GPR50 ligands for use for treating diseases mediated by GPR50

Abstract

GPR50 (also known as MMR, H9 or ML1X), is an orphan G protein-coupled receptor belonging to the melatonin receptor family, but which has lost the ability to bind melatonin during evolution. Its endogenous ligand has not yet been identified. However, the role of GPR50 in the pathophysiology of various diseases has been described, such as major CNS diseases, major depression, and bipolar disorder. Moreover, recent findings showed that the orphan GPR50 receptor promotes constitutive transforming growth factor-β (TGF-β) receptor signaling and protects against cancer development. Herein the inventors identified the first potent agonists of the orphan GPR50 receptor in a functional cAMP assay. Among these agonist, the 5-methoxy-2-methyl indole (Cp15) which displayed a high agonistic activity at GPR50 receptor (EC50 = 0.5 nM (cAMP), Kd = 174nM (MST)). Intriguingly, replacement of the methyl group in the initially screened 5-methoxy-2-methyl indole with a 2-methylamino group (5-methoxy-2-methylamine indole) (Cp32) resulted in also high agonist potency. In another contribution to define SARs, indole nucleus of Cp15 was replaced by benzofuran (Cp34).

Description

[0001] GPR50 LIGANDS FOR USE FOR TREATING DISEASES MEDIATED BY GPR50

[0002] FIELD OF THE INVENTION:

[0003] The present invention relates to novel compounds for use as GPR50 ligands, and their use in the treatment of diseases associated by GPR50.

[0004] BACKGROUND OF THE INVENTION:

[0005] G protein-coupled receptors (GPCRs) represent the largest family of cell membrane proteins with approximately 800 members identified in the human genome. GPCRs are significant therapeutic targets in many pathological conditions including diabetes, neurodegeneration, cardiovascular disease, and psychiatric disorders. GPCRs interact with G proteins in the plasma membrane. Those G proteins are heterotrimeric proteins consisting of three different subunits (a, P and y subunits) with an ability to bind the nucleotides guanosine triphosphate (GTP) and guanosine diphosphate (GDP) depending on whether the protein is active or inactive respectively.1'2G proteins are classified into four families according to their a subunit; Gai / o, Gas, Gal2 / 13, and Gaq / 11.3When a ligand is bound to a GPCR, it causes a conformational change that consequently leads to an interaction between the GPCR and a nearby G protein where GTP physically replaces the GDP bound to the alpha subunit.2'4As a result, the G protein subunits dissociate into two parts: the GTP-a and a GPy dimer. Both parts are no longer bound to the GPCR and they can diffuse to interact with other proteins such as adenylate cyclases, phospholipases and ion channels.2'4Consequently, this affects diverse cellular functions through changes in the concentration of intracellular signaling molecules such as cyclic guanosine monophosphate (cGMP), cyclic adenosine monophosphate (cAMP), diacylglycerol, inositol phosphates, and cytosolic ions.5G proteins remain active as long as their alpha subunits are bound to GTP. G proteins are inactivated when GTP is hydrolysed back to GDP by their intrinsic GTPase activity followed by the reassembly of the inactive GPD- bound G-a and the GPy subunits to form the heterotrimer G protein.4

[0006] However, a significant proportion of GPCRs still lack an identified ligand and the function of a significant number of them remains poorly understood. This is the case for GPR50 (also known as H9 or ML1X), a GPCR belonging to the melatonin receptor family, but which has lost the ability to bind melatonin during evolution. GPR50 homodimerizes and forms heterodimers specifically with MT1 and MT2 melatonin receptors that share the highest sequence homology with GPR50 among all GPCRs. GPR50 has been introduced as a new target for the treatment of major CNS diseases, major depression, and bipolar disorder6'8. GPR50 has been shown to be an important regulator of energy metabolism in GPR50 knockout mice9. A sequence variant study suggested that GPR50 is related to mental disorders10and altered lipid metabolism11. Moreover, recent findings showed that the orphan GPR50 receptor promotes constitutive transforming growth factor-P (TGF-P) receptor signaling and protects against breast cancer development.12The TGF-P signaling pathway plays an important role in cancer development and progression, due to its growth inhibitory function.13TGF-P signaling is initiated by the type I, II TGF-P receptor (TpRI / TpRII) complex.14In the classical mode of action, binding of TGF-B to TBRII promotes the association and phosphorylation of TBRI by TBRII.15'16This induces the so-called “inhibitor to substrate” activator switch, in which the FKBP12 inhibitor (that keeps TpRI in an inactive conformation) dissociates from TBRI leading to the recruitment of Smad2 / 3 (small mothers against decapentaplegic homolog) proteins to TBRI.17Phosphorylation of Smad2 / 3 by the TpRI kinase triggers the formation of heteromeric complexes (mainly composed of two R-Smads2 / 3 and one co-Smad4), they translocate to the nucleus and regulate gene transcription upon DNA binding.18'19However, in the absence of TGF-P, an alternative complex between TpRI and the orphan GPR50 was formed that does not require TpRII. The interaction of GPR50 with TpRI induces spontaneous TpRI-dependent Smad and non-Smad signaling by stabilizing the active TpRI conformation and by competing for the binding of the negative regulator FKBP12.12An upregulation of GPR50 that goes along with a constitutive TGFP signaling activity provides a protective mechanism for a cell against cancer in its early stages. Moreover, GPR50 overexpression in MDA-MB-231 cells mimics the antiproliferative effect of TPRI and decreases tumor growth in a xenograft mouse model. Inversely, targeted deletion of GPR50 in the MMTV / Neu spontaneous mammary cancer model shows decreased survival after tumor onset and increased tumor growth. Low GPR50 expression is associated with poor survival prognosis in human breast cancer (independently of the breast cancer subtype).12

[0007] Thus, there is a need to identify new compound effective for treating disorders and diseases mediated by GPR50, such as neurological metabolic disorders and cancer. More particularly, a need remains to identify new compounds that can be used as agonist ligand to treat diseases mediated by GPR50. SUMMARY OF THE INVENTION:

[0008] The present invention relates to methods and pharmaceutical compositions for the treatment of diseases mediated by GPR50 comprising administering to the subject with a therapeutically effective amount of an GPR50 ligand of formula (I), wherein,

[0009] Ri represents NH or O, and

[0010] R2 represents CH3 or CH3-NH2.

[0011] In particular, the present invention is defined by the claims.

[0012] DETAILED DESCRIPTION OF THE INVENTION:

[0013] The inventors identified the first potent agonists of the orphan GPR50 receptor in a functional cAMP assay. Among these agonists, the 5 -methoxy -2 -methyl indole (Cpl5) displayed a high agonistic activity at the GPR50 receptor (EC50 = 0.5 nM). Intriguingly, replacement of the methyl group in the initially screened 5-methoxy -2 -methyl indole with a 2- methylamino group (5-methoxy-2 -methylamine indole) (Cp32) resulted also in high agonistic potency activity. In another contribution to define SARs, indole nucleus of Cpl5 was replaced by benzofuran (Cp34).

