Use of bile acids and their derivatives in the preparation of GPR39 agonists

Abstract

Use of the bile acids and their derivatives represented by formula (I) in the preparation of GPR39 agonists, and pharmaceutical compositions containing these bile acids and their derivatives as active ingredients, which are used for preventing, treating and / or alleviating diseases related to GPR39 activity.

Description

Technical Field

[0001] This application relates to the field of biomedicine, and particularly to the use of bile acids and their derivatives in the preparation of GPR39 agonists. Background Art

[0002] G protein-coupled receptors (GPCRs) are the largest class of membrane protein receptors in the human genome. This receptor family has a seven-transmembrane structure, with the N-terminus facing the extracellular side and the C-terminus facing the cytoplasm. According to sequence homology, they can be divided into five major classes, namely: the glutamate receptor family (class C), the rhodopsin-like receptor family (class A), the adhesion receptor family, the frizzled receptor family, and the secretin receptor family. Currently, approximately 50% of the drugs on the market target GPCRs.

[0003] GPR39 is a G protein-coupled receptor belonging to the rhodopsin family (class A), the ghrelin / neurotensin subfamily, and is mainly distributed in certain regions of the brain, pancreas, gastrointestinal tract, liver, and kidneys, etc., and is involved in various physiological processes. Currently, the ligand of GPR39 is not clear. Some studies have shown that zinc ions can activate the GCP39 receptor by binding to histidine residues at positions 17 and 19 in the N-terminus of the extracellular domain (H17&H19).

[0004] Bile acids belong to steroid natural products, can promote nutrient absorption, and are endogenous small molecules that regulate lipid and energy metabolism. They are synthesized by the liver and can be divided into primary bile acids and secondary bile acids according to whether they are fermented by intestinal flora. The main primary bile acids in the human body are cholic acid (CA) and chenodeoxycholic acid (CDCA), and the secondary bile acids mainly include lithocholic acid (LCA), deoxycholic acid (DCA), and ursodeoxycholic acid (UDCA), etc. Summary of the Invention

[0005] This application provides the use of bile acids and / or their derivatives in the preparation of GPR39 agonists.

[0006] In certain embodiments, the bile acids and / or their derivatives include primary bile acids and their derivative bile acids, and the primary bile acids and / or their derivatives are selected from the group consisting of: CDCA, CA, and their derivatives.

[0007] In certain embodiments, the bile acid and / or its derivative includes a secondary bile acid and / or its derivative, and the secondary bile acid and / or its derivative is selected from the group consisting of LCA, DCA, UDCA, and their derivatives.

[0008] In certain embodiments, the bile acid and / or its derivative has the structure shown in Formula I,

[0009]

[0010] wherein, R1 is HO or OSO3 - ;

[0011] R2 is H or HO;

[0012] R3 is H or HO;

[0013] R4 is HO, taurine, glycine, or a pharmaceutically acceptable salt thereof.

[0014] In certain embodiments, the R1 is OSO3 - .

[0015] In certain embodiments, the R2 is H.

[0016] In certain embodiments, the R3 is H.

[0017] In certain embodiments, the R4 is HO, taurine, or glycine.

[0018] In certain embodiments, the bile acid and / or its derivative is selected from the group consisting of TLCA, GLCA, LCAS, TLCAS, GLCAS, TDCA, GDCA, DCAS, TDCAS, GDCAS, GUDCA, TUDCA, UDCAS, TUDCAS, GUDCAS, GCDCA, TCDCA, CDCAS, TCDCAS, GCDCAS, TCA, GCA, CAS, TCAS, GCAS, and their derivatives.

[0019] In certain embodiments, the bile acid and / or its derivative is selected from the group consisting of LCAS, TLCAS, and GLCAS.

[0020] In certain embodiments, the GPR39 agonist further includes zinc ions.

[0021] In certain embodiments, the concentration of the zinc ions is about 10 nM - about 10 μM.

[0022] In certain embodiments, the bile acid and / or its derivative can activate the GPR39 without relying on the zinc ion binding site.

[0023] In certain embodiments, the zinc ion binding site comprises H17 and / or H19.

[0024] In certain embodiments, the bile acid and / or its derivative is capable of activating the GPR39 without relying on an activation site selected from the group consisting of E90, F115, E116, and D330.

[0025] In certain embodiments, the GPR39 is human or murine GPR39.

[0026] On the other hand, the present application also provides the use of the bile acid and / or its derivative for the preparation of a medicament for preventing, treating, and / or alleviating a disease or disorder associated with GPR39 activity.

[0027] In certain embodiments, the bile acid and / or its derivative comprises a primary bile acid and its derivative bile acid, and the primary bile acid and / or its derivative is selected from the group consisting of CDCA, CA, and their derivatives.

[0028] In certain embodiments, the bile acid and / or its derivative comprises a secondary bile acid and / or its derivative, and the secondary bile acid and / or its derivative is selected from the group consisting of LCA, DCA, UDCA, and their derivatives.

[0029] In certain embodiments, the bile acid and / or its derivative has the structure shown in Formula I,

[0030]

[0031] wherein, R1 is HO or OSO3 - ;

[0032] R2 is H or HO;

[0033] R3 is H or HO;

[0034] R4 is HO, taurine, glycine, or a pharmaceutically acceptable salt thereof.

[0035] In certain embodiments, the R1 is OSO3 - .

[0036] In certain embodiments, the R2 is H.

[0037] In certain embodiments, the R3 is H.

[0038] In certain embodiments, the R4 is HO, taurine, or glycine.

[0039] In certain embodiments, the bile acid and / or its derivatives are selected from the group consisting of: TLCA, GLCA, LCAS, TLCAS, GLCAS, TDCA, GDCA, DCAS, TDCAS, GDCAS, GUDCA, TUDCA, UDCAS, TUDCAS, GUDCAS, GCDCA, TCDCA, CDCAS, TCDCAS, GCDCAS, TCA, GCA, CAS, TCAS, GCAS, and derivatives thereof.

[0040] In certain embodiments, the bile acid and / or its derivatives are selected from the group consisting of: LCAS, TLCAS, and GLCAS.

[0041] In certain embodiments, the GPR39 agonist further comprises zinc ions.

[0042] In certain embodiments, the concentration of the zinc ions is from about 10 nM to about 10 μM.

[0043] In certain embodiments, the bile acid and / or its derivatives can activate the GPR39 independently of the zinc ion binding site.

[0044] In certain embodiments, the zinc ion binding site comprises H17 and / or H19.

[0045] In certain embodiments, the bile acid and / or its derivatives can activate the GPR39 independently of the activation sites selected from the group consisting of: E90, F115, E116, and D330.

[0046] In certain embodiments, the GPR39 is human or murine GPR39.

[0047] On the other hand, the present application provides a method for treating a disease or disorder associated with GPR39 activity, which comprises the following steps: administering the bile acid and / or its derivatives described in the present application.

[0048] On the other hand, the present application provides a method for activating GPR39, which comprises the following steps: administering the bile acid and / or its derivatives described in the present application.

[0049] In certain embodiments, the method further comprises the following step: administering zinc ions.

[0050] On the other hand, the present application provides a method for stimulating gastric emptying, which comprises the following steps: administering the bile acid and / or its derivatives described in the present application.

