistry program, identified three novel potent Zn²⁺ modulated GPR39 agonists. These agonists were evaluated in acute rodent glucose tolerance tests. The results showed a lack of glucose lowering and insulinotropic effects not only in lean mice, but also in diet-induced obese (DIO) mice and Zucker fatty rats. It is concluded that Zn²⁺ modulated GPR39 agonists do not acutely stimulate insulin release in rodents.     

Physical association of GPR54 C-terminal with protein phosphatase 2A

KiSS1 was discovered as a metastasis suppressor gene and subsequently found to encode kisspeptins (KP), ligands for a G protein coupled receptor (GPCR), GPR54. This ligand-receptor pair was later shown to play a critical role in the neuro-endocrine regulation of puberty. The C-terminal cytoplasmic (C-ter) domain of GPR54 contains a segment rich in proline and arginine residues that corresponds to the primary structure of four overlapping SH3 binding motifs. Yeast two hybrid experiments identified the catalytic subunit of protein phosphatase 2A (PP2A-C) as an interacting protein. Pull-down experiments with GST fusion proteins containing the GPR54 C-ter confirmed binding to PP2A-C in cell lysates and these complexes contained phosphatase activity. The proline arginine rich segment is necessary for these interactions. The GPR54 C-ter bound directly to purified recombinant PP2A-C, indicating the GPR54 C-ter may form complexes involving the catalytic subunit of PP2A that regulate phosphorylation of critical signaling intermediates.     

A Potent Class of GPR40 Full Agonists Engages the EnteroInsular Axis to Promote Glucose Control in Rodents

Type 2 diabetes is characterized by impaired glucose homeostasis due to defects in insulin secretion, insulin resistance and the incretin response. GPR40 (FFAR1 or FFA1) is a G-protein-coupled receptor (GPCR), primarily expressed in insulin-producing pancreatic β-cells and incretin-producing enteroendocrine cells of the small intestine. Several GPR40 agonists, including AMG 837 and TAK-875, have been disclosed, but no GPR40 synthetic agonists have been reported that engage both the insulinogenic and incretinogenic axes. In this report we provide a molecular explanation and describe the discovery of a unique and potent class of GPR40 full agonists that engages the enteroinsular axis to promote dramatic improvement in glucose control in rodents. GPR40 full agonists AM-1638 and AM-6226 stimulate GLP-1 and GIP secretion from intestinal enteroendocrine cells and increase GSIS from pancreatic islets, leading to enhanced glucose control in the high fat fed, streptozotocin treated and NONcNZO10/LtJ mouse models of type 2 diabetes. The improvement in hyperglycemia by AM-1638 was reduced in the presence of the GLP-1 receptor antagonist Ex(9–39)NH₂.     

Synthesis and structure–activity relationship of fused-pyrimidine derivatives as a series of novel GPR119 agonists

A series of fused-pyrimidine derivatives have been discovered as potent and orally active GPR119 agonists. A combination of the fused-pyrimidine structure and 4-chloro-2,5-difluorophenyl group provided the 5,7-dihydrothieno[3,4-d]pyrimidine 6,6-dioxide derivative 14a as a highly potent GPR119 agonist. Further optimization of the amino group at the 4-position in the pyrimidine ring led to the identification of 2-{1-[2-(4-chloro-2,5-difluorophenyl)-6,6-dioxido-5,7-dihydrothieno[3,4-d]pyrimidin-4-yl]piperidin-4-yl}acetamide (16b) as an advanced analog. Compound 16b was found to have extremely potent agonistic activity and improved glucose tolerance at 0.1 mg/kg po in mice. We consider compound 16b and its analogs to have clear utility in exploring the practicality of GPR119 agonists as potential therapeutic agents for the treatment of type 2 diabetes mellitus.     

Design,synthesis, and biological evaluation of aryl N-methoxyamide derivatives as GPR119 agonists

A series of N-methoxyamide derivatives was identified and evaluated as GPR119 agonists. Several N-methoxyamides with thienopyrimidine and pyridine scaffolds showed potent GPR119 agonistic activities. Among them, compound 9c displayed good in vitro activity and potency. Moreover, compound 9c lowered glucose excursion in mice in an oral glucose tolerance test and increased GLP-1 secretion in intestinal cells.     

Discovery and biological evaluation of novel 4-amino-2-phenylpyrimidine derivatives as potent and orally active GPR119 agonists

Novel 4-amino-2-phenylpyrimidine derivatives were synthesized and evaluated as GPR119 agonists. Optimization of the substituents on the phenyl ring at the 2-position and the amino group at the 4-position led to the identification of 3,4-dihalogenated and 2,4,5-trihalogenated phenyl derivatives showing potent GPR119 agonistic activity. The advanced analog (2R)-3-{[2-(4-chloro-2,5-difluorophenyl)-6-ethylpyrimidin-4-yl]amino}propane-1,2-diol (24g) was found to improve glucose tolerance at 1mg/kg po in mice and to show excellent pharmacokinetic profiles in mice and monkeys. Compound 24g also showed an excellent antidiabetic effect in diabetic kk/Ay mice after one week of single daily treatment. These results demonstrate that novel GPR119 agonist 24g improves glucose tolerance not only by enhancing glucose-dependent insulin secretion but also by preserving pancreatic β-cell function.