GPR35 regulator flux screening model and construction method and application thereof

Abstract

The invention belongs to the technical field of drug screening, and particularly relates to a GPR35 regulator flux screening model and a construction method and application thereof. The GPR35 regulator flux screening model is a 293FT cell line for over-expressing GPR35, and the screening model is constructed and obtained through a dual-luciferase reporter gene. The model disclosed by the invention can be used for efficiently detecting candidate drugs with an inhibition function on GPR35, has the advantages of high sensitivity and high flux, can be used for quickly discovering and efficiently identifying a GPR35 regulator, and has a good application prospect in screening GPR35 agonists and inverse agonists. The method has important significance in evaluating compound regulation activity in the early stage of drug research and development, evaluating activity change after structure modification in a drug optimization process and the like, and can also be used for researching a signal transduction mechanism of GPR35.

Description

Technical Field

[0001] The present invention belongs to the technical field of drug screening, and particularly relates to a GPR35 modulator flux screening model and a construction method and application thereof. Background Art

[0002] G protein-coupled receptor 35 (GPR35) is a class A, rhodopsin-like orphan GPCR primarily expressed in colonic epithelial cells. It plays a key role in gastrointestinal diseases, cardiovascular diseases, cancer, and immune regulation, suggesting its potential therapeutic target for a variety of diseases. Recent studies have shown that GPR35 has high intrinsic activity and is highly correlated with the development and progression of colon cancer. The highly active GPR35-T108M mutant receptor, resulting from the single nucleotide polymorphism rs3749171 at its locus, significantly increases susceptibility to colitis and the risk of colorectal cancer.

[0003] Furthermore, dysregulation of the Hippo pathway is closely associated with a variety of tumors, including colorectal cancer. YAP / TAZ, as the main downstream effector molecules of the Hippo pathway, play an important role in colitis-related cancers and liver metastasis of colon cancer. Studies have found that GPR35 intrinsic activity promotes the malignant phenotype of human colon cancer cell lines by inducing abnormal activation of YAP / TAZ, and this process can be inhibited by inverse agonists, indicating that this signaling pathway plays an important regulatory role in tumorigenesis.

[0004] Currently, the screening methods for GPR35 modulators are usually based on the detection of second messengers such as Ca 2+ GPR35 is a highly regulated protein that is expressed in YAP and TAZ signaling pathways. It is a highly regulated protein that is expressed in YAP and TAZ signaling pathways. However, it is not widely used to screen for GPR35 signaling pathways. Summary of the Invention

[0005] The present invention aims to provide a GPR35 regulator flux screening model and its construction method and application.

[0006] To achieve the above purpose, the present invention adopts the following technical solutions:

[0007] A GPR35 modulator flux screening model was developed using a 293FT cell line overexpressing GPR35 and a dual-luciferase reporter gene construct to obtain the screening model.

[0008] The dual luciferase reporter gene is a firefly luciferase reporter gene driven by a TEAD4-binding promoter and a Renilla luciferase as an internal reference gene.

[0009] A method for constructing a GPR35 regulator flux screening model, a 293FT cell line overexpressing GPR35, and a screening model constructed by a dual-luciferase reporter gene detection method.

[0010] Furthermore, the screening model was obtained by transfecting 293FT cells with the GPR35 plasmid, the TEAD4 luciferase reporter gene (pGL4-TEAD4-luc) plasmid, and the Renilla luciferase as an internal reference gene (pRL-TK) plasmid.

[0011] Furthermore, 293FT cells were cultured in DMEM (Dulbecco's Modified Eagle's Medium) medium to a cell density of 30% (i.e., the ratio of the area occupied by the cells in the culture dish), and then culture medium containing TEAD4 luciferase reporter gene plasmid (pGL4-TEAD4-luc), Renilla luciferase internal reference plasmid (pRL-TK) and GPR35 plasmid was added to the culture system in sequence. After mixing, the transfection reagent was added and the cells were cultured again in DMEM medium for 12-24 hours to obtain a screening model.

