URAT1 inhibitor screening method with dibromofluorescein as substrate and application of URAT1 inhibitor screening method

Abstract

The invention belongs to the field of URAT1 inhibitor screening, and discloses a URAT1 inhibitor screening method with dibromofluorescein as a substrate and application of the URAT1 inhibitor screening method. According to the invention, the dibromofluorescein is used as a substrate of a cell model, the color change of cells at the bottom of the 96-well plate can be observed by naked eyes, and the inhibition effect is intuitively presented, so that the screening efficiency is improved.

Description

Technical Field

[0001] The invention belongs to the field of URAT1 inhibitor screening, and particularly relates to a URAT1 inhibitor screening method using dibromofluorescein as a substrate and an application thereof. Background Art

[0002] Hyperuricemia is a metabolic disease caused by purine metabolism disorder in the body, with high serum urate concentration, which is defined as two blood uric acid levels exceeding 420 μmol / L (or 7 mg / dL) on different days. When serum uric acid concentration continues to be too high, urate crystals are deposited in soft tissues or joints, which then cause an inflammatory response and induce gout.

[0003] The excretion process of uric acid includes glomerular filtration, tubular secretion and reabsorption after secretion. Since more than 90% of uric acid will be reabsorbed back into the blood after glomerular filtration, and the reabsorption process is mainly mediated by urate transporter 1 (URAT1), inhibiting the reabsorption function of URAT1 can increase the excretion of uric acid, thereby reducing serum uric acid levels.

[0004] The URAT1 inhibitor benzbromarone was first launched in the 1970s, but due to severe liver toxicity, it was not approved by the FDA and was withdrawn from the European market in 2003. In December 2015, the second URAT1 inhibitor, lesinade, was approved by the FDA for marketing, but due to its potential to increase the risk of acute renal failure, it was subsequently labeled with a black box warning by the FDA and stopped selling in the US market in 2019. In January 2025, Hengrui Medicine's URAT1 inhibitor SHR4640 applied for listing in China. In addition, URAT1 inhibitors developed by several domestic companies have entered the clinical phase II and phase III research stages.

[0005] There is an urgent need to develop new URAT1 inhibitors, and the establishment of a universal in vitro screening method is crucial for the early evaluation of drug development. The existing fluorescence-based in vitro activity evaluation method of URAT1 inhibitors - using 6-carboxyfluorescein as the fluorescent substrate of URAT1, is safer, more economical and more convenient than the radioactive labeling method. However, this method requires the detection of fluorescent signals by an enzyme reader, and it is difficult to observe the inhibitory effect of the test compound with the naked eye. Summary of the invention

[0006] In order to overcome the above-mentioned shortcomings and deficiencies of the prior art, the primary purpose of the present invention is to provide a method for screening URAT1 inhibitors using dibromofluorescein as a substrate.

[0007] The method of the present invention can observe the inhibitory effect with naked eyes.

[0008] Another object of the present invention is to provide application of the above screening method in the in vitro activity evaluation of URAT1 inhibitors.

[0009] The purpose of the present invention is achieved through the following solutions:

[0010] A method for screening URAT1 inhibitors using dibromofluorescein as a substrate comprises the following steps:

[0011] (1) Control group: Add the fluorescent substrate to the culture medium in a container, inoculate the cell model for in vitro activity screening of URAT1 inhibitors to obtain a mixed solution, perform co-culture treatment, and remove the culture medium to obtain cells treated with the control group;

[0012] (2) Test group: Add the test compound and dibromofluorescein to the culture medium in a container, inoculate the cell model for in vitro activity screening of URAT1 inhibitors to obtain a mixed solution, perform co-culture treatment, and remove the culture medium to obtain cells treated with the test group;

[0013] (3) Observe the color of the bottom of the 96-well plate: If there is a significant difference in color between the control group and the test group, the test compound in step (2) is a URAT1 inhibitor, and the greater the color difference, the stronger the inhibitory effect; if there is no significant difference in color between the control group and the test group, the test compound in step (2) is not a URAT1 inhibitor, or it cannot inhibit URAT1 from taking up dibromofluorescein at this concentration, and it is necessary to increase the concentration of the test compound before performing steps (2) and (3).