[0051] In certain embodiments, the bile acid and / or its derivatives include primary bile acids and their derivatives, and the primary bile acids and / or their derivatives are selected from the group consisting of CDCA, CA, and their derivatives.

[0052] In certain embodiments, the bile acid and / or its derivatives include secondary bile acids and / or their derivatives, and the secondary bile acids and / or their derivatives are selected from the group consisting of LCA, DCA, UDCA, and their derivatives.

[0053] In certain embodiments, the bile acid and / or its derivatives have the structure shown in Formula I,

[0054]

[0055] wherein, R1 is HO or OSO3 - ;

[0056] R2 is H or HO;

[0057] R3 is H or HO;

[0058] R4 is HO, taurine or glycine, or a pharmaceutically acceptable salt thereof.

[0059] In certain embodiments, the R1 is OSO3 - .

[0060] In certain embodiments, the R2 is H.

[0061] In certain embodiments, the R3 is H.

[0062] In certain embodiments, the R4 is HO, taurine or glycine.

[0063] In certain embodiments, the bile acid and / or its derivatives are selected from the group consisting of TLCA, GLCA, LCAS, TLCAS, GLCAS, TDCA, GDCA, DCAS, TDCAS, GDCAS, GUDCA, TUDCA, UDCAS, TUDCAS, GUDCAS, GCDCA, TCDCA, CDCAS, TCDCAS, GCDCAS, TCA, GCA, CAS, TCAS, GCAS, and their derivatives.

[0064] In certain embodiments, the bile acid and / or its derivatives are selected from the group consisting of LCAS, TLCAS and GLCAS.

[0065] In certain embodiments, the GPR39 agonist further includes zinc ions.

[0066] In certain embodiments, the concentration of the zinc ions is from about 10 nM to about 10 μM.

[0067] In certain embodiments, the bile acid and / or its derivative is capable of activating the GPR39 without relying on a zinc ion binding site.

[0068] In certain embodiments, the zinc ion binding site includes H17 and / or H19.

[0069] In certain embodiments, the bile acid and / or its derivative is capable of activating the GPR39 without relying on activation sites selected from the group consisting of E90, F115, E116, and D330.

[0070] In certain embodiments, the GPR39 is human or murine GPR39.

[0071] On the other hand, the present application also provides a pharmaceutical composition for activating GPR39, which comprises the bile acid and / or its derivative described in the present application, and a pharmaceutically acceptable carrier.

[0072] Those skilled in the art can easily understand other aspects and advantages of the present application from the following detailed description. Only exemplary embodiments of the present application are shown and described in the following detailed description. As those skilled in the art will recognize, the content of the present application enables those skilled in the art to make changes to the disclosed specific embodiments without departing from the spirit and scope of the invention involved in the present application. Accordingly, the descriptions in the drawings and the specification of the present application are merely exemplary and not restrictive. BRIEF DESCRIPTION OF THE DRAWINGS

[0073] The specific features of the invention involved in the present application are shown as in the appended claims. The features and advantages of the invention involved in the present application can be better understood by referring to the exemplary embodiments and the drawings described in detail below. A brief description of the drawings is as follows:

[0074] Figure 1 Shows the results of a calcium imaging experiment of the bile acid and its derivative described in the present application activating the GPR39 receptor on the m39-20 cell line;

[0075] Figure 2 Shows a bar graph of the activation potency of the bile acid and its derivative described in the present application activating the GPR39 receptor on the m39-20 cell line;

[0076] Figure 3 A-3F shows a dose curve graph of the bile acid and its derivative described in the present application activating the GPR39 receptor on the m39-20 cell line;

[0077] Figure 4A shows the dose-response curve of LCAS activating the GPR39 receptor on the m39-20 cell line after adding zinc ions at different concentrations;

[0078] Figure 4 B shows the dose-response curve of LCAS activating the mutants H17A and H19A of the GPR39 receptor on the m39-20 cell line;

[0079] Figure 5 Shows the map of the plasmid pCMV6-3HA-mGPR39(H17A&H19A)-P2A-mCherry described in this application;

[0080] Figure 6 Shows the dose-response curve of the GPR39 agonists TG-G 1008 and TM-N 1324 activating the GPR39 receptor on the m39-20 cell line;

[0081] Figure 7 Shows the results of calcium imaging experiments of the LCAS activating the site mutants E90A, F115A, E116A, and D330A of the GPR39 receptor on the m39-20 cell line;

[0082] Figure 8 Shows the results of calcium imaging experiments of LCAS activating 6 receptors of the Ghrelin family;

[0083] Figure 9 Shows the results of calcium imaging experiments of LCAS, TLCAS, and GLCAS on the human GPR39-2 cell line;

[0084] Figure 10 Shows the map of the lentiviral vector PLVXK-hGPR39-P2A-mCherry;

[0085] Figure 11 Shows the map of the lentiviral vector PLVXK-mGPR39-P2A-mCherry. Detailed implementation mode

[0086] The following specific embodiments illustrate the implementation mode of the invention of this application. Those skilled in this technology can easily understand other advantages and effects of the invention of this application from the content disclosed in this specification.

[0087] In the present application, the term "receptor" generally refers to a class of special proteins present on the cell membrane or inside the cell, which can specifically recognize and bind to extracellular signal molecules, and then activate a series of intracellular physiological and biochemical reactions, enabling the cell to produce corresponding effects in response to external stimuli. A receptor contains at least two functional regions: namely, a ligand-binding region and an effector region. According to its location, it is divided into membrane receptors and intracellular receptors. The term "ligand" generally refers to any molecule that binds to an anchor protein. Ligands in living organisms can generally be divided into two categories. One category is simple inorganic small molecules or ions; the other category is biological macromolecules such as nucleic acids, proteins, polypeptides, etc.

[0088] In the present application, the term "GPCRs" generally refers to G protein-coupled receptors (GPCR), which is a collective term for the largest membrane protein receptors in the human genome, containing approximately 800 - 1000 family members, accounting for about 3% of the entire genome. According to sequence homology, it can be divided into five major categories, namely: glutamate receptor family (class C), rhodopsin-like receptor family (class A), adhesion receptor family, frizzled receptor family, and secretin receptor family. GPCRs have a wide distribution in tissues and cells and play an important role in various physiological activities of humans by mediating the conversion of extracellular stimulants into intracellular signals. GPCRs are very valuable drug treatment targets. Currently, 30% - 50% of the therapeutic drugs used in the market exert their pharmacological effects through signal pathways mediated by GPCRs.

[0089] In the present application, the term "GPR39" generally refers to G protein-coupled receptor 39, which belongs to the ghrelin / neurotensin subfamily. Studies have shown that GPR39 is involved in gastrointestinal metabolic functions in the human body, and its endogenous ligand is zinc ion. GPR39 in other species can be a homologous protein with a sequence identity of not less than 50% to the human GPR39 (NCBI Reference Sequence: NP_001499.1) or mouse GPR39 (NCBI Reference Sequence: NP_081953.2) obtained by Clustal Omega sequence alignment software. Among them, the sequence homology between human and mouse GPR39 proteins is more than 82%.