[0012] The 293FT cell seeding density was 1.0×10 5 / mL; the final concentration of pGL4-TEAD4-luc plasmid in the system is 0.1-1.0μg / mL; the final concentration of pRL-TK plasmid transfected cells is 0.01-0.5μg / mL; the final concentration of GPR35 expression plasmid is 0.002-0.2μg / mL; the ratio of the total amount of plasmid and PEI transfection reagent in the system is 1:3-6.

[0013] An application of the GPR35 modulator flux screening model, wherein the model is used to screen GPR35 modulators.

[0014] The modulator is an agonist and / or inverse agonist of GPR35.

[0015] A method for screening GPR35 modulators comprises co-incubating the screening model with a substance to be detected, then adding a firefly luciferase substrate and a Renilla luciferase substrate to the system, and reacting for 3-15 seconds; after the reaction, detecting the substance to be detected based on its firefly luciferase activity, and screening to obtain a GPR35 modulator.

[0016] The detection wavelengths are: 560 nm for firefly luciferase and 530 nm for Renilla luciferase.

[0017] Furthermore, the screening model is co-incubated with the test substance, and then a firefly luciferase substrate and a Renilla luciferase substrate are added to the system and reacted for 3-15 seconds. After the reaction, the nature of the test substance is determined based on the firefly luciferase activity of the test substance. If the firefly luciferase activity of the test substance is significantly different from that of the solvent control (DMSO), it is a GPR35 modulator.

[0018] Among them, the firefly luciferase activity of the test substance is greater than that of the solvent control, which is a GPR35 agonist, and the firefly luciferase activity of the test substance is less than that of the solvent control, which is a GPR35 inverse agonist.

[0019] Further,

[0020] The firefly luciferase activity of the test substance is significantly different from that of the standard sample, that is, it is a significant GPR35 modulator; the firefly luciferase activity of the test substance is greater than that of the standard GPR35 agonist, taking GPR35 agonist 2 as an example, it is a GPR35 agonist with more significant activity, and the firefly luciferase activity of the test substance is less than that of the standard GPR35 inverse agonist, taking CID-2745687 as an example, it is a GPR35 inverse agonist with more significant activity.

[0021] The firefly luciferase activity is the ratio of the firefly luciferase value to the Renilla luciferase value of TEAD4 luciferase activity.

[0022] An application of the method is application of the method in screening GPR35 regulators.

[0023] A use of a compound, use of the compound as a GPR35 regulator, wherein the compound is T8531, T11019, T6249, T23101, T3062 or T2251.

[0024] The structural formulas of the compounds are:

[0025]

[0026] The advantages of the present invention are:

[0027] The present invention utilizes GPR35-overexpressing cells and a TEAD4 luciferase reporter gene to construct a model. The resulting model can be used to efficiently detect candidate drugs that have an inhibitory function on GPR35. It has the advantages of high sensitivity and high throughput, and can quickly discover and efficiently identify GPR35 modulators. It is of great significance for screening agonists and inverse agonists of GPR35, as well as for evaluating the regulatory activity of compounds in the early stages of drug development and evaluating activity changes after structural modification during drug optimization. It can also be used to study the signal transduction mechanism of GPR35. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 This is a graph showing changes in TEAD4 luciferase activity in 293FT cells transfected with different concentrations of GPR35 plasmids provided in an embodiment of the present invention.

[0029] Figure 2 This is a diagram for detecting the effects of existing GPR35 agonists, antagonists and inverse agonists provided in an embodiment of the present invention.

[0030] Figure 3 This is a diagram of the preliminary screening of 46 candidate compounds in the compound library provided in an embodiment of the present invention.

[0031] Figure 4 This is a diagram showing the effect of screening compounds with high selectivity for target proteins provided by an embodiment of the present invention.

[0032] Figure 5 This is a dose-dependent effect diagram of the effects of different drug action times and concentrations on TEAD4 luciferase activity provided by an embodiment of the present invention; wherein A is the drug response for 12 hours; B is the drug response for 24 hours. DETAILED DESCRIPTION

[0033] The present invention is further described below with reference to specific examples, which are intended to help those skilled in the art to more fully understand the present invention, but are not intended to limit the present invention in any way.