[0014] The cell model in step (1) and step (2) is HEK293T cells stably expressing hURAT1 (HEK293T / hURAT1 cells).

[0015] The concentration of dibromofluorescein in the mixed solution of step (1) and step (2) is 50-5000 μmol / L, preferably 324.9 μmol / L.

[0016] The inoculation density of the cell model for in vitro activity screening of URAT1 inhibitors in step (1) and step (2) is 4×10 4 ~10×10 4 cells / well.

[0017] The temperature of the co-cultivation treatment in step (1) and step (2) is 25 to 37°C.

[0018] The co-cultivation treatment time of step (1) and step (2) is 15 to 90 minutes, preferably 60 minutes.

[0019] The test compound in step (2) is a compound that has been subjected to cytotoxicity testing, and the test compound is non-toxic to the cell model used for in vitro activity screening of the URAT1 inhibitor.

[0020] The concentration of the compound to be tested in the mixed solution of step (2) is 5 μmol / L to 1000 μmol / L.

[0021] Application of the above screening method in the in vitro activity evaluation of URAT1 inhibitors.

[0022] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0023] The present invention provides a URAT1 inhibitor screening method using dibromofluorescein as a substrate, which can visually present the inhibitory effect by observing the color change of cells at the bottom of a 96-well plate by naked eyes, thereby improving the screening efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The diagrams show the results of hURAT1 transport and kinetic experiments on dibromofluorescein (A: hURAT1 / HEK293T transport experimental results on dibromofluorescein; B: standard curve of amount of substance-fluorescence intensity of dibromofluorescein; C: time-effect curve of hURAT1 uptake of dibromofluorescein; D: saturation curve of hURAT1 uptake of dibromofluorescein with increasing dibromofluorescein concentration when the incubation time is 60 min).

[0025] Figure 2 This is the MTT experiment result of URAT1 inhibitors benzbromarone and lesinade.

[0026] Figure 3 This is a graph showing the inhibition of dibromofluorescein uptake by hURAT1 / HEK293T cells by URAT1 inhibitors benzbromarone and lesinade.

[0027] Figure 4 This image shows the inhibition of dibromofluorescein uptake by hURAT1 / HEK293T cells by URAT1 inhibitors benzbromarone and lesinade. DETAILED DESCRIPTION

[0028] The present invention is further described in detail below with reference to the examples and drawings, but the embodiments of the present invention are not limited thereto. If no specific conditions are specified in the examples, the experiment was carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments is not specified, they are all conventional products that can be purchased from the market.

[0029] Source of reagents:

[0030] DMEM high-glucose medium, fetal bovine serum, trypsin, penicillin / streptomycin were purchased from Gibco, sodium gluconate, potassium gluconate, calcium gluconate, magnesium sulfate, potassium dihydrogen phosphate, HEPES, D-glucose, and NaHCO3 were purchased from Sigma, and dibromofluorescein was purchased from MacLean.

[0031] HEK293T / hURAT1 cells: prepared by the method of Chinese patent publication CN 112877295 A.

[0032] HEK293T / hURAT1 cells and HEK293T / WT cells were grown adherently in DMEM high-glucose medium containing 10% fetal bovine serum and 1% penicillin / streptomycin.

[0033] Preparation of HBSS (Cl-free) solution: Weigh solid sodium gluconate (125 mmol / L), potassium gluconate (4.8 mmol / L), calcium gluconate (1.3 mmol / L), magnesium sulfate (1.2 mmol / L), and potassium dihydrogen phosphate (1.2 mmol / L) in a beaker, dissolve with distilled water, add HEPES (25 mmol / L) and D-glucose (5.6 mmol / L) to 1000 mL, then add NaHCO3 solution to adjust the pH to 7.3-7.4, filter through 0.22 μm nitrocellulose membrane for sterilization, and place in a 4°C refrigerator for use.