[0014] Accordingly, A first object of the present invention is a method for treating diseases mediated by GPR50 comprising administering to the subjects a therapeutically effective amount of an GPR50 ligand.

[0015] In other words, the invention relates to a GPR50 ligand for use for treating a disease mediated by GPR50 in a subject in need thereof.

[0016] As used herein, the term “G protein-coupled receptor” or “GPCR” refers to the largest family of cell membrane proteins with approximately 800 members identified from the human genome. GPCRs were shown to demonstrate signal interactions with G proteins in the plasma membrane. G protein-coupled receptors are found only in eukaryotes, including yeast, and choanoflagellates. Those G proteins are heterotrimeric proteins consisting of three different subunits (a, P and y subunits) with an ability to bind the nucleotides guanosine triphosphate (GTP) and guanosine diphosphate (GDP) depending on whether the protein is active or inactive respectively. There are two principal signal transduction pathways involving GPCRs: the cAMP signal pathway and the phosphatidylinositol signal pathway. An orphan GPCR is a GPCR whose endogenous ligand has not yet been identified. G proteins are classified into four families according to their a subunit; Gai / o, Gas, Gal2 / 13, and Gaq / 11. The G alpha-s subunit (Gs) activates adenylate cyclase, which produces cyclic-AMP (cAMP), leading to the activation of cAMP-dependent protein kinases. G alpha-i / o (Gi / o) proteins classically inhibit the cAMP- dependent pathway through inhibition of adenylate cyclases. The G alpha-z protein (Gz) also inhibits adenylate cyclases but is pertussis toxin insensitive unlike other Gi / o family members. Gz also interacts with the Rapl GTPase activating protein (RAP 1 GAP). G alpha-q / 11 (Gq / 11) activates phospholipase C beta, leading to phosphoinositide hydrolysis, calcium mobilization, and protein kinase C activation. The G-alpha-12 / 13 (Gal2 / 13) family is best known for its involvement in the processes of cell proliferation and morphology, such as stress fibers and focal adhesion formation through the activation of small G proteins such as Rho, Rac, Cdc42.

[0017] As used herein, the term “G protein-coupled receptor 50” or “GPR50” is an orphan GPCR. GPR50 is a melatonin-related receptor that is found exclusively in eutherian mammals and not in fish or birds. It is able to heterodimerize with both the MT1 and MT2 melatonin receptor subtypes. While GPR50 has no effect on MT2 function, GPR50 prevented MT1 from both binding melatonin and coupling to G proteins. GPR50 plays a role in numerous physiological processes including regulation of energy metabolism, neurite outgrowth or cell migration21. GPR50 has been introduced as a new target for the treatment of major CNS diseases, major depression, and bipolar disorder6'8. Moreover, recent findings showed that the orphan GPR50 receptor promotes constitutive transforming growth factor-P (TGF-P) receptor signaling and protects against cancer development.12

[0018] Its Gene ID is 9248. Its Unitprot reference is Q13585. An exemplary amino acid sequence for GPR50 is represented by SEQ ID NO: 1.

[0019] SEQ ID NO : 1 >sp | Q13585 | MTRL1_HUMAN GPR50 0S=Homo sapiens

[0020] MGPTLAVPTPYGCIGCKLPQPEYPPALI I FMFCAMVITIWDLIGNSMVILAVTKNKKLRNSGNI FWS LSVADMLVAIYPYPLMLHAMSIGGWDLSQLQCQMVGFITGLSWGSI FNIVAIAINRYCYICHSLQYERI FSVRN TCIYLVITWIMTVLAVLPNMYIGTIEYDPRTYTCI FNYLNNPVFTVTIVCIHFVLPLLIVGFCYVRIWTKVLAAR DPAGQNPDNQLAEVRNFLTMFVI FLLFAVCWCPINVLTVLVAVSPKEMAGKI PNWLYLAAYFIAYFNSCLNAVIY GLLNENFRREYWTI FHAMRHPI I FFSGLI SDIREMQEARTLARARAHARDQAREQDRAHACPAVEETPMNVRNVP LPGDAAAGHPDRASGHPKPHSRSSSAYRKSASTHHKSVFSHSKAASGHLKPVSGHSKPASGHPKSATVYPKPASV HFKADSVHFKGDSVHFKPDSVHFKPASSNPKPITGHHVSAGSHSKSAFSAATSHPKPTTGHIKPATSHAEPTTAD YPKPATTSHPKPTAADNPELSASHCPEI PAIAHPVSDDSDLPESASSPAAGPTKPAASQLESDTIADLPDPTWT TSTNDYHDVWIDVEDDPDEMAV

[0021] In some embodiments, regarding all the method of the invention, the GPR50 ligand is a GPR50 agonist ligand.

[0022] As used herein, the term “GPR50 agonist ligand” refers to a compound that modulates the activity of GPR50, especially that binds to and initiate / activate the GPR50 activity. Here, the inventors show that the ligand Cpl5 inhibits the cAMP production.

[0023] In some embodiments, the GPR50 agonist ligands inhibit cAMP production.

[0024] In preferred embodiment, the GPR50 agonist ligand directly binds to GPR50.

[0025] As used herein, the term “binding” or “bind” refers to a direct association between two molecules, due to, for example, covalent, electrostatic, hydrophobic, and ionic and / or hydrogenbond interactions, including interactions such as salt bridges and water bridges. In particular, as used herein, the term "binding" in the context of the binding of the compound to a predetermined target molecule typically is a binding with an affinity corresponding to a KD of about 10'5M or less, such as about 10'6M or less, such as about 10'7M or less such as about 10'8M or less, such as about 10'9M or less, about IO'10M or less, or about 10'11M or even less.

[0026] In particular embodiment, the GPR50 agonist ligand is able to inhibit forskolin-induced cAMP production.

[0027] In particular embodiment, the GPR50 agonist ligand is a selective GPR50 agonist.

[0028] In a particular embodiment, the GPR50 agonist ligand is a small organic molecule.

[0029] As used herein, the term “small organic molecule” refers to a molecule of a size comparable to those organic molecules generally used in pharmaceuticals. The term excludes biological macromolecules (e.g., proteins, nucleic acids, etc.). Preferred small organic molecules range in size up to about 5000 Da, more preferably up to 2000 Da, and most preferably up to about 1000 Da.

[0030] In a particular embodiment, the GPR50 agonist ligand is a compound of Formula I or a pharmaceutically acceptable salt thereof, wherein,

[0031] Ri represents NH or O, and

[0032] R2 represents CH3 or CH3-NH2.