[0034] Example 1: Screening Model Optimization of Plasmid Transfection Concentration on 293FT Cells

[0035] First, 1.0 × 10 5293FT cells were seeded at a density of 100 ng / mL and incubated in DMEM medium supplemented with 10% fetal bovine serum in a CO2 incubator for 24 hours. Subsequently, GPR35 plasmids were added to the culture medium at varying concentrations (resulting in final concentrations of 100 ng / mL, 30 ng / mL, 10 ng / mL, 3 ng / mL, 1 ng / mL, 0.3 ng / mL, 0.1 ng / mL, 0.03 ng / mL, 0.01 ng / mL, 0.003 ng / mL, 0.001 ng / mL, and 0 ng / mL), along with a TEAD4 luciferase reporter plasmid (pGL4-TEAD4-luc) and a Renilla luciferase internal reference plasmid (pRL-TK). After gentle mixing, PEI transfection reagent was added at a 1:4 ratio of total plasmid to PEI, and the cells were mixed again before continuing to incubate. After 12 hours of transfection, the culture medium was discarded to create a screening model containing varying concentrations of GPR35.

[0036] Then, DMEM culture medium containing 10 μM GPR35 inverse agonist CID-2745687 was added to the above system, and a solvent control group (0.1% DMSO) was set up at the same time. After drug treatment for 24 hours, the culture medium was discarded, 100 μL dual luciferase reporter gene lysate was added, and lysis was carried out for 10 minutes. 5 μL lysate was added to a white 96-well plate and detected using a dual luciferase reporter gene self-luminescence detector. 25 μL each of firefly luciferase substrate and Renilla luciferase substrate were added sequentially, the reaction time was 5 seconds, and the fluorescence integration time was 5 seconds. TEAD4 luciferase activity was calculated by the ratio of firefly luciferase value to Renilla luciferase value. If the firefly luciferase activity of the test substance is significantly different from that of the solvent control (DMSO), it is a GPR35 modulator. If the firefly luciferase activity of the test substance is greater than that of the solvent control, it is a GPR35 agonist. If the firefly luciferase activity of the test substance is less than that of the solvent control, it is a GPR35 inverse agonist. (See Figure 1 ).

[0037] Depend on Figure 1 The experimental results showed that within a certain range, TEAD4 luciferase activity gradually increased with increasing GPR35 plasmid concentration. The GPR35 inverse agonist CID-2745687 significantly inhibited the GPR35-enhancing effect on TEAD4 luciferase activity. When the GPR35 plasmid concentration was 10 ng / mL, the TEAD4 luciferase activity response was the strongest, effectively reflecting the inhibitory effect of the inverse agonist CID-2745687.

[0038] Example 2: Detection of compound effects

[0039] First, 1.0 × 10 5 293FT cells were inoculated at a density of 100ng / mL and placed in a carbon dioxide incubator for 24 hours. Subsequently, GPR35 plasmid, TEAD4 luciferase reporter gene plasmid (pGL4-TEAD4-luc) and Renilla luciferase internal reference plasmid (pRL-TK) at a concentration of 10ng / mL were added to the culture medium. After gently mixing, PEI transfection reagent was added at a ratio of 1:4 to the total amount of plasmid and PEI in the system, and then placed in an incubator and continued to culture after mixing again. After transfection for 12 hours, the culture medium was discarded to obtain a screening model.

[0040] Then, DMEM medium containing 10 μM of different compounds was added to each well, and DMSO was set as a control. After 24 hours of drug treatment, the medium was discarded and 100 μL of dual luciferase reporter gene lysis buffer was added and lysed for 10 minutes. 5 μL of lysate was added to a white 96-well plate and detected using a dual luciferase reporter gene self-luminescence detector (see Figure 2 ).