[0034] Example 1 Feasibility of dibromofluorescein as a specific transport substrate for URAT1

[0035] (1) HEK293T / hURAT1 cells and HEK293T / WT cells with a confluence greater than 80% were digested from 6-well plates using 0.25% trypsin to prepare 4×10 4 Cell suspension of cells / well was added to 96-well fluorescent plates at 200 μL per well and incubated in a 5% CO2, 37°C cell culture incubator for 48 hours. The control group and the test group were HEK293T / hURAT1 cells, and the blank group was HEK293T / WT cells, with 5 replicate wells in each group.

[0036] (2) After 48 h, remove the old culture medium in the 96-well plate and replace it with HBSS (without Cl - ) solution per well, and then add 200 μL of HBSS (without Cl - ) solution and placed in an incubator for incubation for 10 min.

[0037] (3) During the incubation period, the stock solution of dibromofluorescein (CAS: 596-03-2) was diluted with HBSS (without Cl -) solution was diluted to prepare a working solution with a concentration of 100 μmol / L for use. Benzbromarone was selected as the inhibitor in the drug-dosing group. The prepared benzbromarone stock solution was diluted with prepared HBSS (without Cl - ) solution was diluted to prepare a working solution with a concentration of 100 μmol / L for use. Control group: 100 μmol / L dibromofluorescein working solution was diluted to 50 μmol / L, 100 μL per well, a total of 600 μL. Blank group: 100 μL of prepared HBSS (without Cl - ) solution, a total of 600 μL. Test group: 300 μL of 100 μmol / L dibromofluorescein working solution + 300 μL of 100 μmol / L benzbromarone working solution, a total of 600 μL.

[0038] (4) After incubation in the incubator for 10 min, remove the HBSS (without Cl - ) solution, add the corresponding control group, test group, and blank group solutions, and incubate in the incubator for 1 hour. After 1 hour, aspirate the incubation liquid, and add 200 μL HBSS (without Cl) to each well of the control group, test group, and blank group. - ) solution for a total of 3 times. Finally, add 100 μL of 0.5N NaOH solution to each well and lyse for 30 min at room temperature. Oscillate in an ELISA reader for 10 min and read at an excitation wavelength of 495 nm and an emission wavelength of 530 nm. Figure 1 As shown in Figure A, in the control group using dibromofluorescein as the substrate, the fluorescence intensity was significantly weakened after the addition of benzbromarone, indicating that dibromofluorescein can serve as a specific transport substrate for URAT1.

[0039] Example 2 Determination of Kinetic Parameters of Dibromofluorescein

[0040] 1. Establish a standard curve

[0041] Using dibromofluorescein as substrate, set up a series of concentration gradients, and use the instrument to detect the fluorescence intensity in 0.5N NaOH solution. Plot the amount of dibromofluorescein as the abscissa and the fluorescence intensity as the ordinate to obtain the linear relationship between the fluorescence intensity and the concentration of dibromofluorescein, and establish a standard curve (such as Figure 1 Figure B in the figure).

[0042] 2. Choice of optimal reaction time

[0043] HEK293T / hURAT1 cells with a confluence greater than 80% were digested from a 6-well plate using 0.25% trypsin to prepare 4×10 4Cell suspension of 10 cells / well was added to each well of a 96-well fluorescent plate at 200 μL and incubated in a 5% CO2, 37°C cell culture incubator for 48 h. During the incubation period, the mother solution of dibromofluorescein was diluted with HBSS (without Cl - ) solutions were prepared into working solutions with concentrations of 25 μmol / L, 50 μmol / L, 100 μmol / L, 200 μmol / L, 400 μmol / L, and 800 μmol / L, respectively.

[0044] After incubation in the incubator for 10 min, remove the HBSS (without Cl - ) solution, add the prepared working solution of each concentration, 100 μL per well, set 5 replicate wells for each concentration, and place in the incubator for incubation time of 5 min, 10 min, 15 min, 30 min, 45 min, and 60 min. After the incubation, aspirate the incubation liquid in the well, and add HBSS (without Cl - ) solution 200 μL, a total of 3 times. Figure 1 Figure C in the figure is the time-effect curve of hURAT1 uptake of dibromofluorescein. As can be seen from the curve in the figure, the curve rises almost linearly within 0min-60min, so 60min is the optimal incubation time.