[0033] In another words, the invention relates to is a compound of Formula I or a pharmaceutically acceptable salt thereof for use as GPR50 agonist ligand in the treatment of a subject in need thereof, wherein,

[0034] Ri represents NH or O, and

[0035] R2 represents CH3 or CH3-NH2.

[0036] In particular embodiment, the subject is affected with diseases mediated by GPR50.

[0037] Thus, in particular embodiment, the compound of Formula I or the pharmaceutically acceptable salt thereof of the invention for use as GPR50 agonist ligand in the treatment of disease mediated by GPR50 in a subject in need thereof.

[0038] In preferred embodiment, the compound of Formula I, is chosen from:

[0039] , said compound of Formula I having the structure of Formula II, Formula III or Formula IV. In preferred embodiment, the GPR50 agonist ligand is 5-Methoxy-2-methylindole having the formula (II). (“Cpl5”)

[0040] The compound of Formula II, “2-methyl-5-methoxyindole” also known as “5-Methoxy- 2-methylindole” (Cpl5), is a commercially available indole analog (Ri is -NH and R2 is CH3, in relation to Formula I). Its CAS Number is 1076-74-0.

[0041] In preferred embodiment, the GPR50 agonist ligand is 5-methoxyindolyl-2- methylamine having the formula (III). (“Cp32”)

[0042] The compound of Formula III, “5-methoxyindolyl-2-methylamine” also known as “PIM-35” (Cp32), is an indole derivative with a similar chemical structure to that of serotonin (Ri is -NH and R2 is CH3-NH2, in relation to Formula I). PIM-35 has activity on the serotonergic system as described in Carranza et al22

[0043] In preferred embodiment, the GPR50 agonist ligand is 5-Methoxy-2 -methylbenzofuran having the formula (IV). (“Cp34”)

[0044] The compound of Formula IV, “2-methyl-5-methoxybenzofuran” also known as “5- Methoxy-2 -methylbenzofuran” (Cp34), is a commercially available benzofuran analog of 2- methyl-5-methoxyindols (Ri is -O and R2 is CH3, in relation to Formula I). Its CAS Number is 13391-27-0.

[0045] As defined herein, the term “pharmaceutically acceptable salt” is intended to mean salts which are pharmaceutically acceptable, and which possess the desired pharmacological activity of the parent compound. Such salts are compounds in which the acid proton present in the parent compound is either replaced by a metal ion, for example, an alkali metal ion, an alkaline earth metal ion or an aluminum ion; or is coordinated with a pharmaceutically acceptable organic or inorganic base.

[0046] Acceptable organic bases include diethanolamine, ethanolamine, N-methylglucamine, triethanolamine, tromethamine and the like. Acceptable inorganic bases include aluminum hydroxide, calcium hydroxide, potassium hydroxide, sodium carbonate and sodium hydroxide.

[0047] As used herein, the term "subject" or "subject in need thereof" refers to a human or another mammal (e.g., primate, dog, cat, goat, horse, pig, mouse, rat, rabbit, and the like). In a particular embodiment of the present invention, the subject is a human being. The term "subject" does not denote a particular age, and thus encompasses children, teenagers, and adults. Typically, the patient is afflicted or likely to be afflicted with diseases mediated by GPR50. As used herein, the term "treatment" or "treat" refer to both prophylactic or preventive treatment as well as curative or disease modifying treatment, including treatment of patient at risk of contracting the disease or suspected to have contracted the disease as well as patients who are ill or have been diagnosed as suffering from a disease or medical condition, and includes suppression of clinical relapse. The treatment may be administered to a patient having a medical disorder or who ultimately may acquire the disorder, in order to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of a disorder or recurring disorder, or in order to prolong the survival of a patient beyond that expected in the absence of such treatment. By "therapeutic regimen" is meant the pattern of treatment of an illness, e.g., the pattern of dosing used during therapy. A therapeutic regimen may include an induction regimen and a maintenance regimen. The phrase "induction regimen" or "induction period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the initial treatment of a disease. The general goal of an induction regimen is to provide a high level of drug to a patient during the initial period of a treatment regimen. An induction regimen may employ (in part or in whole) a "loading regimen", which may include administering a greater dose of the drug than a physician would employ during a maintenance regimen, administering a drug more frequently than a physician would administer the drug during a maintenance regimen, or both. The phrase "maintenance regimen" or "maintenance period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the maintenance of a patient during treatment of an illness, e.g., to keep the patient in remission for long periods of time (months or years). A maintenance regimen may employ continuous therapy (e.g., administering a drug at a regular interval, e.g., weekly, monthly, yearly, etc.) or intermittent therapy (e.g., interrupted treatment, intermittent treatment, treatment at relapse, or treatment upon achievement of a particular predetermined criteria [e.g., pain, disease manifestation, etc.]).

[0048] As used herein the term "diseases mediated by GPR50" means any disorder and disease that can be treated and / or prevented by modulating / activating the GPR50 receptors.

[0049] The role of GPR50 in the pathophysiology of various diseases has been described.

[0050] GPR50 is highly expressed in the hypothalamus, pituitary gland, and locus coeruleus, which play critical roles in the regulation of stress, body weight and glucose homeostasis regulation and anxiety-related disorders. GPR50 has been introduced as a new target for the treatment of major CNS diseases, major depression, and bipolar disorder as described in Alavi MS et al6.; Thomson PA et al7.; and MacIntyre DJ et al8. Thus, in some embodiments, the method of the present invention is particularly suitable for treating of central Nervous System (CNS) disorders and / or cognitive and metabolic disorders.

[0051] As used herein the “Cognitive disorders, diseases or dysfunction” include, but are not limited to, Alzheimer's Disease, memory loss, age-dependent memory loss, post-traumatic stress disorder (PTSD), a neuropathy and seizures. The cognitive dysfunction may be stress- related, age-related or a combination thereof. Alternatively, the cognitive dysfunction is associated with a disease or disorder, including but not limited to, Alzheimer's disease (AD), attention deficit hyperactivity disorder (ADHD), autism spectrum disorder (ASD), generalized anxiety disorder (GAD), obsessive compulsive disorder (OCD), Parkinson's Disease (PD), post-traumatic stress disorder (PTSD), Huntington's disease, Rhett Syndrome, Multiple sclerosis (MS), Amyotrophic lateral sclerosis (ALS or motor neuron disease), Schizophrenia, Bipolar disorder; and major depression.

[0052] In some embodiments, the cognitive disorders are multiple sclerosis (MS), bipolar disorder, major depression, Parkinson and Alzheimer's disease.

[0053] In some embodiments, the method of the present invention is particularly suitable for treating of anxiety or stress-related disorders.