[0041] The above-mentioned different compounds are all reported GPR35 agonists, antagonists and inverse agonists, purchased from Beijing Taoshu Biotechnology Co., Ltd., namely CID-2745687 (Cas No. 264233-05-8, Product No. T21874), ML314 (Cas No. 1448895-09-7, Product No. TQ0075), YE120 (Cas No. 383124-82-1, Product No. T23542), ML193 (Cas No. 713121-80-3, Product No. T22103), ML184 (Cas No. 794572-10-4, Product No. T8571), Tryphostin A25 (Cas No. 118409-58-8, Product No. T21622), ML145 (Cas No. 1164500-72-4, Product No. T12074), CID-1231538 (Cas No. 354126-20-8, Product No. T9162), GPR35 agonist 2 (Cas No. 494191-73-0, Product No. T23434), GPR35 agonist 3 (Cas No. 123021-85-2, Product No. T72755), Zaprinast (Cas No. 37762-06-4, Product No. T2129), Lithocholic acid (Cas No. 434-13-9, Product No. T2202), 5-HIAA (Cas No. 54-16-0, Product No. T4744), Pamoic acid acid (Cas No. 6640-22-8, Product No. T23120), Kynurenic acid (Cas No. 492-27-3, Product No. T65135), Compound 15 (Cas No. 2978694-22-1, Product No. T72414).

[0042] Depend on Figure 2Results from the experiment showed that addition of the well-characterized inverse agonists CID-2745687, ML145, and ML314 significantly reduced TEAD4 luciferase activity, demonstrating that the screening model effectively identifies GPR35 inverse agonists. Lithocholic acid, kynurenic acid, and 5-HIAA are potential endogenous ligands (the endogenous ligand and function of GPR35 are not yet clearly defined). In this model, 5-HIAA significantly reduced TEAD4 luciferase activity, suggesting that it has some GPR35 inverse agonist activity. Other compounds, all GPR35 agonists, did not exhibit significant effects in this screening model. However, GPR35 agonist 2, the most potent commercially available GPR35 agonist, significantly increased TEAD4 luciferase activity, demonstrating the potential of this screening model to identify highly potent GPR35 agonists.

[0043] Example 3: High-throughput screening of compound libraries

[0044] First, 1.0 × 10 5 293FT cells were inoculated at a density of 100 μg / mL and placed in a carbon dioxide incubator for 24 hours. Subsequently, GPR35 plasmid, TEAD4 luciferase reporter gene plasmid (pGL4-TEAD4-luc) and Renilla luciferase internal reference plasmid (pRL-TK) at a concentration of 10 ng / mL were added to the culture medium, and a group of empty transfection plasmids (10 ng / mL) was set as a control. After gently mixing, the ratio of the total amount of plasmid to PEI in the system was 1:4. PEI transfection reagent was added, and after mixing again, it was placed in an incubator and continued to be cultured. After transfection for 12 hours, the culture medium was discarded to obtain a screening model.

[0045] Fresh culture medium containing 10 μM of different candidate compounds was then added to each well. A positive control group (10 μM CID-2745687) and a solvent control group (0.1% DMSO) were also set up. After 24 hours of drug treatment, the culture medium was discarded and 100 μL of dual-luciferase reporter gene lysis buffer was added and lysed for 10 minutes. 5 μL of lysate was added to a white 96-well plate and detected using a dual-luciferase reporter gene luminescence detector (see Figure 3 ).

