Cell screening model of unmarked membrane receptor GPR139 and application of cell screening model

Abstract

The invention relates to drug screening of diseases closely related to a GPR139 receptor, in particular to a cell screening model of an unmarked membrane receptor GPR139 and application of the cell screening model. The cell screening model of the unmarked membrane receptor GPR139 is a stably transfected cell strain CHO-K1-GPR139 with high expression of the GPR139 receptor. The invention also discloses application of the cell screening model in screening drugs for diseases closely related to the GPR139 receptor through an unmarked cell integration pharmacology technology. When the GPR139 cell screening model constructed by the invention is used for screening, fluorescence labeling is not needed, an indicator does not need to be additionally added in the detection process, and the GPR139 cell screening model has the characteristics of target path integration response, no damage to cells, reliable detection result, high sensitivity, high screening flux, short period, simplicity and convenience in operation and the like. The method is used for searching high-activity ligands of the GPR139 receptor from a natural product library, a metabolite library and a combinatorial chemistry library, and provides guidance for drug screening of diseases closely related to the GPR139 receptor. The GPR139 receptor agonist compound A is obtained by screening through a GPR139 cell screening model established in the invention.

Description

Technical Field

[0001] This invention relates to drug screening for diseases closely related to the GPR139 receptor, specifically to a cell screening model for the label-free membrane receptor GPR139 and its application. Background Technology

[0002] G protein-coupled receptors (GPCRs) are seven-transmembrane receptors and are among the most important targets in drug development. Currently, over 30% of marketed drugs target GPCRs. However, to date, these developed GPCRs represent only a small fraction of all known GPCRs, indicating a vast untapped potential for drug discovery in this field. GPR139 is an orphan GPCR expressed in the central nervous system, highly expressed in the thalamus, amygdala, and spinal cord. It has been shown to play a crucial role in motor regulation and food intake, demonstrating its significant importance in the nervous system. Furthermore, it exhibits a high correlation with the expression of μ-opioid receptors and dopamine receptors (D2Rs) and participates in fundamental life activities such as regulation and metabolism. Studies have shown that GPR139 is closely related to the occurrence and development of numerous central nervous system diseases, including schizophrenia, Parkinson's disease, and alcohol addiction. Currently, the small molecule agonist TAK-041 targeting GPR139 has entered Phase I clinical trials for the treatment of negative symptoms of schizophrenia. Simultaneously, studies have also found that GPR139 has a negative regulatory effect on μ-opioid receptor signaling, and GPR139 antagonists hold promise for improving the safety of opioid medications. Therefore, research on the ligand regulation and signal transduction molecular mechanisms of GPR139 has significant guiding implications for future basic and translational research. Thus, the search for and development of highly active GPR139 ligands is essential. Liu C, Bonaventure P, Lee G, Nepomuceno D, Kuei C, Wu J, Li Q, Joseph V, Sutton SW, Eckert W, Yao X, Yieh L, DvorakC, Carruthers N, Coate H, Yun S, Dugovic C, Harrington A, Lovenberg TW.GPR139,anOrphan Receptor Highly Enriched in the Habenula and Septum,Is Activated by the Essential Amino Acids L-Tryptophan and L-Phenylalanine.MolPharmacol.2015Nov;88(5):911-25;Gloriam DE, HB,Fredriksson R.Nine newhuman Rhodopsin family G-protein coupled receptors: identification, sequencecharacterisation and evolutionary relationship.Biochim Biophys Acta.2005Apr15;1722(3):235-46;Wang J,Zhu LY,Liu Q,Hentzer M,Smith GP,Wang MW.High-throughput screening ofantagonists for the orphan G-protein coupled receptorGPR139.Acta Pharmacol Sin.2015;36(7):874-878;

[0003] Yali Zhou,Henrik Daver,Boris Trapkov,Lijie Wu,Meng Wu,Kasper Patrick R Gentry,Kaiwen Liu,Marina Larionova,Junlin Liu,Na Chen,Hans David E Gloriam,Tian Hua,Zhi-Jie Liu.Molecular insights into ligand recognition and G protein coupling of the neuromodulatory orphanreceptor GPR139.Nature.2022,32(2):210-213.