[0090] In the present application, the term "agonist" generally refers to a substance that can enhance the activity of another molecule or other sites of a receptor. According to the source, it can be divided into endogenous agonists and exogenous agonists. According to efficacy, it can be divided into full agonists, superagonists, partial agonists, inverse agonists, etc. An agonist is a substance that can bind to a receptor, change the receptor state, and trigger a receptor biological response. In the present application, a GPR39 agonist refers to a substance that can bind to the GPR39 receptor and enhance its activity, such as bile acids and their derivatives, zinc ions, the known GPR39 agonist TC-G 1008 (Peukert et al, 2014), and TM-N 1324 (Frimurer et al, 2017).

[0091] In the present application, the term "bile acid" generally refers to a class of endogenous steroid natural products composed of a rigid steroid skeleton containing 1-3 hydroxyl groups and an alkane side chain with a terminal carboxyl group, which is the main component of bile, accounting for 50%-60% of the total bile volume. Bile acids are mainly synthesized in the liver from cholesterol and secreted in the duodenum. Approximately 40%-50% of cholesterol in the human body is converted into bile acids. Farnesoid X receptor (FXR) and pregnane X receptor (PXR) of bile acids are currently known specific nuclear receptors of bile acids. Bile acids can regulate the absorption of fats in food and the synthesis of bile acids in the human body by activating FXR and PXR.

[0092] The basic structure of bile acids is a 24-carbon carboxylic acid cholane series with cyclopentane polyhydrophenanthrene as the core, as shown in Formula I. In the present application, R1 can be HO or OSO3 - ; R2 can be H or HO; R3 can be H or HO; R4 can be HO, taurine (CAS: 107-35-7), or glycine (CAS: 56-40-6). Among them, in some embodiments, OSO3 - can bind protons; in other embodiments, OSO3 - can also bind other ions to form salts. More than 90% of bile acids in the human body exist in the conjugated form. Bile acids combine with glycine or taurine through peptide bonds to form tauro-conjugated bile acids or glyco-conjugated bile acids, and the content of free bile acids is extremely small.

[0093]

[0094] According to the source, bile acids can be divided into two categories: primary bile acids and secondary bile acids. Bile acids directly synthesized from cholesterol in hepatocytes are called primary bile acids, including:

[0095] 1) Cholic acid (CA, CAS: 81-25-4): R1 is HO, R 2α is HO, R 2β is H, R3 is HO, R4 is HO;

[0096] 2) Chenodeoxycholic acid (CDCA, CAS: 474-25-9): R1 is HO, R 2α is HO, R 2β is H, R3 is H, R4 is HO.

[0097] The structural difference between cholic acid (CA) and chenodeoxycholic acid (UDCA) is only the number of hydroxyl groups contained in the cholane skeleton. Cholic acid (CA) contains 3 hydroxyl groups (3α, 7α, 12α), while chenodeoxycholic acid contains 2 hydroxyl groups (3α, 7α).

[0098] Primary bile acids are acted on by bacteria in the intestine, and the bile acids generated by the 7α-dehydroxylation reaction are called secondary bile acids, including:

[0099] 1) Deoxycholic acid (DCA, CAS: 83-44-3): R1 is HO, R 2α is H, R 2β is H, R3 is HO, R4 is HO;

[0100] 2) Lithocholic acid (LCA, CAS: 434-13-9): R1 is HO, R 2α is H, R 2β is H, R3 is H, R4 is HO;

[0101] 3) Ursodeoxycholic acid (UDCA, CAS: 128-13-2): R1 is HO, R 2α is H, R 2β is HO, R3 is H, R4 is HO.

[0102] In this application, the term "bile acid derivative" generally refers to a compound that has a common core four-ring structure with cholic acid (CA) and is substituted by various substituents.

[0103] In this application, the derivatives of cholic acid (CA) include:

[0104] 1) TCA (CAS: 145-42-6): R1 is HO, R 2α is HO, R 2βR1 is H, R3 is HO, and R4 is taurine;

[0105] 2) GCA (CAS: 863-57-0): R1 is HO, R 2α is HO, R 2β is H, R3 is HO, and R4 is glycine;

[0106] 3) TCAS (CAS: 67030-62-0): R1 is OSO3 - 、R 2α is HO, R 2β is H, R3 is HO, and R4 is taurine.

[0107] In this application, the derivatives of chenodeoxycholic acid (CDCA) include:

[0108] 1) GCDCA (CAS: 16564-43-5): R1 is HO, R 2α is HO, R 2β is H, R3 is H, and R4 is glycine;

[0109] 2) TCDCAS (CAS: 67030-59-5): R1 is OSO3 - 、R 2α is HO, R 2β is H, R3 is H, and R4 is taurine;

[0110] 3) GCDCAS (CAS: 66874-09-7): R1 is OSO3 - 、R 2α is HO, R 2β is H, R3 is H, and R4 is glycine.

[0111] In this application, the derivatives of deoxycholic acid (DCA) include:

[0112] 1) TDCA (CAS: 1180-95-6): R1 is HO, R 2α is H, R 2β is H, R3 is HO, and R4 is taurine;

[0113] 2) GDCA (CAS: 16409-34-0): R1 is HO, R 2α is H, R 2β is H, R3 is HO, and R4 is glycine;

[0114] 3) DCAS (CAS: 60237-35-6): R1 is OSO3 - 、R 2α is H, R2β R1 is H, R3 is HO, and R4 is HO;

[0115] 4) TDCAS (CAS: not available yet): R1 is OSO3 - , R 2α is H, R 2β is H, R3 is HO, and R4 is taurine;

[0116] 5) GDCAS (CAS: 66874-09-7): R1 is OSO3 - , R 2α is H, R 2β is H, R3 is HO, and R4 is glycine.

[0117] In this application, the derivatives of lithocholic acid (LCA) include:

[0118] 1) TLCA (CAS: 6042-32-6): R1 is HO, R 2α is H, R 2β is H, R3 is H, and R4 is taurine;

[0119] 2) GLCA (CAS: 24404-83-9): R1 is HO, R 2α is H, R 2β is H, R3 is H, and R4 is glycine;

[0120] 3) LCAS (CAS: 64936-81-8): R1 is OSO3 - , R 2α is H, R 2β is H, R3 is H, and R4 is HO;

[0121] 4) TLCAS (CAS: 64936-83-0): R1 is OSO3 - , R 2α is H, R 2β is H, R3 is H, and R4 is taurine;

[0122] 5) GLCAS (CAS: 64936-82-9): R1 is OSO3 - , R 2α is H, R 2β is H, R3 is H, and R4 is glycine.

[0123] In this application, the derivatives of ursodeoxycholic acid (UDCA) include:

[0124] 1) GUDCA (CAS: 64480-66-6): R1 is HO, R 2α is H, R2β R1 is HO, R3 is H, and R4 is glycine;

[0125] 2) UDCAS (CAS: 68780-73-4): R1 is OSO3 - and R 2α is H, R 2β is HO, R3 is H, and R4 is HO;

[0126] 3) TUDCAS (CAS: not available yet): R1 is OSO3 - and R 2α is H, R 2β is HO, R3 is H, and R4 is taurine.