[0045] 3. Selection of optimal substrate concentration

[0046] At the optimal incubation time, the concentration-absorption rate curve was obtained by setting the concentration gradient. The specific operation was as follows: HEK293T / hURAT1 cells with a confluence of more than 80% were digested with 0.25% trypsin to prepare about 4×10 4 200 μL of cell suspension per well was added to a 96-well fluorescent plate and replaced with new HBSS (without Cl - ) solution and incubate in an incubator for 10 min. - ) solution, add 25μmol / L, 50μmol / L, 100μmol / L, 200μmol / L, 400μmol / L, 800μmol / L dibromofluorescein working solution, 100μL per well, set 5 replicates for each concentration, and place in the incubator for the optimal incubation time of 60min. After 1h, aspirate the incubation liquid and add HBSS (without Cl - ) solution, and then washed with 200 μL of 0.5 N NaOH solution for 3 times. After that, 100 μL of 0.5 N NaOH solution was added to each well, and the cells were lysed at room temperature for 30 min. The cells were shaken in a microplate reader for 10 min and the readings were read. The dose-effect curve (e.g., with the concentration of dibromofluorescein as the horizontal axis and the absorption rate of dibromofluorescein as the vertical axis) was plotted. Figure 1The Km value of dibromofluorescein was calculated to be 324.9 μmol / L.

[0047] Example 3 Methodology Verification

[0048] The optimal reaction time and incubation concentration of dibromofluorescein were obtained based on the time-effect curve and the dose-effect curve. This parameter can be used as the basis for the URAT1 inhibitor screening method. The URAT1 inhibitors benzbromarone and lesinade were used as positive compounds to verify this method.

[0049] 1. MTT assay

[0050] (1) HEK293T / hURAT1 cells with a confluence greater than 80% were digested from a 6-well plate using 0.25% trypsin to prepare 4×10 4 Cell suspension of cells / well was added to 96-well fluorescent plates at 200 μL per well. Test group, control group and zeroing group were set up respectively. HEK293T / hURAT1 cells were plated in the test group and control group, and no cells were added to the zeroing group. Five replicate wells were set up in each group. 200 μL per well was added to 96-well fluorescent plates and incubated in a 5% CO2, 37°C cell culture incubator for 48 hours.

[0051] After 48 h, the old culture medium in the well was removed and HBSS (without Cl - ) solution per well, and then add 200 μL of HBSS (without Cl - ) solution and placed in an incubator for incubation for 10 min.

[0052] (2) Preparation of the working solution of the test compound: Test group: dibromofluorescein and benzbromarone were mixed in equal volumes, so that the final concentration gradient of benzbromarone was 3.9 μmol / L, 7.8 μmol / L, 15.6 μmol / L, 31.3 μmol / L, 62.5 μmol / L, and 125.0 μmol / L. After mixing evenly, 100 μL of the mixed solution was added to each well; dibromofluorescein and lesinade were mixed in equal volumes, so that the final concentration gradient of lesinade was 31.3 μmol / L, 62.5 μmol / L, 125.0 μmol / L, 250.0 μmol / L, 500.0 μmol / L, and 1000.0 μmol / L. After mixing evenly, 100 μL of the mixed solution was added to each well; Control group: 100 μL of 324.9 μmol / L dibromofluorescein solution per well; Zero adjustment group: HBSS (without Cl - ) solution 100 μL per well.

[0053] After incubation in the incubator for 10 min, remove the HBSS (without Cl -) solution, add the prepared solutions of the corresponding test group, control group and zero adjustment group respectively, and then incubate in an incubator for 1 hour.

[0054] (3) After 1 hour, remove the incubation solution and add HBSS (without Cl) to each well. - ) solution for a total of 3 times. Use DMEM complete medium containing 2% fetal bovine serum to prepare MTT solution (5 mg / mL diluted to 0.5 mg / mL), and add 100 μL to each well. Place in an incubator and incubate for 4 hours. After incubation for 4 hours, carefully aspirate the MTT solution, add 200 μL DMSO to each well with a spray gun, shake in a microplate reader for 10 minutes, read at an ultraviolet wavelength of 490 nm, obtain the fluorescence intensity value of each group, and calculate the survival rate of the cells (survival rate (%) = (fluorescence intensity of the test group - fluorescence intensity of the zero-adjustment group) / (fluorescence intensity of the control group - fluorescence intensity of the zero-adjustment group) × 100%).