[0004] Currently, high-throughput screening is commonly used for G protein-coupled receptor (GPCR) cell screening. Methods include traditional radioligand-receptor binding assays, GTPγS binding assays, cyclic adenosine monophosphate (cAMP) assays, calcium flux assays, reporter gene assays, receptor endocytosis assays, and β-arrestin recruitment assays. However, these methods have limitations. Most GPCR detection methods target only one signaling pathway and cannot simultaneously detect the activation of multiple pathways. Furthermore, they require the addition of fluorescent labels or additional indicators, making the process cumbersome, and the indicators can also have some impact on the cells.

[0005] Therefore, how to obtain label-free integrative pharmacology technology to construct a label-free high-throughput screening model for the GPR139 receptor is an important issue to greatly improve the efficiency of GPR139 ligand screening. It will be of great significance to elucidating the pharmacological and physiological functions of GPR139 and provide guidance for drug screening of diseases closely related to the GPR139 receptor (Thomsen W, Frazer J, Unett D. Functional assays for screening GPCR targets. Curr Opin Biotechnol. 2005, 16(6): 655-65; Mayr LM, Bojanic D. Novel trends in high-throughput screening. Curr Opin Pharmacol. 2009, 9(5): 580-8; Morse, M., Sun, H., Tran, E. et al. Label-free integrative pharmacology on-target of opioid ligands at the opioid receptor family. BMC Pharmacol Toxicol 2013, 14, 17). Summary of the Invention

[0006] The purpose of this invention is to provide a cell screening model for the label-free membrane receptor GPR139 and its application.

[0007] To achieve the above objectives, the technical solution of the present invention is as follows:

[0008] A cell screening model for the label-free membrane receptor GPR139, wherein the cell screening model is the stable cell line CHO-K1-GPR139 that highly expresses the GPR139 receptor.

[0009] The stable cell line CHO-K1-GPR139 was obtained by transfecting the GPR139 plasmid into CHO-K1 cells to obtain a cell line CHO-K1-GPR139 that highly expresses the GPR139 receptor. The ratio of GPR139 plasmid to transfection reagent was 1:3; the GPR139 plasmid was GPR139-pcDNA3.1(+) / zeo.

[0010] An application of the cell screening model of the label-free membrane receptor GPR139, and the application of the cell screening model in screening drugs for diseases closely related to the GPR139 receptor using label-free cell integration pharmacology technology.

[0011] The application of the cell screening model in screening drugs for the prevention and / or treatment of diseases by activating or antagonizing GPR139 receptors using label-free cell integration pharmacology techniques.

[0012] The disease in question is a central nervous system disorder. Specifically, it refers to a central nervous system disorder used to treat schizophrenia, Parkinson's disease, and alcohol addiction.

[0013] A method for screening drugs for diseases closely related to the GPR139 receptor, using the cell screening model and label-free cell integration pharmacology technology to screen for drugs for diseases closely related to the GPR139 receptor.

[0014] Based on label-free cell integrated pharmacology, using the stable GPR139-expressing cell line CHO-K1-GPR139, the agonist or antagonist activity of the test sample with the GPR139 receptor is determined by the similarity and specificity of the DMR signal spectrum of the test sample with the DMR characteristic signal spectra of standard known agonists and antagonists.

[0015] The similarity of the DMR characteristic signal spectrum of the agonist is that the DMR signal spectrum of the test sample or the agonist on CHO-K1-GPR139 cells is similar; the specificity is that the above DMR response signal can be antagonized by the GPR139 receptor antagonist in a dose-dependent manner.

[0016] The DMR characteristic signal spectrum of the antagonist is specific in that the test sample or antagonist can antagonize the DMR response signal of the GPR139 receptor agonist in a dose-dependent manner.

[0017] The known agonists and antagonists for the above standards are TC-O9311 for the GPR139 receptor agonist and NCRW0005F05 for the GPR139 antagonist.