[0127] In the present application, the "salt" in the term "pharmaceutically acceptable salt" refers to a product formed by an ionic bond with the bile acid, and the salt is generally prepared by reacting the bile acid and / or its derivative with an acid or a base, where the acid or the base is suitable for administration to a subject. For example, the acids include, but are not limited to, ammonium chloride, hydrochloric acid, sulfuric acid, phosphoric acid, acetic acid, citric acid, tartaric acid, ascorbic acid, benzoic acid, and common amino acids such as glycine, alanine, phenylalanine, arginine, etc.; the bases include, but are not limited to, sodium carbonate, sodium bicarbonate, ammonia water, ethanolamine, N-methylglucosamine, etc.

[0128] In the present application, the term "about" generally refers to a variation within a range of 0.5% - 10% above or below the specified value, for example, within a range of 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% above or below the specified value.

[0129] Use

[0130] On the one hand, the present application provides the use of bile acids and / or their derivatives in the preparation of GPR39 agonists. The bile acids and / or their derivatives include primary bile acids and their derivative bile acids, and secondary bile acids and their derivative bile acids.

[0131] GPR39 is a G protein-coupled receptor whose sequence is similar to that of the growth hormone secretagogue receptor (GHSR) and neurotensin receptors 1 and 2 (NTSR1 and NTSR2). According to bioinformatics speculation, the GPR39 protein is composed of 453 amino acid residues and contains 7 transmembrane regions characteristic of growth hormone secretagogues (GHSR). GPR39 has a wide tissue distribution, such as in the brain tissue, stomach and small intestine, pancreas, thyroid gland, and colon, etc.

[0132] In this application, the GPR39 can be from murine, human, and other species. The murine GPR39 protein sequence can be determined according to NCBI Reference Sequence: NP_081953.2; the human GPR39 protein sequence can be determined according to NCBI Reference Sequence: NP_001499.1; the GPR39 protein sequences of other species are homologous proteins with a sequence identity of not less than 50% obtained by aligning with human GPR39 or murine GPR39 using the Clustal Omega sequence alignment software. For example, the GPR39 protein can be from porcine, bovine, ovine, rabbit, or monkey sources. In this application, the GPR39 can also be a cell line stably expressing GPR39, such as the murine m39-20 cell line, the human human GPR39-2 cell line, and cell lines from other species that can express GPR39. In this application, the GPR39 can also be cultured cells that transiently overexpress human or murine GPR39 by transfection of 293T cells using the lipofectamine 3000 transfection kit (Invitrogen).

[0133] In this application, the bile acid and / or its derivatives include primary bile acids and their derivative bile acids. The primary bile acids and / or their derivatives can be selected from, but not limited to, the following group: CDCA, CA, and their derivatives. For example, the derivatives of primary bile acids can be derivatives of CA such as TCA, GCA, TCAS, derivatives of CDCA such as GCDCA, TCDCAS, GCDCAS, or derivative bile acids that have a common core tetracyclic structure with CA and CDCA and are substituted with different groups. The substituting groups can be alkyl, alkenyl, alkynyl, halogen, trifluoromethyl, aryl, mercapto, cyano, alkoxy, etc.

[0134] The bile acids and / or their derivatives include secondary bile acids and / or their derivatives, which may be selected from, but not limited to, the following group: LCA, DCA, UDCA, and their derivatives. For example, the derivatives of secondary bile acids may be derivatives of LCA such as TLCA, GLCA, LCAS, TLCAS, GLCAS, derivatives of DCA such as TDCA, GDCA, DCAS, TDCAS, GDCAS, derivatives of UDCA such as GUDCA, UDCAS, TUDCAS, or derivatives of bile acids having a common core tetracyclic structure with LCA, DCA, UDCA and substituted by different groups. The substituents may be alkyl, alkenyl, alkynyl, halogen, trifluoromethyl, aryl, mercapto, cyano, alkoxy, etc.

[0135] Intracellular calcium imaging technology is a conventional technique for studying cell functions. Its basic principle is to use calcium ion indicators to detect the calcium ion concentration in tissues or cells, and then reflect certain reactions in tissues or cells. In this application, the calcium fluorescent indicator Fluo8-AM was used to detect the intracellular calcium release results of stably or transiently expressed GPR39 cell lines to determine the activation effect of bile acids and their derivatives on GPR39 receptors.

[0136] For example, in the murine m39-20 cell line stably expressing GPR39, TLCA, GLCA, LCAS, TLCAS, GLCAS, TDCA, GDCA, DCAS, TDCAS, GDCAS, GUDCA, UDCAS, TUDCAS, GCDCA, TCDCAS, GCDCAS, TCA, GCA, TCAS can all cause intracellular calcium signals of varying degrees ( Figure 1 ); while in 293T cells that do not express GPR39, only DCA and CDCA can cause observable calcium signals, and other bile acid derivatives cannot cause intracellular calcium signals in 293T cells, indicating that bile acid derivatives bind to and activate GPR39 receptors and then trigger intracellular calcium signals.

[0137] Positive allosteric regulation of bile acids and their derivatives by zinc ions to activate GPR39

[0138] In this application, the GPR39 agonist also includes zinc ions. Zn 2+ has been reported to be a ligand of GPR39. Using Zn 2 +Stimulating cell lines that transiently or stably express GPR39 can detect intracellular calcium release (Yasuda, Miyazaki et al, 2007), inositol phosphate accumulation (EC50 = 22 μM), and cAMP elevation (EC50 = 7.4 μM) (Holst, Egerod et al, 2007). For example, zinc ions can bind to GPR39 in the m39-20 cell line and activate GPR39, thereby inducing an intracellular calcium signal ( Figure 1 ).

[0139] In the present application, the zinc ion binding site includes H17 and / or H19. The human zinc ion binding sites H17 and H19 are determined according to NCBI Reference Sequence: NP_001499.1. H17 and H19 are the 17th histidine and the 19th histidine counted from the N-terminus of the GPR39 protein sequence, respectively. The GPR39 protein sequences of other species can be aligned with the reference sequence NP_001499.1 by the ClustalOmega sequence alignment software to obtain the corresponding H17 and H19 zinc ion binding sites. For example, the zinc ion binding sites corresponding to the mouse GPR39 protein are the 17th histidine and the 19th histidine counted from the N-terminus of the mouse GPR39 protein sequence.

[0140] In the present application, the bile acid and / or its derivative can activate the GPR39 independently of the zinc ion binding site. For example, in some embodiments, the mouse wild-type (WT) GPR39 or the zinc ion binding site mutant (H17A and H19A) GPR39 is transiently expressed in 293T cells using the lipofectamine3000 transfection kit. The activation effect of LCAS on the wild-type (WT) and mutant (H17A and H19A) was detected by calcium imaging. The results showed that LCAS could activate the mutant (H17A and H19A) GPR39 and trigger an intracellular calcium signal, and its EC50 value was 24 ± 0.8 μM, which was less than the EC50 value of 33.1 ± 5.1 μM for LCAS to activate the wild-type (WT) GPR39, indicating that the bile acid and / or its derivative activates GPR39 independently of the zinc ion binding sites H17 and H19.

[0141] Allosteric regulation refers to the change in the spatial conformation of a receptor protein after a substance acts on it, which in turn causes a change in the function of the receptor protein. A positive allosteric modulator can promote the signal transduction of the receptor protein, and conversely, a negative modulator inhibits the signal transduction of the receptor protein. Allosteric regulation plays an important regulatory role in the function of the receptor, but the specific mechanism of allosteric regulation is not yet clear. It is generally believed that there are multiple sites on the receptor. When one site binds a ligand, its own conformation changes, which in turn affects the binding activity of other sites.