[0046] The above compounds were purchased from Beijing Taoshu Biotechnology Co., Ltd., namely T2869 (Cas No. 518-82-1, trade name Emodin), T9377 (Cas No. 2541792-70-3, trade name ASK1-IN-2), T20714 (Cas No. 138530-95-7, trade name Levolansoprazole), T1240 (Cas No. 78755-81-4, trade name Flumazenil), T12235 (Cas No. 307519-88-6, trade name NMDI14), T12432 (Cas No. 2378173-15-8, trade name PF-06928215), T417 0 (Cas No. 94164-88-2, trade name PKM2-IN-1), T6434 (Cas No. 883050-24-6, trade name CCG-50014), T28430 (Cas No. 200266-76-8, trade name PNU-107484A), TN6740 (Cas No. 491-60-1, trade name Emodinanthrone), T67776 (Cas No. 2882035-56-3, trade name PCSK9-IN-11), T26397 (Cas No. 5539-66-2, trade name 6PGD-IN-S3), T0703 (Cas No. 3717-88-2, trade name Flavoxate hydrochloride), T1731 (Cas No. 1784-03-8, trade name TAME hydrochloride), T26632 (Cas No. 241809-12-1, trade name AnnH31), T13671 (Cas No. 1948273-03-7, trade name (S, R,S)-AHPC-Me hydrochloride), T2005 (Cas No. 1341200-45-0, trade name Dubermatinib), T20623 (Cas No. 79277-27-3, trade name Thifensulfuron-methyl), T1098 (Cas No. 3734-33-6, trade name Denatonium benzoate), T13747 (Cas No. 1800465-47-7, trade name KRN5), T10084 (Cas No. 1379445-54-1, trade name CRTh2 antagonist 1), T8957L (Cas No. 2308510-39-4, trade name PKD-IN-1dihydrochloride), T9632 (Cas No. 748146-89-6, trade name CDK9-IN-30), T20556L (Cas No. 1050-28-8, trade name Tyrosyltyrosine acetate), T67727 (Cas No. 2878360-80-4, trade name MCT1-IN-3), T77605 (Cas No. 550301-63-8, trade name WAY-323061), T40009 (Cas No. 2225940-48-5, trade name Pomalidomide-C4-COOH), T12232 (Cas No. 47439-36-1, trade name Name Nitroxazepine), T9816 (Cas No. 136145-07-8, trade name Arofylline), TQ0117 (Cas No. 1000279-69-5, trade name A-867744), T68147 (Cas No. 261944-52-9, trade name T-900607), T10659 (Cas No. 1402821-24-2, trade name Ca, 2+channel agonist 1), T13424 (Cas No. 1263273-14-8, trade name (1R,2S)-VU0155041), T8482 (Cas No. 1442472-39-0, trade name Ripretinib), T2061 (Cas No. 1232221-74-7, trade name APY0201), T7890 (Cas No. 1448440-52-5, trade name SP-13786), T7667 (Cas No. 449811-92-1, trade name R1487), TQ0219 (Cas No. 1001917-37-8, trade name MK-8033) , CID2745687, T8531 (Cas No. 200933-14-8, trade name m-3M3FBS), T11019 (Cas No. 1800296-63-2, trade name DHODH-IN-1), T6249 (Cas No. 1146699-66-2, trade name Avagacestat), T23101 (Cas No. 313981-55-4, trade name o-3M3FBS), T3062 (Cas No. 838818-26-1, trade name WIKI4), T2251 (Cas No. 761439-42-3, trade name NVP-TAE 684).

[0047] Depend on Figure 3 ,The experimental results showed that the TEAD4 luciferase activity of compounds TQ0219, T8531, T11019, T6249, T23101, T3062, and T2251 was better than that of the positive control CID-2745687.

[0048] Example 4: Screening of highly selective GPR35 inverse agonists

[0049] First, set up two 96-well plates and add 1.0 × 10 5 293FT cells were seeded at a density of 100 μg / mL and cultured in a CO2 incubator for 24 hours. Subsequently, two culture media containing different plasmids were prepared. The TEAD4 luciferase reporter gene plasmid (pGL4-TEAD4-luc) and the Renilla luciferase internal reference plasmid (pRL-TK) were added to the culture medium. The GPR35 plasmid and the empty vector plasmid were added at a concentration of 10 ng / mL, respectively. After gentle mixing, PEI transfection reagent was added, mixed again, and the cells were placed in the incubator for continued culture. After 12 hours of transfection, the culture medium was discarded to obtain the screening model.

[0050] Fresh culture medium containing 10 μM candidate compounds (TQ0219, T6249, T3062) was then added to each well. A positive control group (10 μM CID-2745687) and a solvent control group (0.1% DMSO) were also set up. After 24 hours of drug treatment, the culture medium was discarded and 100 μL of lysis buffer was added for lysis. The luciferase reporter gene autoluminescence detector was used for detection (see Figure 4 ).

[0051] Depend on Figure 4 Experimental results showed that compounds TQ0219, T6249, and T3062 have high selectivity for the target protein GPR35.

[0052] Example 5: Plotting drug IC50 curves and evaluating compound time-dependence and concentration-dependence

[0053] First, set up two 96-well plates and add 1.0 × 10 5 293FT cells were seeded at a density of 100 μg / mL and cultured in a CO2 incubator for 24 hours. Subsequently, two culture media containing different plasmids were prepared. The TEAD4 luciferase reporter gene plasmid (pGL4-TEAD4-luc) and the Renilla luciferase internal reference plasmid (pRL-TK) were added to the culture medium. The GPR35 plasmid and the empty vector plasmid were added at a concentration of 10 ng / mL, respectively. After gentle mixing, PEI transfection reagent was added, mixed again, and the cells were placed in the incubator for continued culture. After 12 hours of transfection, the culture medium was discarded to obtain the screening model.