[0018] The label-free cell integrated pharmacology technology utilizes a resonant waveguide grating (RWG) biosensor to convert the dynamic redistribution of intracellular components caused by drugs into a holistic, dynamic wavelength shift response signal. This signal is the response value (pm) of wavelength change, which is realized through an Epic optical biosensor 384 microplate.

[0019] The cell screening model for the GPR139 receptor involved seeding CHO-K1-GPR139 cells into 384-well microplates with optical biosensing capabilities at a cell-compatible density of 1.5 × 10⁻⁶ cells / well. 4 Cells per well, with a cell culture medium volume of 40 μL per well, and a cell culture time of 18–24 h after seeding.

[0020] For methods involving drugs with agonistic activity against the GPR139 receptor, the screening steps for the test samples, considering sensitivity, saturation, and specificity, are as follows:

[0021] (1) The GPR139 receptor agonist TC-O9311 dissolved in HBSS buffer salt was added to 384 microplates seeded with CHO-K1-GPR139 cells. The concentrations of the GPR139 receptor agonist TC-O9311 were 0.00003 μM, 0.00009 μM, 0.00028 μM, 0.00085 μM, 0.0025 μM, 0.008 μM, 0.023 μM, 0.07 μM, 0.21 μM, 0.62 μM, 1.85 μM, 5.56 μM, 16.67 μM, and 50 μM. Its DMR characteristic signal spectrum was detected.

[0022] (2) The GPR139 antagonist NCRW0005F05 dissolved in HBSS buffer salt was added to 384-well microplates seeded with CHO-K1-GPR139 cells. The concentrations of the GPR139 antagonist NCRW0005F05 were 0.00016μM, 0.00047μM, 0.0014μM, 0.0042μM, 0.013μM, 0.038μM, 0.11μM, 0.34μM, 1.03μM, 3.09μM, 9.26μM, 27.78μM, 83.33μM, and 250μM. Its DMR characteristic signal spectrum was detected.

[0023] (3) Add the lowest concentration of TC-O9311 corresponding to the highest response intensity to the 384-well plates of CHO-K1-GPR139 cells that were added to the GPR139 receptor agonist TC-O9311 and the GPR139 antagonist NCRW0005F05 in steps (1) and (2), respectively, and detect their DMR characteristic signal spectrum.

[0024] (4) All the obtained DMR characteristic signal spectra have a concentration-response dependence and are sensitive, saturated and specific.

[0025] The method for screening test samples with agonistic activity related to the GPR139 receptor is as follows:

[0026] (1) The GPR139 receptor agonist TC-O9311 dissolved in HBSS buffer salt was added to 384-well microplates seeded with CHO-K1-GPR139 cells. The concentrations of the GPR139 receptor agonist TC-O9311 were 50000 nM, 16666.67 nM, 5555.56 nM, 1851.85 nM, 617.28 nM, 205.76 nM, 68.59 nM, 22.86 nM, 7.62 nM, 2.54 nM, 0.85 nM, 0.28 nM, 0.09 nM, and 0.03 nM. Its DMR characteristic signal spectrum was detected.

[0027] (2) Samples with concentrations of 0.01 μM, 0.24 μM, 0.05 μM, 0.10 μM, 0.20 μM, 0.39 μM, 0.78 μM, 1.56 μM, 3.13 μM, 6.25 μM, 12.5 μM, 25 μM, 50 μM, and 100 μM were added to microplates inoculated with CHO-K1-GPR139 cells, and their DMR characteristic signal spectra were detected.

[0028] (3) Correlation analysis of the DMR feature signal spectrum in steps (1) and (2), if the DMR feature signal spectrum in step (2) has contour similarity with the DMR feature spectrum in (1);

[0029] (4) Add the GPR139 antagonist NCRW0005F05 (concentrations of 250μM, 90μM, 30μM, 10μM, 3.33μM, 1.11μM, 0.37μM, 0.12μM, 0.04μM, 0.01μM, 4.57nM, 1.53nM, 0.51nM, and 0.17nM) to microplates inoculated with CHO-K1-GPR139 cells, pretreat for 5–90 min, and add the same concentration (EC) as in step (2). 80 -EC 100 The DMR characteristic signal of the sample to be tested is detected. If the intensity of the DMR characteristic signal is lower than that of the DMR characteristic signal in step (2), the sample is determined to be an agonist of the GPR139 receptor.