[0142] EC50, the concentration for 50% of maximal effect, refers to the concentration that can cause 50% of the maximal effect. In this application, EC50 refers to the concentration of bile acids and their derivatives and zinc ions that can cause 50% of the maximal effect of the GPR39 receptor. Efficacy represents the potency of an agonist. In some embodiments, after adding zinc ions, the efficacy of GLCA, TDCA, GDCA, DCAS, TDCAS, GDCAS, GUDCA, UDCAS, TUDCAS, GCDCA, TCDCAS, GCDCAS, TCA, GCA, TCAS in activating GPR39 in the murine m39-20 cell line was significantly increased (Table 1, Figure 2 ), and the dose curve of bile acids activating the GPR39 receptor shifted to the left, indicating that zinc ions play a positive allosteric regulatory role in the activation of the GPR39 receptor by bile acids and their derivatives. The concentration of the zinc ions can be 0.01 μM, 0.1 μM, 0.7 μM, 3 μM, 20 μM; while the efficacy of LCAS, TLCAS, GLCAS at a concentration of 200 μM in activating GPR39 in the murine m39-20 cell line was relatively high compared to other bile acids and their derivatives before adding zinc ions, and the increase in their efficacy after adding zinc ions was small. Analyzing the reason, it may be that when R1 at the 3rd carbon position of the cholate molecule is OSO3 - , LCA has a relatively high efficacy in activating GPR39.

[0143] On the other hand, the present application also provides the use of the bile acid and / or its derivative for preparing a drug for preventing, treating and / or alleviating diseases or disorders related to GPR39 activity. Since GPR39 is highly expressed in various cells and tissues such as human jejunum, ileum, duodenum, stomach, liver, adipose tissue, retinal pigment epithelial cells and pituitary gland, it has become one of the targets for intervening drug action and has attracted more and more attention. In the present application, the drug may be a pharmaceutical composition containing a safe and effective dose of the bile acid and / or its derivative described in the present application and a pharmaceutically acceptable carrier or excipient. The carriers include but are not limited to saline, glucose, buffer solution, water, glycerol, ethanol and their combinations. The pharmaceutical composition described in the present application can be made into forms such as injections, tablets, capsules, pills, etc. The excipients include but are not limited to, for example, binders, fillers, disintegrants, lubricants in tablets; wine, vinegar, medicinal juices, etc. in traditional Chinese medicine pills; the matrix part in semi-solid preparations such as ointments and creams; preservatives, antioxidants, flavoring agents, fragrances, solubilizing agents, emulsifying agents, solubilizers, osmotic pressure regulators, coloring agents, etc. in liquid preparations.

[0144] Method

[0145] On the other hand, the present application also provides a method for treating diseases or disorders related to GPR39 activity, which includes the following steps: administering the bile acid and / or its derivative described in the present application. In certain embodiments, a pharmaceutical composition containing a safe and effective dose of the bile acid and / or its derivative described in the present application and a pharmaceutically acceptable carrier or excipient can be administered, and through various different administration routes, such as oral, subcutaneous, intravenous injection, intramuscular injection, inhalation, rectal, etc. for treating diseases or disorders related to GPR39 activity.

[0146] On the other hand, the present application also provides a method for activating GPR39, which includes the following steps: administering the bile acid and / or its derivative described in the present application. The subjects to be administered can be mammals, such as humans, rats, mice; or can also be isolated tissues, organs or cells, etc.

[0147] On the other hand, the present application also provides a method for stimulating gastric emptying, which includes the following steps: administering the bile acid and / or its derivative described in the present application. In the present application, the term "gastric emptying" generally refers to the process of food emptying from the stomach into the duodenum. Existing studies have shown that GPR39 is involved in gastrointestinal motility and digestive juice secretion in vivo. Therefore, the GPR39 receptor may become a target for treating gastrointestinal diseases, which usually affect gastric motility, such as functional dyspepsia, diabetic gastroparesis, gastric spasm; diseases caused by abnormal colorectal motility, such as irritable bowel syndrome, diarrhea, chronic constipation, etc.

[0148] In certain embodiments, the bile acid and / or its derivatives are capable of activating the GPR39 independent of activation sites selected from the group consisting of E90, F115, E116, and D330. In certain embodiments, bile acids can elicit intracellular calcium signaling in mutants E90A, F115A, E116A, and D330A of GPR39, indicating that the activation of GPR39 by bile acids is independent of the known agonist binding sites: E90, F115, E116, and D330. Additionally, in certain embodiments, the bile acid and / or its derivatives are capable of activating independent of activation sites selected from the group consisting of E90, F115, E116, and E330. In certain embodiments, bile acids can elicit intracellular calcium signaling in mutants E90A, F115A, E116A, and E330A of GPR39, indicating that the activation of GPR39 by bile acids is independent of the known agonist binding sites: E90, F115, E116, and E330.

[0149] Without wishing to be bound by any theory, the examples below are merely for illustrating the bile acids and their derivatives, preparation methods, and uses of the present application, and are not intended to limit the scope of the invention of the present application.

[0150] Examples

[0151] Example 1 Construction of a cell line m39-20 stably expressing the murine GPR39 gene

[0152] 1.1 Cell culture

[0153] Cell culture was performed using DMEM medium (gibco), supplemented with 10% dialyzed fetal bovine serum (gibco), 100 U / mL penicillin, and streptomycin. Cells were cultured at 37 °C in a 5% CO2 atmosphere.

[0154] 1.2 Lentiviral vector construction

[0155] The target gene murine GPR39 cDNA sequence (sequence as shown in SEQ ID NO:11) was amplified from a murine duodenum cDNA library using primers mGPR39-F (whose sequence is as shown in SEQ ID NO:12) and mGPR39-R (whose sequence is as shown in SEQ ID NO:13), and the obtained target sequence was cloned into the lentiviral vector PLVXK-mGPR39-P2A-mCherry (see Figure 11 ). Positive monoclonal clones were purified for later use after sequencing verification.

[0156] 1.3 Cell transfection and puromycin selection

[0157] HEK293T cells (purchased from ATCC) were seeded at an appropriate density (about 0.5 - 2×105 Inoculate (into the wells) the cells in a 6-well plate (from Corning). When the cell density reaches over 30%, use Lipofectamine 3000 (from Invitrogen) to transfect the plasmid PLVXK-mGPR39-P2A-mCherry, with the plasmid transfection concentration being 2 μg per well. After 24 hours of transfection, passage the cells and add puromycin (from gibco) to the medium at a concentration of 2 μg / mL for antibiotic screening of the cells. After continuously screening for 2 - 4 days, digest the cells with puromycin resistance with trypsin and screen for monoclonal cells with red fluorescence by flow cytometry. Then inoculate the monoclonal cells into a 96-well plate and culture them in the medium without added puromycin for about 10 days. Select the cell colonies with red fluorescence under a fluorescence microscope, digest them with trypsin and passage and amplify them in a 6-well plate. Through the above method, a cell line m39-20 stably expressing murine GPR39 is obtained.