[0054] Fresh culture medium containing different concentrations of candidate compounds (i.e., T3062, T6249) was then added. The concentration of each compound was set to 10 μM, 3 μM, 1 μM, 0.3 μM, 0.1 μM, 0.03 μM, 0.01 μM, 0.003 μM, 0.001 μM, and 0 μM. A positive control group (CID-2745687) and a solvent control group (0.1% DMSO) were also set up. After 12 hours and 24 hours of drug treatment, the culture medium was discarded and 100 μL of lysis buffer was added for lysis. The cells were detected using a dual-luciferase reporter gene luminescence detector, and the data were processed using the formula (WT treatment group - empty treatment group) / (WT DMSO group - empty DMSO group) * 100% (see Figure 5 ).

[0055] Depend on Figure 5 The experimental results showed that after the compounds interacted with the target protein GPR35 for 12 hours, the IC50 of the compounds were: T3062 IC 50 :0.02151μΜ, IC of T6249 50:5.047μΜ, IC of CID-2745687 50 :0.04506μΜ; After the compound was treated with the target protein GPR35 for 24 hours, the IC50 values ​​of the compounds were: IC 50 :0.007966μΜ, C of T6249 50 : 7.031μM, C of CID-2745687 50 Comparison of the inhibitory effects at different exposure times showed that T3062 had a significant inhibitory effect on the target protein GPR35, and its inhibitory effect was superior to that of the positive control CID-2745687. The effect was even better when the drug was exposed to the target protein GPR35 for 24 hours.

Claims

1. A GPR35 modulator flux screening model, characterized in that: The 293FT cell line overexpressing GPR35 was used, and a screening model was obtained by constructing a dual-luciferase reporter gene.

2. The GPR35 modulator flux screening model according to claim 1, characterized in that: The dual luciferase reporter gene is a firefly luciferase reporter gene driven by a TEAD4-binding promoter and a Renilla luciferase as an internal reference gene.

3. A method for constructing a GPR35 modulator flux screening model according to claim 1, characterized in that: A 293FT cell line overexpressing GPR35 was constructed and a screening model was obtained using a dual-luciferase reporter gene assay.

4. The method for constructing a GPR35 modulator flux screening model according to claim 3, wherein: The screening model was obtained by transfecting 293FT cells with GPR35 plasmid, TEAD4 luciferase reporter gene (pGL4-TEAD4-luc) plasmid, and Renilla luciferase as an internal reference gene (pRL-TK) plasmid.

5. The method for constructing a GPR35 modulator flux screening model according to claim 4, characterized in that: 293FT cells were cultured in DMEM medium to a cell density of 30%, and then culture medium containing TEAD4 luciferase reporter gene plasmid (pGL4-TEAD4-luc), Renilla luciferase internal reference plasmid (pRL-TK) and GPR35 plasmid was added to the culture system in sequence. After mixing, the transfection reagent was added and the cells were cultured again in DMEM medium for 12-24 hours to obtain a screening model.

6. A use of the GPR35 modulator flux screening model according to claim 1, characterized in that: Application of the model in screening GPR35 modulators.

7. The use of the GPR35 modulator flux screening model according to claim 1, characterized in that: The modulator is an agonist and / or inverse agonist of GPR35.

8. A method for screening a GPR35 modulator, characterized in that: The screening model according to claim 1 is co-incubated with the substance to be detected, and then a firefly luciferase substrate and a Renilla luciferase substrate are added to the system and reacted for 3-15 seconds; after the reaction, the property of the substance to be detected is determined according to the firefly luciferase activity of the substance to be detected, and a GPR35 modulator is screened.

9. An application of the method according to claim 8, characterized in that: The method is used in screening GPR35 regulators.

10. A use of a compound, characterized in that: The invention relates to the use of a compound as a GPR35 regulator, wherein the compound is T8531, T11019, T6249, T23101, T3062, or T2251.

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