[0030] For methods involving drugs with antagonistic activity against the GPR139 receptor, the screening steps for test samples with antagonistic activity are as follows:

[0031] (1) The test samples and TC-O9311 were added to microplates seeded with CHO-K1-GPR139 cells, respectively. The concentrations of the test samples were 0.01 μM, 0.24 μM, 0.05 μM, 0.10 μM, 0.20 μM, 0.39 μM, 0.78 μM, 1.56 μM, 3.13 μM, 6.25 μM, 12.5 μM, 25 μM, 50 μM, and 1 μM. DMR signal spectra were detected at TC-O9311 concentrations of 50000 nM, 16666.67 nM, 5555.56 nM, 1851.85 nM, 617.28 nM, 205.76 nM, 68.59 nM, 22.86 nM, 7.62 nM, 2.54 nM, 0.85 nM, 0.28 nM, 0.09 nM, and 0.03 nM.

[0032] (2) If the sample to be tested in step (1) does not cause a DMR signal spectrum, add TC-O9311 at the same concentration as in step (1) to the cell plate in which the sample to be tested was added in step (1), and detect the DMR characteristic signal spectrum; if this DMR characteristic signal is weaker than the signal of TC-O9311 in step (1), and its DMR characteristic signal spectrum is similar to the DMR spectrum outline of TC-O9311 in step (1), it can be determined that the sample to be tested is an antagonist of GPR139 receptor.

[0033] The rising phase is 1–30 min and the plateau phase is 30–90 min.

[0034] The method screened GPR139 receptor agonists to identify compound A, which has the following structural formula:

[0035]

[0036] The use of compound A in the preparation of a medicament for the prevention and / or treatment of diseases by activating GPR139 receptors, wherein the following compound A is used in the preparation of a medicament for the prevention and / or treatment of diseases by activating GPR139 receptors;

[0037]

[0038] The GPR139 receptor agonist is one or more active ingredients comprising compound A and pharmaceutically acceptable salts of compound A, and may also contain pharmaceutically acceptable carriers or excipients such as starch, sodium chloride, microcrystalline cellulose, sorbic acid, and / or mannitol. The composition can be administered via, but is not limited to, intravenous injection, oral administration, intramuscular administration, subcutaneous administration, or local injection. Its dosage form can be, but is not limited to, injection solutions, lyophilized powder for injection, injection microspheres, liposomes, tablets, capsules, aqueous solutions, powders, pastes, sprays, granules, soft capsules, pellets, gels, patches, and ointments, with injection solutions, lyophilized powders, tablets, and capsules being preferred.

[0039] The use of compound A of the present invention in the preparation of drugs for the prevention and / or treatment of diseases such as schizophrenia, Parkinson's disease and alcohol addiction, wherein compound A and one or more of the corresponding pharmaceutically acceptable salts are included.

[0040] The drug is an active ingredient consisting of one or more of compound A and its corresponding pharmaceutically acceptable salt, and may also contain a pharmaceutically acceptable carrier or excipient.

[0041] The GPR139 receptor agonist is an active ingredient consisting of one or more of compound A and pharmaceutically acceptable salts of compound A, and may also contain a pharmaceutically acceptable carrier or excipient.

[0042] The advantages and beneficial effects of this invention are as follows:

[0043] The label-free GPR139 cell model established in this invention can be used for high-throughput screening of commercial small molecule libraries, self-prepared natural product extracts, component or compound libraries and chemical modifiers to obtain highly active ligands of the GPR139 receptor.

[0044] In this invention, compound A, obtained by screening through a label-free GPR139 cell model, acts on the GPR139 receptor, which is a G protein-coupled receptor. The GPR139 receptor is closely related to the occurrence and development of many central nervous system diseases, such as schizophrenia, Parkinson's disease, and alcohol addiction. Based on the correlation between the target and the disease, the clinical application scope of this compound can be broadened.