[0158] Example 2 FLIPR Calcium Imaging

[0159] 2.1 Cell Culture

[0160] Before the calcium imaging experiment, first inoculate the m39-20 cells from Example 1 at an appropriate density (about 3×10 4 cells / well) into a black-walled clear-bottom 96-well plate coated with PDL (from Corning), and culture them at 37 °C under 5% CO2 for at least 24 h to make the cell density reach 90% - 100%.

[0161] 2.2 Pre-incubate Cells with Calcium Dye

[0162] Dissolve the calcium dye Fluo8-AM (from ATT bioquest) in DMSO solvent to prepare a stock solution of 1 μg / μL, and then dilute the calcium dye stock solution 500-fold with 4K solution (NaCl 150 mM, KCl 4 mM, MgCl2 2 mM, CaCl2 2 mM, glucose 11 mM, HEPES 10 mM, pH = 7.4) as the calcium dye working solution. Add 50 μL of the calcium dye working solution to the m39-20 cells in Example 2.1 per well. After incubating at room temperature for 25 min, replace the calcium dye working solution (50 μL per well) with 4K solution, and perform the calcium imaging experiment after adapting at room temperature for 5 min.

[0163] 2.3 FLIPR Calcium Imaging

[0164] Dissolve the bile acids and their derivatives shown in Table 1 below in dimethyl sulfoxide (DMSO, Sigma) solvent to prepare a 50 mM stock solution and store it at -20 °C. When performing calcium imaging using FLIPR, dilute the drug to a working concentration of 400 μM with the above 4K solution and dispense it into another 96-well plate. Place the drug solution and the cell samples in Example 2.2 at the designated positions on the FLIPR instrument (Molecular Devices). Set the instrument parameters so that 50 μL of the drug solution can be added to the culture well within 2 s. The fluorescence recording results within the first 30 s are used as the baseline value. Set the program so that the FLIPR robotic arm automatically adds bile acids and their derivatives to the cell sample culture well at 31 s. Use 1% DMSO solvent as the blank control.

[0165] The results of the FLIPR calcium imaging experiment for bile acids and their derivatives are as Figure 1 shown, where the abscissa represents time and the ordinate represents relative fluorescence intensity. From the results of the calcium imaging experiment, it can be seen that: 1) LCAS, TLCAS, and GLCAS can also cause a strong intracellular calcium signal response in m39-20 cells in the absence of zinc ions, while the 1% DMSO blank control group cannot cause an observable calcium signal in m39-20 cells, indicating that the activation of the GPR39 receptor depends on bile acids and their derivatives; 2) Only DCA and CDCA can cause an observable calcium signal in HEK293T cells, and other bile acids and their derivatives cannot cause an observable calcium signal in 293T cells, indicating that the intracellular calcium signal induced by bile acids and their derivatives depends on the GPR39 receptor.

[0166] 2.4 Allosteric regulation experiment of zinc ions

[0167] Pre-incubate the cells with the calcium dye according to the method described in Example 2.2. After pre-incubating with the calcium dye, replace the calcium dye working solution (50 μL per well) with a 4K solution containing 4 μM zinc ions and incubate at room temperature for 5 min. Prepare a solution of bile acids and their derivatives according to the method described in Example 2.3 and then add 4 μM zinc ions to it. Then perform a calcium imaging experiment, and the results are as Figure 1 shown, where the ordinate represents relative fluorescence intensity and the abscissa represents time. From the results of the calcium imaging experiment, it can be seen that: 1) Under the condition of adding 4 μM zinc ions, various bile acids and their derivatives can cause different degrees of intracellular calcium signal responses in m39-20 cells; 2) Under the condition of adding 4 μM zinc ions, the calcium signals caused by DCA and CDCA in the m39-20 cell line are very different from those in the 293T cell line, indicating that DCA and CDCA can activate the GPR39 receptor in the presence of zinc ions.

[0168]

[0169] Table 1

[0170]

[0171]

[0172] Detection of the half-maximal effective concentration (EC50) and activation efficacy of Example 3

[0173] The half-maximal effective concentration (EC50) and activation efficacy of bile acids and their derivatives in activating the GPR39 receptor on the m39-20 cell line were detected using FLIPR respectively. The detection results are summarized in Table 1 above. The bar chart of the activation efficacy of bile acids and their derivatives in activating the GPR39 receptor is as shown in Figure 2 shown, and the dose curve is as shown in Figure 3 A-3F. The results show that bile acids and their derivatives have a dose effect on the activation of the GPR39 receptor. Among them, LCAS, TLCAS, and GLCAS have the strongest activation ability, and zinc ions can shift the dose curve of bile acids and their derivatives in activating GPR39 to the left, indicating that zinc ions have a positive allosteric regulatory effect on the activation of GPR39 by bile acids and their derivatives.

[0174] Example 4 Positive allosteric regulation of different concentrations of zinc ions on the activation of the GPR39 receptor by bile acids

[0175] Taking LCAS as an example, according to the method described in Example 3, the effects of adding different concentrations of zinc ions on the half-maximal effective concentration (EC50) of activating the GPR39 receptor on the m39-20 cell line were detected respectively. The detection results are summarized in Table 2 below, and the dose curve is as shown in Figure 4 A. The results show that: 1) When the zinc ion concentration is lower than 0.1 μM, the positive allosteric regulatory effect of zinc ions disappears; 2) When the zinc ion concentration is higher than 1 μM, the positive allosteric regulatory ability on GPR39 is more significant.

[0176] Table 2

[0177] Zinc ion (μM) EC50 (μM) 4 0.7±0.03 2 1.9±0.03 1 6.4±0.67 0.4 20.1±0.67 0.2 25.4±1.1 0.1 30.6±2 0.05 30.7±2.4 0 31±2.1

[0178] Example 5 Activation of mutants H17A and H19A of GPR39 by bile acids

[0179] 5.1 Construction of site-directed mutation vectors

[0180] Select the pCMV6-3HA-mGPR39-P2A-mCherry vector with the murine GPR39 gene and a total length of approximately 7 Kbp as the PCR template. Obtain the site-directed mutation vector by PCR site-directed mutagenesis. Design the forward primer m39-H17A-F and the reverse primer m39-H17A-R by extending approximately 15 bp forward and backward from the mutation site as the center. Their sequences are shown in Table 3 below. Design a 15-bp complementary sequence with the forward primer at 1 bp upstream of the mutation site, and design the length of the reverse primer to maintain the primer annealing temperature at 60 °C. Use high-fidelity DNA polymerase (Phanta Max Super-fidelity DNA polymerase, Vazyme) for site-directed PCR. After adding 5 μL of cutsmart buffer (NEB) and 1 μL of restriction endonuclease DpnI (NEB) to the PCR product and incubating overnight for digestion, take 10 μL of the product and transfer it into 50 μL of DH5α for heat shock transformation. Select the corresponding resistant solid LB medium for coating and culturing, grow overnight at 37 °C, pick single colonies for inoculation, shake the bacteria to extract plasmids for sequencing verification. Select the monoclonal vector with correct sequencing and denote it as pCMV6-3HA-mGPR39(H17A&H19A)-P2A-mCherry, and its structure is as Figure 5 shown.