[0054] As used herein the term “Anxiety” refers to a common psychiatric disorder. Often, anxiety disorders involve repeated episodes of sudden feelings of intense anxiety and fear or terror that reach a peak within minutes (panic attacks). Generalized anxiety disorder, social anxiety disorder, panic disorder, and agoraphobia are the most common types of anxiety.

[0055] Recent study shows also that GPR50 is an important regulator of energy metabolism26that can attenuate inflammation and regulate insulin resistance through the regulation of the insulin signaling pathway, and is introduced as new target for the treatment of Type 2 diabetes (T2D), as disclosed in Yao Z et al23.

[0056] In some embodiments, the method of the present invention is particularly suitable for treating of insulin resistance disorders or metabolic disorders.

[0057] As used herein the term “metabolic disorders” refers to conditions that affect any aspect of metabolism. Examples of metabolic disorders include gaucher’s disease, hemochromatosis, diabetes, obesity.

[0058] As used herein the term “Insulin resistance” refers to an impaired biologic response to insulin stimulation of target tissues, primarily involves liver, muscle, and adipose tissue. Insulin resistance impairs glucose disposal, resulting in a compensatory increase in beta-cell insulin production and hyperinsulinemia. The metabolic consequences of insulin resistance can result in hyperglycemia, hypertension, dyslipidemia, hyperuricemia, elevated inflammatory markers, endothelial dysfunction, and a prothrombotic state. The predominant consequence of insulin resistance is type 2 diabetes (T2D).

[0059] In some embodiment, the insulin resistance disorders are hyperglycemia, hypertension, dyslipidemia, hyperuricemia, or T2D.

[0060] Moreover, recent findings showed that the orphan GPR50 receptor promotes constitutive transforming growth factor-P (TGF-P) receptor signaling and protects against cancer development as described in Wojciech S, et al12. This study shows that GPR50 overexpression in MDA-MB-231 cells mimics the antiproliferative effect of T RI and decreases tumor growth in a xenograft mouse model. Inversely, targeted deletion of GPR50 in the MMTV / Neu spontaneous mammary cancer model shows decreased survival after tumor onset and increased tumor growth. Low GPR50 expression is associated with poor survival prognosis in human breast cancer (independently of the breast cancer subtype). Another study shows the impact of GPR50 in hepatocellular carcinoma (HCC)24. GPR50 was found to directly interact with ADAMI 7 regulating the activation of Notch signaling. GPR50 was also found to regulate ADAMI 7 transcription and translation via the AKT / SP1 axis24

[0061] In particular embodiment, the GPR50 agonist ligand is able to activate GPR50 to form a complex with TpRI. Thus, in particular embodiment, the GPR50 agonist ligand is able to promotes TGF-P receptor signaling. In particular embodiment, the GPR50 agonist ligand is able to decrease tumor growth.

[0062] In some embodiments, the method of the present invention is particularly suitable for treating of cancer.

[0063] As used herein, the terms "cancer" and "tumors" refer to or describe the pathological condition in mammals that is typically characterized by unregulated cell growth. More precisely, in the use of the invention, diseases, namely cancer that expresses / secretes TGF-P are most likely to respond to the GPR50 agonist ligand of the invention. More precisely, in the use of the invention, diseases, namely cancer that expresses / secretes GPR50 are most likely to respond to the GPR50 agonist ligand of the invention.

[0064] The cancer that may be treated by methods and compositions of the invention include, but are not limited to cancer cells from the bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, gastrointestinal, gum, head, kidney, liver, lung, nasopharynx, neck, ovary, prostate, pancreas, skin, stomach, testis, tongue, or uterus. In addition, the cancer may specifically be of the following histological type, though it is not limited to these: neoplasm, malignant; carcinoma; carcinoma, undifferentiated; giant and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatrix carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyp; adenocarcinoma, familial polyposis coli; solid carcinoma; carcinoid tumor, malignant; branchioloalveolar adenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma; acidophil carcinoma; oxyphilic adenocarcinoma; basophil carcinoma; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; papillary and follicular adenocarcinoma; nonencapsulating sclerosing carcinoma; adrenal cortical carcinoma; endometroid carcinoma; skin appendage carcinoma; apocrine adenocarcinoma; sebaceous adenocarcinoma; ceruminous; adenocarcinoma; mucoepidermoid carcinoma; cystadenocarcinoma; papillary cystadenocarcinoma; papillary serous cystadenocarcinoma; mucinous cystadenocarcinoma; mucinous adenocarcinoma; signet ring cell carcinoma; infiltrating duct carcinoma; medullary carcinoma; lobular carcinoma; inflammatory carcinoma; paget's disease, mammary; acinar cell carcinoma; adenosquamous carcinoma; adenocarcinoma w / squamous metaplasia; thymoma, malignant; ovarian stromal tumor, malignant; thecoma, malignant; granulosa cell tumor, malignant; and roblastoma, malignant; Sertoli cell carcinoma; leydig cell tumor, malignant; lipid cell tumor, malignant; paraganglioma, malignant; extra-mammary paraganglioma, malignant; pheochromocytoma; glomangiosarcoma; malignant melanoma; amelanotic melanoma; superficial spreading melanoma; malign melanoma in giant pigmented nevus; epithelioid cell melanoma; blue nevus, malignant; sarcoma; fibrosarcoma; fibrous histiocytoma, malignant; myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma; embryonal rhabdomyosarcoma; alveolar rhabdomyosarcoma; stromal sarcoma; mixed tumor, malignant; mullerian mixed tumor; nephroblastoma; hepatoblastoma; carcinosarcoma; mesenchymoma, malignant; brenner tumor, malignant; phyllodes tumor, malignant; synovial sarcoma; mesothelioma, malignant; dysgerminoma; embryonal carcinoma; teratoma, malignant; struma ovarii, malignant; choriocarcinoma; mesonephroma, malignant; hemangiosarcoma; hemangioendothelioma, malignant; kaposi's sarcoma; hemangiopericytoma, malignant; lymphangiosarcoma; osteosarcoma; juxtacortical osteosarcoma; chondrosarcoma; chondroblastoma, malignant; mesenchymal chondrosarcoma; giant cell tumor of bone; ewing's sarcoma; odontogenic tumor, malignant; ameloblastic odontosarcoma; ameloblastoma, malignant; ameloblastic fibrosarcoma; pinealoma, malignant; chordoma; glioma, malignant; ependymoma; astrocytoma; protoplasmic astrocytoma; fibrillary astrocytoma; astroblastoma; glioblastoma; oligodendroglioma; oligodendroblastoma; primitive neuroectodermal; cerebellar sarcoma; ganglioneuroblastoma; neuroblastoma; retinoblastoma; olfactory neurogenic tumor; meningioma, malignant; neurofibrosarcoma; neurilemmoma, malignant; granular cell tumor, malignant; malignant lymphoma; Hodgkin's disease; Hodgkin's lymphoma; paragranuloma; malignant lymphoma, small lymphocytic; malignant lymphoma, large cell, diffuse; malignant lymphoma, follicular; mycosis fungoides; other specified non-Hodgkin's lymphomas; malignant histiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferative small intestinal disease; leukemia; lymphoid leukemia; plasma cell leukemia; erythroleukemia; lymphosarcoma cell leukemia; myeloid leukemia; basophilic leukemia; eosinophilic leukemia; monocytic leukemia; mast cell leukemia; megakaryoblastic leukemia; myeloid sarcoma; and hairy cell leukemia.