[0045] By utilizing a novel label-free cell integrative pharmacology technique, a high-throughput screening cell model for the GPR139 receptor is provided for efficient and rapid screening of highly active ligands of the GPR139 receptor, providing guidance for research related to highly active GPR139 ligands and targeted therapy for related diseases. Attached Figure Description

[0046] Figure 1(A) DMR characteristics of different concentrations of TC-O9311 in CHO-K1-GPR139 cells

[0047] Signal spectrum;

[0048] (B) Concentration-response dependent curves of different concentrations of TC-O9311 on CHO-K1-GPR139 cells.

[0049] Figure 2 (A) CHO-K1-GPR139 cells were pretreated with different concentrations of TC-O9311 for 90 min, and then...

[0050] DMR signal spectrum of TC-O9311 at a constant concentration;

[0051] (B) Concentration-response dependent curves of DMR signal spectra of fixed concentration of TC-O9311 after CHO-K1-GPR139 cells were pretreated with different concentrations of TC-O9311 for 90 min.

[0052] Figure 3 Different concentrations of the GPR139 antagonist NCRW0005F05 in CHO-K1-GPR139 cells

[0053] DMR characteristic signal spectrum;

[0054] Figure 4 (A) DMR signal spectra of CHO-K1-GPR139 cells after pretreatment with different concentrations of the GPR139 antagonist NCRW0005F05 for 90 min, with a fixed concentration of TC-O9311.

[0055] (B) Concentration-response dependent curves of DMR signal spectra corresponding to a fixed concentration of TC-O9311 after CHO-K1-GPR139 cells were pretreated with different concentrations of the GPR139 antagonist NCRW0005F05 for 90 min.

[0056] Figure 5 : The activating, desensitizing, and antagonizing signal values ​​of compound A and its ligands on the GPR139 receptor in CHO-K1-GPR139 cells. Detailed Implementation

[0057] The present invention will now be further illustrated with examples. These examples are merely illustrative and not intended to limit the scope of the invention.

[0058] The novel label-free cell-integrated pharmacology technology used in this model is based on label-free resonant waveguide grating (RWG) biosensors. It transforms the dynamic mass redistribution of intracellular components caused by drugs into a holistic, dynamic wavelength shift response signal, called dynamic mass reset (DMR) signal. It features non-invasiveness, high spatiotemporal resolution, high sensitivity, high throughput, target-pathway integration research, simple operation, and short experimental cycle. The detection process does not require labeling or the addition of additional indicators, and more realistically responds to the effects of drugs at the whole level of living cells.

[0059] In the following examples, CHO-K1 cells were purchased from the Chinese Academy of Sciences Type Culture Collection Committee, catalog number SCSP-507. TC-O9311 (catalog number: 4255) was purchased from TOCRIS, and the GPR139 antagonist NCRW0005F05 (catalog number: A16184) was purchased from AdooQ Bioscience. Culture medium F12 (catalog number: C11765500BT), fetal bovine serum (FBS) (catalog number: 10099141), Zeocin (catalog number: R25001), Lipofectamine 3000 transfection reagent (catalog number: L3000015), balanced salt solution HBSS (catalog number: 14065-056), and HEPES (catalog number: 15630-080) were purchased from Gibco. The cell culture plates were Epic Optical Biosensor 384 microplates, purchased from Corning. The detection platform was Corning third-generation... The imaging system, purchased from Corning, detects the wavelength shift caused by dynamic mass resetting (DMR) of cells.

[0060] Example 1: The cell screening model was the stable cell line CHO-K1-GPR139, which highly expresses the GPR139 receptor.

[0061] Specifically, it is constructed as follows:

[0062] The CHO-K1-GPR139 ovarian cells from Chinese hamsters were derived from and constructed in our laboratory. Specific transfection methods are as follows:

[0063] 1) The GPR139 plasmid was transfected into CHO-K1 cells at a ratio of 1:3 (8 μg GPR139 plasmid, 1 μg / μL: transfection reagent, 24 μL). The GPR139 plasmid was GPR139-pcDNA3.1(+) / zeo, and the transfection reagent was Lipofectamine 3000 (Thermo). A cell line, CHO-K1-GPR139, expressing the GPR139 receptor was obtained.