[0181] Table 3

[0182] Name SEQ ID NO m39-H17A-F 1

[0183] m39-H17A-R 2

[0184] 5.2 Transient transfection cell system

[0185] Inoculate HEK293T cells at an appropriate density (about 3×10 4 cells / well) into a 96-well plate coated with PDL (coring company), and culture them under the conditions of 37 °C and 5% CO2. After 24 h of inoculation, when the cell density reaches 60%-70%, use Lipofectamine 3000 (Invitrogen) for transfection. The plasmid transfection concentration is 50 ng per well. After 24 h of transfection, detect the half-maximal effective concentration (EC50) of LCAS activating the corresponding receptor on wild-type (WT) GPR39 and its mutant transiently transfected cells according to the method described in Example 3. The results are shown in Table 4 and Figure 4 Figure B below. The results show that: 1) The activation of GPR39 by bile acids and their derivatives does not depend on the previously reported zinc ion binding sites H17 and H19, and its mechanism of activating GPR39 is different from that of zinc ions. 2) Zinc ions have a strong positive allosteric regulatory ability on the LCAS-activated GPR39 mutants H17A and H19A.

[0186] Table 4

[0187]

[0188] Example 6 Comparison of bile acids with known GPR39 agonists

[0189] The half-maximal effective concentration (EC50) of the known GPR39 agonists TC-G 1008 (cat. no 5355, Tocris) and TM-N 1324 (cat. no 6325, Tocris) for activating GPR39 was detected on the m39-20 cell line according to the method described in Example 3. The detection results are shown in Table 5 below, and the dose curves are as Figure 6 shown. Comparing with the detection results in Example 3, in the presence of 4 μM zinc ions, the EC50 value of the bile acid derivative LCAS was 0.88 ± 0.16 μM, which was less than the EC50 value of TC-G 1008 (1.5 μM) under the same conditions and less than the EC50 value of TM-N 1324 (3.5 μM) under the same conditions, indicating that LCAS was more potent than the known GPR39 agonists TC-G 1008 and TM-N 1324 in activating GPR39.

[0190] Table 5

[0191]

[0192] Example 7 Calcium imaging of bile acids on known GPR39 activation site mutants

[0193] First, the GPR39 mutants E90A, F115A, E116A, and D330A were constructed according to the method described in Example 5. The sequences of the forward and reverse primers used for constructing each mutant are shown in Table 6 below:

[0194] Table 6

[0195] Name SEQ ID NO m39-E90A-F 3 m39-E90A-R 4 m39-F115A-F 5 m39-F115A-R 6 m39-E116A-F 7 m39-E116A-R 8 m39-D330A-F 9 m39-D330A-R 10

[0196] Then, according to the method described in Example 5.2, calcium imaging experiments of LCAS activating the GPR39 activation site mutants E90A, F115A, E116A, and D330A were carried out on the Lipofectamine 3000 transient transfection cell system. The detection results are as Figure 7 shown. The results showed that: 1) The GPR39 activation site mutants E90A, F115A, E116A, and D330A could all be activated by LCAS; 2) The activation of GPR39 by bile acids was independent of the known agonist binding sites.

[0197] Example 8 Calcium Imaging of Bile Acids on the Ghrelin Receptor Family

[0198] GPR39 is one of the G protein-coupled receptors belonging to the Ghrelin family, which has a total of 7 members. In addition to GPR39, it also includes the Ghrelin receptor (GHSR, NP_940799.1), Motilin receptor (MLNR, NP_001498.1), Neuromedin-U receptor 1 (NMUR1, NP_006047.3), Neuromedin-U receptor 2 (NMUR2, NP_064552.3), Neurotensin receptor 1 (NTSR1, NP_002522.2), and Neurotensin receptor 2 (NTSR2, NP_036476.2).

[0199] According to the transient transfection method described in Example 5.2, 6 receptors of the Ghrelin family except GPR39 (the expression vectors of these 6 receptors are from the research group of Rao Yi at Peking University) were overexpressed in HEK293T cells respectively, and the calcium dye was incubated. An inverted laser confocal microscope (Leica) was used to detect the activation of the 6 receptors of the Ghrelin family by LCAS. The specific operation was as follows: the cells to be detected were placed on the microscope stage, the fluorescence signal value of the calcium dye Fluo8 was detected using the 488 nm channel, and the fluorescence signal value of mCherry was detected using the 543 nm channel. The detection results are as Figure 8 shown. All receptors were fused and expressed with the red fluorescent protein mCherry, and the expression of the receptors was determined by detecting the red fluorescence emitted by mCherry. The results showed that only GPR39 could be activated by 200 μM LCAS, and the remaining 6 receptors of the Ghrelin family could not be activated by 200 μM LCAS.

[0200] Example 9 Activation of the Cell Line human GPR39-2 Stably Expressing the Human GPR39 Gene by Bile Acids

[0201] The human GPR39 cDNA vector was purchased from Weizhen Biotech in Shandong. After PCR amplification using the primers hGPR39-F (whose sequence is shown in SEQ ID NO:15) and hGPR39-R (whose sequence is shown in SEQ ID NO:16), it was cloned into the lentiviral vector PLVXK-hGPR39-P2A-mCherry( Figure 10 ), where the sequence of the target gene human GPR39 cDNA is shown in SEQ ID NO:14. According to the method described in Example 1, the cell line human GPR39-2 stably expressing the human GPR39 gene was constructed.

[0202] Detect the calcium imaging of LCAS, TLCAS, and GLCAS on the human GPR39-2 cell line, the half-maximal effective concentration (EC50) for activating the GPR39 receptor, and the regulatory effect of zinc ions on the activation of the human GPR39 receptor according to the methods of Example 2 and Example 3 above.

[0203] The detection results are shown in Table 7 below. Figure 9 As shown, the results indicate that LCAS, TLCAS, and GLCAS can all induce an intracellular calcium signal response in human GPR39-2, and have a concentration-dependent effect, and zinc ions also have a positive allosteric regulatory effect on the human GPR39 receptor.

[0204] Table 7

[0205]