[0065] In a particular embodiment, the cancer is a solid cancer.

[0066] In a particular embodiment, the cancer is a cancer that expresses / secretes GPR50.

[0067] In a particular embodiment, the cancer is a cancer that expresses / secretes TGFR1 and / or TGF-p.

[0068] In a particular embodiment, the cancer is in early stage.

[0069] In a particular embodiment, the cancer is selected from the group consisting of head and neck cancer, renal cancer, liver cancer (such as hepatocellular carcinoma), breast cancer, bladder cancer or lung cancer.

[0070] As used herein, a “therapeutically effective amount” is intended for a minimal amount of active agent (ie, the GPR50 agonist ligand of the invention) which is necessary to impart therapeutic benefit to a patient. For example, a “therapeutically effective amount of the active agent” to a patient is an amount of the active agent that induces, ameliorates or causes an improvement in the pathological symptoms, disease progression, or physical conditions associated with the disease affecting the patient. It will be understood that the total daily usage of the compounds and compositions of the present invention will be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular patient will depend upon a variety of factors including the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific polypeptide employed; and like factors well known in the medical arts. For example, it is well known within the skill of the art to start doses of the compound at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. However, the daily dosage of the products may be varied over a wide range from 0.01 to 1,000 mg per adult per day. Preferably, the compositions contain 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100, 250 and 500 mg of the active ingredient for the symptomatic adjustment of the dosage to the patient to be treated. A medicament typically contains from about 0.01 mg to about 500 mg of the active ingredient, preferably from 1 mg to about 100 mg of the active ingredient. An effective amount of the drug is ordinarily supplied at a dosage level from 0.0002 mg / kg to about 100 mg / kg of body weight per day.

[0071] As used herein the terms “administering” or “administration” refer to the act of injecting or otherwise physically delivering a substance as it exists outside the body (e.g., the agonist ligand of the invention) into the subject, such as by oral, mucosal, intradermal, intravenous, subcutaneous, intramuscular delivery and / or any other method of physical delivery described herein or known in the art. When a disease, or a symptom thereof, is being treated, administration of the substance typically occurs after the onset of the disease or symptoms thereof. When a disease or symptoms thereof, are being prevented, administration of the substance typically occurs before the onset of the disease or symptoms thereof. In particular embodiment, the agonist ligand of the invention is intravenously administered.

[0072] In particular embodiment, the GPR50 agonist of the invention is typically combined with pharmaceutically acceptable excipients, and optionally sustained-release matrices, such as biodegradable polymers, to be administered in the form of a pharmaceutical composition.

[0073] As used herein, the term "Pharmaceutically" or "pharmaceutically acceptable" refer to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to a mammal, especially a human, as appropriate. A pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. In the pharmaceutical compositions of the present invention for oral, sublingual, subcutaneous, intramuscular, intravenous, transdermal, local or rectal administration, the active principle, alone or in combination with another active principle, can be administered in a unit administration form, as a mixture with conventional pharmaceutical supports, to animals and human beings. Suitable unit administration forms comprise oral-route forms such as tablets, gel capsules, powders, granules and oral suspensions or solutions, sublingual and buccal administration forms, aerosols, implants, subcutaneous, transdermal, topical, intraperitoneal, intramuscular, intravenous, subdermal, transdermal, intrathecal and intranasal administration forms and rectal administration forms.

[0074] Thus, a second object of the present invention relates to a pharmaceutical composition comprising a GPR50 agonist ligand as previously defined, or a pharmaceutically acceptable salt thereof.

[0075] In particular embodiment, the present invention relates to a pharmaceutical composition comprising a GPR50 agonist ligand as previously defined for use in therapy.

[0076] In particular embodiment, the present invention relates to a pharmaceutical composition comprising a GPR50 agonist ligand as previously defined, or a pharmaceutically acceptable salt thereof for use for treating disorder mediated with GPR50.

[0077] As used herein, the term “pharmaceutical composition” refers to a composition described herein, or pharmaceutically acceptable salts thereof, with other agents such as carriers and / or excipients. The pharmaceutical compositions as provided herewith typically include a pharmaceutically acceptable carrier.

[0078] Typically, the pharmaceutical compositions contain vehicles which are pharmaceutically acceptable for a formulation capable of being injected. These may be in particular isotonic, sterile, saline solutions (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride and the like or mixtures of such salts), or dry, especially freeze-dried compositions which upon addition, depending on the case, of sterilized water or physiological saline, permit the constitution of injectable solutions. The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be fluid to the extent that easy syringeability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. Solutions comprising compounds of the invention as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. The GPR50 agonist ligand of the invention can be formulated into a composition in a neutral or salt form. Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like. The carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetables oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminium monostearate and gelatin. Sterile injectable solutions are prepared by incorporating the active agonist ligand in the required amount in the appropriate solvent with several of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuumdrying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above, but drug release capsules and the like can also be employed. For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this connection, sterile aqueous media which can be employed will be known to those of skill in the art in light of the present disclosure. For example, one dosage could be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion. Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject.

[0079] A third object of the present invention relates to a method of screening an GPR50 agonist ligand comprising: i) providing a test compound, ii) determining the ability of said test compound to modulate GPR50 activity

[0080] In some embodiments, regarding all the method of screening, the methods comprise first determining the ability of the test compound to bind to GPR50.

[0081] Any biological assay well known in the art could be suitable for determining the ability of the test compound to bind GPR50 defined above.

[0082] For example, MicroScale Thermophoresis (MST) can be used for quantifying the strength of test compounds to bind to GPR50.

[0083] MST is a fluorescence-based biophysical technique that builds upon the principle that the chemical environment around a fluorophore bound to a target molecule changes when the target molecule interacts with a ligand, causing a variation in the intensity of the fluorescence. During MST measurements a brief and precise laser-induced temperature increase is applied to amplify the change in fluorescence intensity which is related to the amount of compound bound.

[0084] In some embodiments, regarding all the method of screening, the test compound is based structurally on a compound having formula (I).