[0064] 2) Eight hours after transfection, replace with 10 mL of fresh F12 complete culture medium (10% FBS).

[0065] 3) 24 h after transfection, replace with 12 mL of fresh F12 complete medium containing 400 μg / μL Zeocin.

[0066] 4) Culture the cells for 7-10 days, replacing 12 mL of fresh F12 complete medium containing 400 μg / μL Zeocin every 2-3 days to obtain a stable cell line CHO-K1-GPR139 that highly expresses the GPR139 receptor.

[0067] Example 2: DMR characteristic signal spectrum of GPR139 receptor agonist TC-O9311 in CHO-K1-GPR139 cells

[0068] CHO-K1-GPR139 cells in logarithmic growth phase were seeded in cell-compatible 384-well microplates using F12K culture medium. The seeding volume per well was 40 μL, and the seeding density was 1.5 × 10⁻⁶. 4 Incubate the seeded cells in a cell culture incubator for 20–24 hours until cell confluence reaches approximately 95%, then perform cell viability testing. Replace the cell culture medium in the microplate with Hank's balanced salt solution (HBSS, containing 20 mM HEPES), adding 30 μL to each well. After adding the solution, place the plate in an incubator. After equilibration on the imaging instrument for 90 min, the baseline was rescanned for 2 min. TC-O9311 was added to each well in a microplate at a volume of 10 μL, with concentrations of 50000 nM, 16666.67 nM, 5555.56 nM, 1851.85 nM, 617.28 nM, 205.76 nM, 68.59 nM, 22.86 nM, 7.62 nM, 2.54 nM, 0.85 nM, 0.28 nM, 0.09 nM, and 0.03 nM, repeated four times. The DMR signal was monitored in real time on an Epic instrument for 90 min. The EC50 of TC-O9311 was calculated based on the characteristic DMR response values ​​within 90 min after TC-O9311 treatment. 50 Values, results are shown below Figure 1 Experimental results showed that TC-O9311 activated the GPR139 receptor to produce a dose-dependent DMR signal response. The dose-response curves were monophasic "S"-shaped and all reached saturation, with the highest DMR response value reaching approximately 250 pm. Its EC50... 50 The value is 5.16±0.93μM.

[0069] Example 3: Desensitized DMR characteristic signal spectrum of CHO-K1-GPR139 cells

[0070] CHO-K1-GPR139 cells in logarithmic growth phase were seeded in cell-compatible 384-well microplates using F12K culture medium. The seeding volume per well was 40 μL, and the seeding density was 1.5 × 10⁻⁶. 4 Incubate the seeded cells in a cell culture incubator for 20–24 hours until cell confluence reaches approximately 95%, then perform cell viability testing. Replace the cell culture medium in the microplate with Hank's balanced salt solution (HBSS, containing 20 mM HEPES), adding 30 μL to each well. After adding the solution, place the plate in an incubator. The cells were equilibrated on an imaging instrument for 90 min. Different concentrations of TC-O9311 were added to microplates to pretreat CHO-K1-GPR139 cells for 90 min: 50000 nM, 16666.67 nM, 5555.56 nM, 1851.85 nM, 617.28 nM, 205.76 nM, 68.59 nM, 22.86 nM, 7.62 nM, 2.54 nM, 0.85 nM, 0.28 nM, 0.09 nM, and 0.03 nM, in quadruplicate. The baseline was rescanned for 2 min. A fixed concentration of 20 μM TC-O9311 was added to each well (10 μL), in quadruplicate. The cells were then placed on an Epic instrument for real-time DMR monitoring for 90 min. The IC50 was calculated based on the characteristic DMR response values ​​within 90 min after TC-O9311 treatment. 50 Values, results are shown below Figure 2 Experimental results showed that TC-O9311 dose-dependently desensitized GPR139 receptors, with a dose-response curve exhibiting a monophasic "S" shape and reaching saturation response in all cases. Its IC50 value... 50 The value was 7.86 ± 2.51 μM.