[0206] The foregoing detailed description is provided by way of explanation and example, and is not intended to limit the scope of the appended claims. Various changes to the embodiments recited in the present application are obvious to those of ordinary skill in the art and are within the scope of the appended claims and their equivalents. Sequence Listing <110> Peking University <120> Use of Bile Acids and Their Derivatives in the Preparation of GPR39 Agonists <130> 0122-PA-020CN <160> 16 <170> PatentIn version 3.5 <210> 1 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> m39-H17A-F <400> 1 tcccgtgtca tcgatgccag cgctgttcct gaatttgag 39 <210> 2 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> m39-H17A-R <400> 2 atcgatgaca cgggagcaga tg 22 <210> 3 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> m39-E90A-F <400> 3 attggcatgc ccatggcgtt ctacagcatc atttg 35 <210> 4 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> m39-E90A-R <400> 4 catgggcatg ccaatcaaaa agacc 25 <210> 5 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> m39-F115A-F <400> 5 tccacacgtt cctcgctgag acgtgcagct ac 32 <210> 6 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> m39-F115A-R <400> 6 gaggaacgtg tggagcttac aggac 25 <210> 7 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> m39-E116A-F <400> 7 cacacgttcc tctttgcgac gtgcagctac gcc 33 <210> 8 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> m39-E116A-R <400> 8 aaagaggaac gtgtggagct tacag 25 <210> 9 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> m39-D330A-F <400> 9 tcctgccctt ctctgctacc ttcttctacc tc 32 <210> 10 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> m39-D330A-R <400> 10 agagaagggc aggaggatca tgtatg 26 <210> 11 <211> 1371 <212> DNA <213> Artificial Sequence <220> <223> Mouse GPR39 cDNA <400> 11 atggcttcat ccagtggctc caaccacatc tgctcccgtg tcatcgatca cagccatgtt 60 cctgaatttg aggtggccac ttggatcaaa atcaccctca tcttggtgta cctgatcatc 120 tttgtggtag gcatcttggg caacagcgtc accatcaggg ttacgcaggt attgcagaag 180 aagggctatt tgcagaagga ggtgacagat cacatggtca gtttggcttg ttcagatatc 240 ttggtctttt tgattggcat gcccatggag ttctacagca tcatttggaa ccccctgacc 300 acacccagct atgctctgtc ctgtaagctc cacacgttcc tctttgagac gtgcagctac 360 gccacactgc tgcacgtgct gaccctcagc tttgagcgct acattgccat ttgtcatccc 420 ttcaagtata aagcagtgtc tggacctcgc caggtgaaac tgctgattgg ctttgtatgg 480 gtcacctccg ccctggtggc actgcctttg ctctttgcca tgggtatcga gtaccctctg 540 gtaaacgtac ccactcacaa gggactcaac tgcaacctct ctcgcacccg ccaccacgat 600 gaacctggaa actccaatat gtccatctgc acgaacctct ccaaccgttg ggaggtcttc 660 cagtccagca tctttggggc ctttgctgtt tacctggtgg tcctggcgtc tgtggctttc 720 atgtgttgga atatgatgaa agtgctaatg aagagcaagc agggcactct tgcagggacc 780 gggccacagc tccagctgag gaagtcagag agtgaggaga gccggacagc aagaagacag 840 accatcatat tcctgagact gattgtggtg acgttggccg tgtgttggat gcccaatcag 900 atccgacgga tcatggctgc agcaaaaccc aaacatgact ggaccagaac gtacttcagg 960 gcatacatga tcctcctgcc cttctctgat accttcttct acctcagctc tgtggtcaac 1020 cctctcctct acaacgtgtc ctctcagcag ttccggaagg tgttctggca ggtgctctgc 1080 tgccgcctga ctctgcagca tgccaaccaa gagaaacgcc agcgtgcccg cttcatctcc 1140 accaaggaca gcaccagctc agcccgcagc cccctcatct tcctagcctc ccggcgcagt 1200 aactcttcct ccaggagaac taacaaggtt ttcttaagca cttttcagac tgaggccaag 1260 cctggagagg ctaagcccca gcccttgagt cctgagtcac cacagactgg ctcagagacc 1320 aaaccagctg ggtccacccc agaaaatagt ttacaggagc aggaagtatg a 1371 <210> 12 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> mGPR39-F <400> 12 atggcttcat ccagtggctc c 21 <210> 13 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> mGPR39-R <400> 13 tcatacttcc tgctcctgta aactattttc t 31 <210> 14 <211> 13,62 <212> DNA <213> Artificial Sequence <220> <223> Human GPR39 cDNA <400> 14 atggcttcac ccagcctccc gggcagtgac tgctcccaaa tcattgatca cagtcatgtc 60 cccgagtttg aggtggccac ctggatcaaa atcaccctta ttctggtgta cctgatcatc 120 ttcgtgatgg gccttctggg gaacagcgcc accattcggg tcacccaggt gctgcagaag 180 aaaggatact tgcagaagga ggtgacagac cacatggtga gtttggcttg ctcggacatc 240 ttggtgttcc tcatcggcat gcccatggag ttctacagca tcatctggaa tcccctgacc 300 acgtccagct acaccctgtc ctgcaagctg cacactttcc tcttcgaggc ctgcagctac 360 gctacgctgc tgcacgtgct gacactcagc tttgagcgct acatcgccat ctgtcacccc 420 ttcaggtaca aggctgtgtc gggaccttgc caggtgaagc tgctgattgg cttcgtctgg 480 gtcacctccg ccctggtggc actgcccttg ctgtttgcca tgggtactga gtaccccctg 540 gtgaacgtgc ccagccaccg gggtctcact tgcaaccgct ccagcacccg ccaccacgag 600 cagcccgaga cctccaatat gtccatctgt accaacctct ccagccgctg gaccgtgttc 660 cagtccagca tcttcggcgc cttcgtggtc tacctcgtgg tcctgctctc cgtagccttc 720 atgtgctgga acatgatgca ggtgctcatg aaaagccaga agggctcgct ggccgggggc 780 acgcggcctc cgcagctgag gaagtccgag agcgaagaga gcaggaccgc caggaggcag 840 accatcatct tcctgaggct gattgttgtg acattggccg tatgctggat gcccaaccag 900 attcggagga tcatggctgc ggccaaaccc aagcacgact ggacgaggtc ctacttccgg 960 gcgtacatga tcctcctccc cttctcggag acgtttttct acctcagctc ggtcatcaac 1020 ccgctcctgt acacggtgtc ctcgcagcag tttcggcggg tgttcgtgca ggtgctgtgc 1080 tgccgcctgt cgctgcagca cgccaaccac gagaagcgcc tgcgcgtaca tgcgcactcc 1140 accaccgaca gcgcccgctt tgtgcagcgc ccgttgctct tcgcgtcccg gcgccagtcc 1200 tctgcaagga gaactgagaa gattttctta agcacttttc agagcgaggc cgagccccag 1260 tctaagtccc agtcattgag tctcgagtca ctagagccca actcaggcgc gaaaccagcc 1320 aattctgctg cagagaatgg ttttcaggag catgaagttt ga 1362 <210> 15 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> hGPR39-F <400> 15 atggcttcac ccagcctcc 19 <210> 16 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> hGPR39-R <400> 16 tcaaacttca tgctcctgaa aaccattctc 30

Claims

1. Use of a bile acid derivative in the preparation of a medicament for preventing, treating and / or alleviating a disease or disorder associated with GPR39 activity, said bile acid derivative being selected from the group consisting of LCAS, TLCAS and GLCAS, wherein, The CAS number of LCAS is 64936-81-8, the CAS number of TLCAS is 64936-83-0, the CAS number of GLCAS is 64936-82-9, and the disease or disorder related to GPR39 activity is a gastrointestinal disease.

2. The use according to claim 1, wherein the drug further comprises zinc ions.

3. The use according to claim 2, wherein the concentration of the zinc ions is 10 nM - 10 μM.

4. The use according to claim 1, wherein the GPR39 is human GPR39 or mouse GPR39.

5. Use of a bile acid derivative in the preparation of a medicament for preventing, treating and / or alleviating a disease or disorder associated with GPR39 activity, said bile acid derivative being selected from the group consisting of UDCAS, TCDCAS and GCDCAS, wherein, The CAS number of UDCAS is 68780-73-4, the CAS number of TCDCAS is 67030-59-5, the CAS number of GCDCAS is 66874-09-7, and the disease or disorder related to GPR39 activity is a gastrointestinal disease, the drug further comprises zinc ions, and the concentration of the zinc ions is 10 nM - 10 μM.

6. The use according to claim 5, wherein the GPR39 is human GPR39 or mouse GPR39.

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