[0085] In some embodiments, regarding all the method of screening, the methods comprise also determining the ability of the test compound to compete with the compound having formula (I ) of the invention for binding to GPR50. In some embodiments, the methods comprise also determining the ability of the test compound to compete with the compound having formula (II) (Cpl5) of the invention for binding to GPR50.

[0086] In other words, the methods comprise also determining the ability of the test compound to bind to the same or overlapping GPR50-binding site of the compound having formula (II) (Cpl5). Any biological assay well known in the art could be suitable for determining the ability of the test compound to compete with the compound having formula (II) (Cpl5) to bind GPR50 defined above.

[0087] Thus, in particular embodiment, the present invention relates to a method of screening an GPR50 agonist ligand comprising i) providing a test compound, ii) determining the ability of said test compound to bind to the same or overlapping GPR50-binding site of the compound having formula (I) and iii) determining the ability of said test compound to modulate GPR50 activity.

[0088] As used herein, the term “GPR50 activity” refers to various signaling pathways induced by GPR50. GPR50, as other GPCRs, induce the cAMP signal pathway. The inventors previously found that interaction of GPR50 with TpRI induces spontaneous TpRI-dependent Smad and non-Smad signalling by stabilizing the active TpRI conformation and by competing for the binding of the negative regulator FKBP12. GPR50 increased Smad2 and Smad3 phosphorylation. They also found that GPR50 promotes non-canonical TGFP signaling pathways such as MAPKp38 and AKT and to a lesser extent ERK1 / 2. GPR50 was found to directly interact with ADAMI 7 regulating the activation of Notch signaling. GPR50 was also found to regulate ADAMI 7 activity and translation via the AKT / SP1 axis24. Recent study shows also that GPR50 regulates the IRS1 / AKT signaling pathway and modulate PPAR-y expression23. GPR50 also regulated the NF-KB-Notch signaling pathway27

[0089] As used herein the term “compound able to modulate GPR50 activity” or “compound able to modulate signalling pathways” refers to a compound able to increase (i.e to screen an agonist ligand) or to decrease (i.e to screen an antagonist ligand) GPR50 activity.

[0090] Thus, in particular embodiment, the ability of said test compound to modulate GPR50 activity is the ability to inhibit cAMP production.

[0091] Any biological assay well known in the art could be suitable for determining the ability of the test compound to inhibit cAMP production. cAMP productions assays are well known in the art as described in Wang T, Li Z, Cvijic ME, et al. Measurement of cAMP for Gas- and Gai Protein-Coupled Receptors (GPCRs) 2017 Nov 20. In: Markossian S, Grossman A, Brimacombe K, et al. Assay Guidance Manual. Forskolin, a labdane diterpene, increases intracellular cAMP level through activation of adenylyl cyclase in various cell types. In particular embodiment, it is determined in sept ii) the ability of the test compound to inhibit cAMP production induced by forskolin.

[0092] In some embodiments, the assay also comprises determining that the ability of the test compound to inhibit cAMP production is not mediated by Gi / o proteins. In some embodiments, the assay also comprises determining that the ability of the test compound to inhibit cAMP production is mediated by Pertussin toxin-insensitive proteins.

[0093] In some embodiments, the assay also comprises determining the ability of the test compound to inhibit cAMP production in presence of Pertussin toxin (PTX).

[0094] In some embodiments, a population of cells is then selected and activated so as to determine the ability of the test compound to inhibit cAMP production

[0095] Thus, in particular embodiment, the ability of said test compound to modulate GPR50 activity is the ability to modulate TpRI-signalling pathways.

[0096] Any biological assay well known in the art could be suitable for determining the ability of the test compound to modulate TpRI-signalling pathways, as described in Wojchiech et al12.

[0097] Thus, In particular embodiment, it is determined that the test compound modulate Smad2 and Smad3 phosphorylation. In particular embodiment, it is determined that the test compound modulate MAPKp38, AKT and / or ERK1 / 2.

[0098] Thus, in particular embodiment, the ability of said test compound to modulate GPR50 activity is the ability to modulate NF-kB -signalling pathways.

[0099] Any biological assay well known in the art could be suitable for determining the ability of the test compound to modulate NF-kB -signalling pathways, as described in Biswas et al27.

[0100] Thus, in particular embodiment, the ability of said test compound to modulate GPR50 activity is the ability to modulate ADAMI 7 activity.

[0101] ADAMI 7 is a proteolytic enzyme that cleaves the Notch receptor. Thus, in particular embodiment, it is determined that the test compound modulate Notch signalling pathways.

[0102] Any biological assay well known in the art could be suitable for determining the ability of the test compound to modulate Notch signalling pathways, as described in Biswas et al24.

[0103] In some embodiments, a population of cells is then selected and activated so as to determine the ability of the test compound to modulate GPR50 activity. In particular, the effect triggered by the test compound is determined relative to that of a population of cells incubated in parallel in the absence of the test compound or in the presence of a control agent either of which is analogous to a negative control condition. The term "control substance", "control agent", or "control compound" as used herein refers to a molecule that is inert or has no activity relating to an ability to modulate a biological activity or expression

[0104] It is to be understood that test compounds capable to modulate GPR50 activity, as determined using in vitro methods described herein, are likely to exhibit similar modulatory capacity in applications in vivo. Typically, the test compound is small organic molecules. For example, the test compound according to the invention may be selected from a library of compounds previously synthesised, or a library of compounds for which the structure is determined in a database, or from a library of compounds that have been synthesised de novo. In some embodiments, the test compound may be selected form small organic molecules.

[0105] The invention will be further illustrated by the following figures and examples. However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention.

[0106] FIGURES:

[0107] Figure 1: Structures of indole analogs screened for activity on cells transfected with GPR50 receptors (HEK293T cells).

[0108] Figure 2 : Inhibition of forskolin-induced cAMP production by A. compounds 15 and B. 29 in mock-transfected HEK293 cells and HEK293 cell expressing GPR50WT. EC50 values are indicated for compound 15.

[0109] Figure 3: Synthetic approach towards GPR50 target ligands. Reagents and reaction conditions: i) Cone. H2SO4, CH3OH, reflux Ihr; 11) L1AIH4, THF; 111) NH4Q, DMF, EDCI.HC1, DIPEA, HOBt; iv) CsCOi, CH3I, acetonitrile; v) LiAlH4, THF, reflux 2 hrs

[0110] Figure 4: Inhibition of forskolin-induced cAMP production by compounds 15 in mock- transfected HEK293 cells and HEK293 cell expressing GPR50WT that were pretreated or not over night with pertussis toxin (PTX) (lOOng / mL). Mean curves of 4-5 biologically independent experiments are shown.

[0111] Figure 5: Binding Check assay of Compound 15 and Compound 32 by Microscale Thermophoresis.