[0071] Example 4: DMR characteristic signal spectrum of GPR139 antagonist NCRW0005F05 in CHO-K1-GPR139 cells

[0072] CHO-K1-GPR139 cells in logarithmic growth phase were seeded in cell-compatible 384-well microplates using F12K culture medium. The seeding volume per well was 40 μL, and the seeding density was 1.5 × 10⁻⁶. 4 Incubate the seeded cells in a cell culture incubator for 20–24 hours until cell confluence reaches approximately 95%, then perform cell viability testing. Replace the cell culture medium in the microplate with Hank's balanced salt solution (HBSS, containing 20 mM HEPES), adding 30 μL to each well. After adding the solution, place the plate in an incubator. The plate was equilibrated on the imaging instrument for 90 min; the baseline was rescanned for 2 min, and different concentrations of the GPR139 antagonist NCRW0005F05 were added to the microplates. The volume added to each well was 10 μL, with concentrations of 250 μM, 90 μM, 30 μM, 10 μM, 3.33 μM, 1.11 μM, 0.37 μM, 0.12 μM, 0.04 μM, 0.01 μM, 4.57 nM, 1.53 nM, 0.51 nM, and 0.17 nM, in quadruplicate. The plate was placed on an Epic instrument to monitor the DMR signal in real time for 90 min. The results are shown in [Figure number missing]. Figure 3 Experimental results show that the DMR response signals of different concentrations of the GPR139 antagonist NCRW0005F05 are close to zero.

[0073] Example 5: Antagonistic DMR Characteristic Signal Spectrum of CHO-K1-GPR139 Cells

[0074] CHO-K1-GPR139 cells in logarithmic growth phase were seeded in cell-compatible 384-well microplates using F12K culture medium. The seeding volume per well was 40 μL, and the seeding density was 1.5 × 10⁻⁶. 4 Incubate the seeded cells in a cell culture incubator for 20–24 hours until cell confluence reaches approximately 95%, then perform cell viability testing. Replace the cell culture medium in the microplate with Hank's balanced salt solution (HBSS, containing 20 mM HEPES), adding 30 μL to each well. After adding the solution, place the plate in an incubator. Cells were equilibrated on an imaging instrument for 90 min. Different concentrations of the GPR139 antagonist NCRW0005F05 were added to microplates for 90 min pretreatment. Each well contained 10 μL of the antagonist at concentrations of 250 μM, 90 μM, 30 μM, 10 μM, 3.33 μM, 1.11 μM, 0.37 μM, 0.12 μM, 0.04 μM, 0.01 μM, 4.57 nM, 1.53 nM, 0.51 nM, and 0.17 nM, in quadruplicate. The baseline was rescanned for 2 min. Then, a fixed concentration of 20 μM TC-O9311 was added to each well (10 μL per well), in quadruplicate. The microplates were placed on an Epic instrument for real-time DMR monitoring for 90 min. The IC50 was calculated based on the characteristic DMR response values ​​within 90 min after TC-O9311 treatment. 50 Values, results are shown below Figure 4 Experimental results showed that the GPR139 antagonist NCRW0005F05 exhibited dose-dependent antagonism of the GPR139 receptor, with a monophasic "S"-shaped dose-response curve that reached saturation response. Its IC50 value was [missing information]. 50 The value is 1.88±0.89μM.