[0055] like Figure 2 As shown, when the concentration of benzbromarone was 125.0 μmol / L, the cell survival rate could still reach about 90%, indicating that the compound had no obvious toxicity to cells within 125.0 μmol / L; when the concentration of lesinade was 1000.0 μmol / L, the cell survival rate could still reach about 90%, indicating that the compound had no obvious toxicity to cells within 1000.0 μmol / L.

[0056] 2. Fluorescent substrate uptake experiment:

[0057] (1) HEK293T / hURAT1 cells with a coverage rate of about 80% were digested from the culture dish with 0.25% trypsin to prepare a cell suspension. 200 μL per well was added to a 96-well plate. A control group, a test group, and a blank group were set up. HEK293T / hURAT1 cells were plated in the control group and the test group, and no cells were added to the blank group. Five replicate wells were set up for each group. After 48 hours, new HBSS (without Cl) was replaced. - ) solution and incubate in an incubator for 10 min.

[0058] (2) During the incubation period, the working solution of the test compound was prepared. Test group: Dibromofluorescein and benzbromarone were mixed in equal volumes, so that the final concentration gradient of benzbromarone was 3.9μmol / L, 7.8μmol / L, 15.6μmol / L, 31.3μmol / L, 62.5μmol / L, 125.0μmol / L. After mixing evenly, 100μL of the mixed solution was added to each well; Dibromofluorescein and lesinade were mixed in equal volumes, so that the final concentration gradient of lesinade was 31.3μmol / L, 62.5μmol / L, 125.0μmol / L, 250.0μmol / L, 500.0μmol / L, 1000.0μmol / L. After mixing evenly, 100μL of the mixed solution was added to each well. Control group: 324.9μmol / L of dibromofluorescein without inhibitor was added, 100μL per well. Blank group: HBSS (without Cl - ) solution 100mL per well.

[0059] After incubation in the incubator for 10 min, remove the HBSS (without Cl - ) solution, and added the prepared solutions of the control group, test group, and blank group respectively, and incubated in the incubator for 1 hour.

[0060] (3) After 1 hour, remove the incubation liquid and add 200 μL HBSS (without Cl - ) solution for 3 times to remove the unabsorbed substrate. Before the fluorescence measurement, the inhibitory effect can be preliminarily evaluated by visually observing the color change of the substrate in the 96-well plate. Finally, 100 μL of 0.5N NaOH solution was added to each well, lysed at room temperature for 30 minutes, and after shaking in the microplate reader for 10 minutes, the microplate reader read the fluorescence intensity value of each group and calculated the inhibition rate (inhibition rate (%) = (fluorescence intensity of the control group - fluorescence intensity of the test group) / (fluorescence intensity of the control group - fluorescence intensity of the blank group) × 100%).

[0061] Figure 3 The inhibitory effects of URAT1 inhibitors benzbromarone and lesinade on dibromofluorescein uptake in hURAT1 / HEK293T cells are shown. Figure 3 The ordinate Uptake (% of control) represents the dibromofluorescein uptake of the test group relative to the control group, expressed as a percentage. The abscissa represents the six concentrations of benzbromarone / lesinade set for the test group in the uptake experiment. If the dibromofluorescein uptake of the test group relative to the control group gradually decreases with the increase of the concentration of the test group, it means that the inhibitor has an inhibitory effect on the uptake of dibromofluorescein by HEK293T / hURAT1 cells.

[0062] In the fluorescent substrate uptake experiment, the six concentrations of the benzbromarone test group were 3.9μmol / L, 7.8μmol / L, 15.6μmol / L, 31.3μmol / L, 62.5μmol / L, and 125.0μmol / L, from low to high. Figure 3 Calculated IC of benzbromarone 50 The values ​​were 19.64 μmol / L, corresponding to Figure 4 It was observed that the color of the third concentration of 15.6 μmol / L in the benzbromarone test group was about half of the color of the control group. The six concentrations of the Resinade test group were 31.3 μmol / L, 62.5 μmol / L, 125.0 μmol / L, 250.0 μmol / L, 500.0 μmol / L, and 1000.0 μmol / L from low to high. Figure 3 Calculate the IC of Resinade 50 The values ​​were 539.80 μmol / L, corresponding to Figure 4 It was observed that the color of the fourth concentration of 500.0 μmol / L in the Resinade test group was approximately half of that of the control group.