[0112] Figure 6: Binding affinity of Cpdl5, 32 and 34 measured by microscale thermophoresis. Lysats of CHO cells stably expressing GPR50-Dcter-8His labeled with Red- tris-NTA were incubated with concentration response of Cpdl5 (A), Cpd32 (B) or Cpd34 (C). Figure 7: Proliferation of breast cancer cells expressing GPR50: 4T1 murine mammary carcinoma cells expressing GPR50 were stimulated with compound 15 (lOOnM) (triangle) or not (circle) every 24 hours. Cell proliferation was monitored using an Incucyte (Sartorius) by measuring the confluence of the cells in each well. 3 wells per condition were analyzed with 4 images captured per well (n=l).

[0113] Figure 8: Lipolysis in Ex Vivo Adipose Tissue: Inguinal adipose tissue from humanized knock-in mice expressing human GPR50 was treated with isoproterenol (ISO), cpl5, or a combination of both at the indicated concentrations for 90 minutes at 37°C. Lipolysis, measured as glycerol release into the medium, is expressed as fold change (FC) relative to untreated condi ti on (n= 1 ) .

[0114] Table 1 : Derivatives of Cpdl5 in cAMP inhibition assay in HEK293 cells expressing

[0115] GPR50

[0116] Table 2: Binding Check assay of compounds on GPR50-DCter-YFP. EXAMPLE

[0117] Material & Methods

[0118] Design approach towards Ligands for the Orphan GPR50 Receptor

[0119] Synthesis of the potential GPR50 ligands :

[0120] Different derivatives of simple 2-substituted 5-methoxyindole were prepared starting from the commercially available 5-methoxyindole-2-carboxylic acid (Cp28) as following:

[0121] 1. An esterification reaction was carried out using a standard procedure, wherein the acid Cp28 dissolved in methanol was treated with catalytic amount of cone. H2SO4 that act as desiccant as well, were heated under reflux for 1 hr to give ester Cp29 in 87 % yield after being purified using silica gel chromatography.

[0122] 2. A second 2-substituted 5-methoxyindole derivative was prepared by subjecting Cp28 to a reduction using excess of LiAlH4 in THF. Reaction workup was afforded by quenching the reaction with slow addition of 80 % aqueous EtOAc solution to give Cp31 in 94 % yield after being purified by column chromatography using an eluent mixture of ethyl acetate and cyclohexane (1.25 : 1). Of note, either quenching the reaction during the workup with 80% aqueous MeOH or performing a column chromatography with an eluent mixture containing methanol, leads to the product Cp31 decomposition. Afterward, an excess of CsCO3 was used to deprotonate Cp31 that subsequently can be reacted with methyl iodide to substitute the iodine atom in a nucleophilic displacement reaction. Thereafter, Cp48 was obtained in 76 % yield after purifications by silica gel chromatography.

[0123] 3. To achieve a third 2-substituted 5-methoxyindole derivative, the amidation of 5- methoxyindole 2-carboxylic acid (Cp28) was carried out in dry DMF using EDCI.HC1, HOBt, and NH4C1 to yield Cp30 in 87 % after purification using silica gel chromatography. It is noteworthy that the replacement of EDCI.HC1 with another coupling reagents, such as PyBOP or HBTU resulted in an extremely low yielded reaction (-15-20 % yield). Compound 30 was subjected to a reduction using LiAlH4 in THF to give Cp32. Reaction workup was afforded by quenching the reaction with slow addition of 80 % aqueous MeOH solution to give 50 in 91 % yield after purifications by column chromatography (CHC13:CH3OH:NH3; 10:2:0.2).

[0124] 4. In an attempt to extend the series of 2-substituted 5-methoxyindole derivatives, Cp32 was subjected to a acylation and a cyclization reaction. Cp32 was subsequently acylated using acetic anhydride and tri ethylamine in THF to give Cp33 in 43 % yield after purifications using silica gel chromatography.

[0125] Synthesis of the potential GPR50 ligands (Cp28-33) is shown in Figure 3. cAMP functional assay.

[0126] The obtained monomeric analogs were tested for agonistic activity at GPR50 receptor using cAMP functional assay. The cAMP assay was performed as previously described.20Briefly, HEK293 cells expressing GPR50 wild type were stimulated with 2 pM forskolin in the presence of increasing concentrations of monomeric analogs.

[0127] Microscale thermophoresis (MST).

[0128] MST was conducted using a NT.115 Pico MST instrument (Nano Temper Technologies GmbH) equipped with red and blue filter sets. The lysate containing the GPR50 DCter-YFP or or GPR50 DCter-8xHis labeled with RED-tris-NTA (NanoTemper) fusion proteins, was diluted in PBS-T buffer (supplied by vendor).

[0129] Ligands (50 pM) were diluted in TEM buffer (75 mM Tris-HCl, 5 mM EDTA, 12.5 mM MgC12, pH 7.4.) with a serial 1 : 1 ratio of 16 gradients. Then the labeled protein and ligands were mix with 1 : 1 ratio and incubated at room temperature in the dark for 10 min. Capillaries are then filled individually and loaded into instrument. Data were acquired using high MST power and 80 % LED for GPR50 DCter-YFP protein. Data were analyzed using MO Control Software (Nano Temper). MST figures were rendered using MO Affinity Analysis (Nano Temper) for for Binding Check assay, and using Prism GraphPad for concentration-response experiments.

[0130] Prolifereation Assay:

[0131] Cell proliferation was measured by monitoring cell confluence over time using an Incucyte (Stratorius). 4T1 mammary carcinoma cell line expressing GPR50 were stimulated every 24 hours with Cpl5 (lOOnM) or complete medium. Confluence was monitored every 2 hours during 72 hours. Data were analyzed using GraphPad Prism 10.

[0132] Lipolysis assay:

[0133] Inguinal adipose tissue was dissected from humanized knock-in mice expressing human GPR50 under the control of the mouse GPR50 promoter. 50mg of adipose tissue pieces cultured in HBSS-HEPES-BSA2%, were untreated or treated with isoproterenol (lOOnM, luM, lOuM), or cpl5 (lOuM), or with a combination of lOOnM ISO + cpl5 lOuM, for 90 minutes at 37°C. Glycerol released into the medium, reflecting lipolysis, was measured with Glycerol-Glo (Promega) (n=l).

[0134] Results

[0135] No GPR50 ligands have been reported until now. Initial screening of simple commercially available indole analogs (Cpl5, Cp23-27, Cp34, Cp35) was performed (Fig. 1). Only two compounds, namely 5-methoxy -2 -methyl indole (Cpl5) and the corresponding benzofuran analog (Cp34) displayed an agonistic activity at GPR50 receptor (i.e inhibiting AMPc production) in HEK293T cells transiently expressing GPR50 (Fig. 2, Table 1). In particular, the 5 -methoxy -2 -methyl indole (Cpl5) displayed a high agonistic activity at GPR50 receptor (EC 50 = 0.5 nM) (Fig. 2).