[0075] Example 6: Evaluation of GPR139 receptor agonist activity of compound A

[0076] CHO-K1-GPR139 cells in logarithmic growth phase were seeded in cell-compatible 384-well microplates using F12K culture medium. The seeding volume per well was 40 μL, and the seeding density was 1.5 × 10⁻⁶. 4 Incubate the seeded cells in a cell culture incubator for 20–24 hours until cell confluence reaches approximately 95%, then perform cell viability testing. Replace the cell culture medium in the microplate with Hank's balanced salt solution (HBSS, containing 20 mM HEPES), adding 30 μL to each well. After adding the solution, place the plate in an incubator. Cells were equilibrated on the imaging instrument for 90 min. Compound A (50 μM), GPR139 agonist TC-O9311 (20 μM), and the control group (HBSS buffer containing 0.1% DMSO) were added to microplates for 90 min pretreatment, repeated in quadruplicate. The baseline was rescanned for 2 min, and 10 μL of TC-O9311 at a fixed concentration of 20 μM was added to each well of the microplate, repeated in quadruplicate. The microplates were then placed on an Epic instrument for real-time monitoring of DMR signals for 90 min. The percentage of response signal was calculated based on the characteristic DMR response values ​​within 90 min after TC-O9311 treatment. Results are shown in […]. Figure 5 Experimental results showed that compound A exhibited DMR activating signal (57%) in CHO-K1-GPR139 cells and had a desensitizing effect on the DMR of the GPR139 receptor agonist TC-O9311 (55%), indicating that it has GPR139 receptor activating activity.

[0077] In summary, the GPR139 cell screening model constructed in this invention does not require fluorescent labeling or additional indicators during screening, and features target pathway integration response, no cell damage, reliable detection results, high sensitivity, high screening throughput, short cycle, and simple operation. It can be used to search for highly active ligands of the GPR139 receptor from natural product libraries, metabolite libraries, and combinatorial chemistry libraries, providing guidance for drug screening of diseases closely related to the GPR139 receptor. The GPR139 receptor agonist compound A was screened using the GPR139 cell screening model established in this invention. Compound A, screened using this model, can be used for the prevention and / or treatment of diseases treated by activating or antagonizing the GPR139 receptor, such as schizophrenia, Parkinson's disease, and alcohol addiction. Current research indicates that the GPR139 receptor is closely related to the occurrence and development of many central nervous system diseases such as schizophrenia, Parkinson's disease, and alcohol addiction, thus providing highly active new ligands with clearly defined targets for these diseases.

[0078] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A cell screening model for the label-free membrane receptor GPR139, characterized in that: The cell selection model was the stable cell line CHO-K1-GPR139, which highly expresses the GPR139 receptor.

2. The application of the cell screening model of the label-free membrane receptor GPR139 as described in claim 1, characterized in that: The application of the cell screening model in screening drugs for diseases closely related to the GPR139 receptor using label-free cell integration pharmacology.

3. The application of the cell screening model of the label-free membrane receptor GPR139 according to claim 2, characterized in that: The application of the cell screening model in screening drugs for the prevention and / or treatment of diseases by activating or antagonizing GPR139 receptors using label-free cell integration pharmacology techniques.

4. The application of the cell screening model for the label-free membrane receptor GPR139 according to claim 3, characterized in that: The disease in question is a central nervous system disease.

5. A method for screening drugs for diseases closely related to the GPR139 receptor, characterized in that: Using the cell screening model of the label-free membrane receptor GPR139 as described in claim 1, drugs for diseases closely related to the GPR139 receptor can be screened through label-free cell integration pharmacology technology.

6. The method for screening drugs for diseases closely related to the GPR139 receptor according to claim 5, characterized in that: Based on label-free cell integrative pharmacology, using the CHO-K1-GPR139 cell line stably expressing GPR139 as described in claim 1, the agonist or antagonist activity of the test sample with the GPR139 receptor is determined according to the similarity and specificity of the DMR signal spectrum of the test sample with the DMR characteristic signal spectra of standard known agonists and antagonists.

7. The method for screening drugs for diseases closely related to the GPR139 receptor according to claim 6, characterized in that: The agonist generates a DMR response signal in CHO-K1-GPR139 cells, and this DMR signal can be antagonized in a dose-dependent manner by a GPR139 receptor antagonist; the antagonist can inhibit the generation of DMR agonist signals in CHO-K1-GPR139 cells in a dose-dependent manner by the GPR139 receptor agonist.

8. The method for screening drugs for diseases closely related to the GPR139 receptor according to claim 6, characterized in that: The method screened GPR139 receptor agonists to identify compound A, which has the following structural formula:

9. The use of compound A in the preparation of a medicament for the prevention and / or treatment of diseases by activating the GPR139 receptor, characterized in that: The use of the following compound A in the preparation of medicaments for the prevention and / or treatment of diseases by activating GPR139 receptors;

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