[0063] The inhibitory activity of benzbromarone and lecithin on URAT1 measured by the method using dibromofluorescein as a substrate was as follows: benzbromarone > lecithin, which was consistent with the order of the detection method using 6-carboxyfluorescein as a substrate (as shown in Table 1), indicating that the method of using dibromofluorescein as a substrate to evaluate the activity of URAT1 inhibitors in vitro is feasible.

[0064] Table 1 IC measured by fluorescence method using 6-carboxyfluorescein and dibromofluorescein as substrates 50 value

[0065]

[0066] As can be seen from the above examples, the URAT1 inhibitor screening method using dibromofluorescein as a substrate can effectively evaluate the activity of URAT1 inhibitors and can be used to screen active compounds targeting URAT1. Compared with the method using 6-carboxyfluorescein as a substrate, the relative activity of the inhibition of the method of the present invention is at the same order of magnitude, and has the advantage of intuitive visualization of the inhibitory effect.

[0067] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A method for screening URAT1 inhibitors using dibromofluorescein as a substrate, characterized in that: The following steps are involved: (1) Control group: Add the fluorescent substrate to the culture medium in a container, inoculate the cell model for in vitro activity screening of URAT1 inhibitors to obtain a mixed solution, perform co-culture treatment, and remove the culture medium to obtain cells treated with the control group; (2) Test group: Add the test compound and dibromofluorescein to the culture medium in a container, inoculate the cell model for in vitro activity screening of URAT1 inhibitors to obtain a mixed solution, perform co-culture treatment, and remove the culture medium to obtain cells treated with the test group; (3) Observe the color of the bottom of the 96-well plate: If there is a significant difference in color between the control group and the test group, the test compound in step (2) is a URAT1 inhibitor, and the greater the color difference, the stronger the inhibitory effect; if there is no significant difference in color between the control group and the test group, the test compound in step (2) is not a URAT1 inhibitor, or it cannot inhibit URAT1 from taking up dibromofluorescein at this concentration, and it is necessary to increase the concentration of the test compound before performing steps (2) and (3).

2. The method for screening URAT1 inhibitors using dibromofluorescein as a substrate according to claim 1, characterized in that: The cell models in step (1) and step (2) are HEK293T cells that stably express hURAT1.

3. The method for screening URAT1 inhibitors using dibromofluorescein as a substrate according to claim 1, characterized in that: The concentration of dibromofluorescein in the mixed solution of step (1) and step (2) is 50-5000 μmol / L, preferably 324.9 μmol / L.

4. The method for screening URAT1 inhibitors using dibromofluorescein as a substrate according to claim 1, characterized in that: The inoculation density of the cell model for in vitro activity screening of URAT1 inhibitors in step (1) and step (2) is 4×10 4 ~10×10 4 cells / well.

5. The method for screening URAT1 inhibitors using dibromofluorescein as a substrate according to claim 1, characterized in that: The temperature of the co-cultivation treatment in step (1) and step (2) is 25 to 37°C.

6. The method for screening URAT1 inhibitors using dibromofluorescein as a substrate according to claim 1, characterized in that: The co-cultivation treatment time of step (1) and step (2) is 15 to 90 minutes, preferably 60 minutes.

7. The method for screening URAT1 inhibitors using dibromofluorescein as a substrate according to claim 1, characterized in that: The test compound in step (2) is a compound that has been subjected to cytotoxicity testing, and the test compound is non-toxic to the cell model used for in vitro activity screening of the URAT1 inhibitor.

8. The method for screening URAT1 inhibitors using dibromofluorescein as a substrate according to claim 1, characterized in that: The concentration of the compound to be tested in the mixed solution of step (2) is 5 μmol / L to 1000 μmol / L.

9. Use of the screening method according to any one of claims 1 to 8 in the in vitro activity evaluation of URAT1 inhibitors.

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