[0136] In Cpl5 and Cp34, the indole or the bioisosteric furane ring bears a methyl group in position 2. In order to probe the 2-methyl binding region of the GPR50 receptors, a series of simple 2-substituted 5-methoxyindole analogs were prepared and screened in the functional cAMP assay. The structure modifications include acidic (Cp28), basic (Cp32), and non- ionizable groups at physiological pH (Cp29, Cp48 and Cp30).

[0137] The obtained monomeric analogs were tested for agonistic activity at GPR50 receptor using cAMP functional assay. Among the tested compounds 29, 28, 30, 31, 32, 33, only compound 32 (replacement of the methyl group in the initially screened 5 -methoxy -2 -methyl indole with a 2-methylamino group (5-methoxy-2-methylamine indole)) demonstrated liganddependent activation (agonistic activity) of the GPR50 receptor which resulted in cAMP inhibition in cells expressing GPR50 receptors (HEK293T cells) (Table 1). Compounds 28, 29, 30, 31, and 33 showed no differences in cAMP inhibition between both of HEK293T cells and Mock cells (without GPR50) as exemplified by the dose-response curves for compound 29 (Fig.2, Table 1)

[0138] To determine whether the inhibition of cAMP production by compound 15 is mediated by Gi / o proteins we pretreated mock cells or cells expressing GPR50WTwith pertussis toxin (PTX), an inhibitor of Gi / o proteins. The pretreatment was without any effect suggesting that different from MT1 and MT2 receptors the effect of GPR50WT is mediated by PTX-insensitive proteins such as the Gz protein (Fig. 4)

[0139] For a Binding Check assay, the temperature-related intensity change (TRIC) signal of the target GPR50 deleted of its carboxyl terminal domain (ACter) + / - Gz protein overexpression in the absence and presence of compound 15 are compared to asses binding in a qualitative manner. Under these conditions, compound 15 (10 pM) interacts with the GPR50ACter protein resulting in a fluorescence amplitude difference of 6.2 (AFnorm) + / - compound 15 (Fig. 5 A, Table 2). This interaction was strengthened by the presence of Gz, since the response amplitude increased from 6.2 to 116.6, with a 30-fold increase in the signal -to-noise ratio (Fig. 5B, Table 2). These results indicate a strong affinity of compound 15 for GPR50ACter in the presence or absence of Gz. Melatonin (10 pM) did not have any notable effect (amplitude lower than 4) in this assay indicating that the binding site is different from melatonin binding site of MT1 and MT2 receptors (Fig. 5C-D, Table 2).

[0140] Compound 32 (10 pM) interacts with the GPR50ACter protein resulting in a fluorescence amplitude difference of 4.8 (AFnorm) + / - compound 15 (Fig. 5E; Table 2) and 5 4 (Fig. 5F; Table 2)

[0141] Using GPR50-DCter-8xHis construct labeled with RED-tris-NTA, more suitable to MST technology setup, binding affinities were determined with increasing concentrations of Compounds 15 (Kd=174 nM), 32 (Kd=70 nM), 34 (Kd=200 nM) (Figure 6A-C).

[0142] The functional impact of compound 15 was evaluated ex vivo or in cells in two distinct disease models: cancer and metabolic diseases.

[0143] Considering the protective role of GPR50 expression in breast cancer development and progression, the effect of Cpl5 on cancer cells proliferation was measured. Stimulation of mammary carcinoma cell line expressing GPR50 with Cpl5 reduced their proliferation rate (Figure 7).

[0144] In obesity, unregulated lipolysis may contribute to the development of insulin resistance. The excessive accumulation of free fatty acids (FFAs) resulting from unregulated lipolysis leads to lipotoxicity in non-adipose tissues. Conversely, inhibiting lipolysis within adipose cells can help mitigate insulin resistance. GPR50 is expressed in the adipose tissue. Interestingly, cpl5 exhibits anti -lipolytic properties potentially mitigating excessive lipolysis (Figure 8).

[0145] REFERENCES:

[0146] Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure.

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Claims

CLAIMS:

1. A method for treating diseases mediated by GPR50 comprising administering to the subject with a therapeutically effective amount of an GPR50 agonist ligand, wherein the GPR50 agonist ligand is a compound of Formula I or a pharmaceutically acceptable salt thereof,wherein,Ri represents NH or O, andR2 represents CH3 or CH3-NH2.

2. The method according to claim 1, wherein the GPR50 agonist ligand directly bind to GPR50.

3. The method according to claim 1 or 2, wherein the compound of Formula I, is chosen from:, said compound of Formula I having the structure of Formula II, Formula III or Formula IV.

4. The method according to claim 1 to 3, wherein the tGPR50 agonist ligand is 5- Methoxy-2-methylindole having the formula (II).

5. The method according to claim 1 to 4, wherein the diseases mediated by GPR50 is central Nervous System (CNS) disorders and / or cognitive disorders, anxiety or stress-related disorders, metabolic disorders, insulin resistance disorders or cancer.

6. The method according to claim 5, wherein the cognitive disorders is multiple sclerosis (MS), bipolar disorder, major depression, Parkinson and Alzheimer’s disease.

7. The method according to claim 5, wherein the cancer is breast cancer.

8. A pharmaceutical composition comprising a GPR50 agonist ligand, or a pharmaceutically acceptable salt thereof, , wherein the GPR50 agonist ligand is a compound of Formula I or a pharmaceutically acceptable salt thereof,wherein,Ri represents NH or O, andR2 represents CH3 or CH3-NH2.

9. The pharmaceutical composition according to claim 8, wherein the compound of Formula I, is chosen from:, said compound of Formula I having the structure of Formula II, Formula III or Formula IV.

10. The pharmaceutical composition according to claim 8 or 9 for use in therapy.

11. The pharmaceutical composition according to claim 8 or 9 for use for treating disorder mediated with GPR5012. A method of screening an GPR50 ligand comprising i) providing a test compound, ii) determining the ability of said test compound to modulate GPR50 activity.

13. The method of screening according to claim 12, wherein the methods comprises first determining the ability of the test compound to bind to GPR50.

14. The method of screening according to claim 13, wherein the methods comprises determining the ability of the test compound to bind to the same or overlapping GPR50-binding site of the compound having formula (II).

15. The method of screening according to claim 12 to 13, wherein the ability of said test compound to modulate GPR50 activity is selected among : the ability to inhibit cAMP production, the ability to modulate TpRI-signalling pathways, the ability to modulate NF-kB - signalling pathways, and / or the ability to modulate ADAM17 activity.

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