Screening model of PROTAC protein degradation drug targeting FGFR4 (Fibroblast Growth Factor Receptor 4)

Abstract

The invention relates to the technical field of drug research and development, and discloses a screening model for high-throughput rapid screening of PROTAC protein degradation drugs. According to the model, a 96-well plate for cell culture is selected, the intensity of green fluorescence carried by an exogenous target protein is detected through a microplate reader, and the protein degradation activity of the PROTAC compound is rapidly screened. When the model is used for detecting the degradation activity of a PROTAC compound taking FGFR4 as a target spot, the method comprises the following steps: (1) inserting an FGFR4 gene into a Turbo GFP-Vector plasmid with a green fluorescent label; (2) instantaneously transfecting HEK293T cells for 24 hours; (3) adding a PROTAC compound to treat the cells for 24 hours; and (4) detecting a fluorescence value at the wave band of Ex482nm / Em502nm by a microplate reader so as to judge the activity of the PROATC compound for specifically degrading the expression of the exogenous FGFR4 protein. The 96-well plate for cell culture adopted by the model can achieve high-throughput live cell screening, and the exogenous target protein is expressed by using a transient transfection plasmid method, so that the method is more time-saving, simple, convenient and economic, and has strong repeatability.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of drug research and development, and particularly relates to a high-throughput screening model of a PROTAC protein degradation drug targeting FGFR4. BACKGROUND

[0002] Targeted Protein Degradation (TPD) is a technology that degrades target proteins by ubiquitination, and utilizes the cell's own protein degradation mechanism (e.g., proteasome) to complete the degradation of target proteins. Proteolysis targeting chimeras (PROTAC) is one of the major categories, which connects the ligand of E3 ligase and the binding ligand of the target protein (Protein Of Interest, POI) together through a linker to form a heterobifunctional molecule, i.e., "E3 ligase-linker-target protein". Once the PROTAC compound recognizes and anchors the E3 ligase and the target protein, the degradation mechanism is immediately started, which is the advantage of PROTAC in targeting and degrading target proteins. However, the screening of drugs, especially high-throughput screening of drugs, is a difficulty.

[0003] The screening of PROTAC protein degradation drugs currently mainly relies on western blot technology to directly detect the changes in the expression content of target proteins, which is very effective but cannot achieve the purpose of high-throughput screening. Native Mass Spectrometry (nMS) can detect the instantaneous binding of PROTAC compounds and target proteins through the principle of in vitro protein-protein interaction, achieving the purpose of high-throughput screening. However, this screening model requires protein purification and is relatively expensive. In addition, the in vitro screening model cannot reflect the recognition, binding, and ultimate degradation of PROTAC compounds and target proteins in the complex environment of cells. In addition, Promega Company has developed a detection technology based on the bioluminescent peptide tag HiBiT, which can achieve the purpose of high-throughput screening of PROTAC compounds. However, based on the demand for large-scale screening of PROTAC compounds, this model is obviously relatively expensive.

[0004] Therefore, it is particularly urgent to design and develop a high-throughput screening model of protein degradation drugs that is efficient, simple, economical, and highly reproducible. SUMMARY

[0005] The first technical purpose of the present application is to provide a screening model that can high-throughput and rapidly screen PROTAC protein degradation drugs.

[0006] The second technical objective of this invention is its use in the rapid screening of PROTAC compounds targeting FGFR4 using the screening model.

[0007] To solve the technical problem of this invention, the present invention provides the following technical solution:

[0008] A first aspect of the present invention provides a high-throughput rapid screening model for PROTAC protein degradation drugs, comprising the following steps:

[0009] 1) The target gene was inserted into a blank plasmid vector with a green fluorescent tag and then introduced into E. coli.

[0010] Amplification and extraction of plasmid DNA in Coli;

[0011] 2) Seed cells in 96-well plates for cell culture;

[0012] 3) Transfect plasmid transiently and incubate in a cell culture incubator for 24 hours;

[0013] 4) Set a concentration gradient according to the IC50 value of the PROTAC protein degradation drug, add it to the corresponding cell culture wells, and continue culturing for 24 hours;

[0014] 5) Detect the intensity of the green fluorescence signal using an enzyme-linked immunosorbent assay (ELISA) reader.

[0015] Preferably, the model is established based on the use of 96-well plates for cell culture with black walls / transparent bottoms, which can reduce the fluorescence background signal and increase the signal-to-noise ratio.

[0016] In a specific implementation, in step 1), when constructing the plasmid, try to select the longest mRNA (longest isoform) of the target gene, unless the target gene to be screened contains a mutation site, in which case the corresponding mutant plasmid needs to be constructed.

[0017] In a specific implementation, step 1) uses DH5α competent cells of Escherichia coli (E. coli).

[0018] In a specific implementation, in step 1), Qiagen's plasmid extraction kit (HiSpeed ​​PlasmidMaxi Kit (10), Cat#12662) is preferred, which can obtain high-purity plasmids suitable for transient transfection experiments.

[0019] In a specific implementation, in step 2), preferably one day before the transfection experiment, HEK293T cells are seeded at 0.32 x 10⁻⁶ cells per well in a 96-well cell culture plate with black walls and a clear bottom. 5 indivual.

[0020] In a specific embodiment, in step 3), Lipofectamine 2000 transfection reagent (Thermofisher, Cat#11668019) is used to perform transient plasmid transfection experiments. The preferred transfection ratio is 0.1 μg of plasmid DNA per well in a 96-well plate for cell culture: 0.48 μL of Lipofectamine 2000 transfection reagent.

[0021] In a specific implementation, in step 3), the empty vector plasmid TurboGFP-Vctor must be used as a control group, transfected in parallel, and the drug concentration must be consistent with that of the model group, with each concentration dose repeated in three wells.

[0022] In a specific implementation, in step 3), since HEK293T cells do not adhere well, the drug needs to be added extremely slowly to avoid the cells leaving the bottom of the culture plate, causing cell death and affecting the experimental results.

[0023] In a specific implementation, in step 4), the IC50 value of the PROTAC compound to be tested is determined through preliminary experiments, and the concentration gradient is extended to both sides with the IC50 value as the midpoint.

[0024] In a specific implementation, in step 4), the PROTAC compound screening concentration is preferably set at a maximum initial concentration of 5 μM, and then diluted 10-fold to a minimum concentration of 0.5 nM (compound #0334) or 5 nM (compound #0541).

[0025] In a specific implementation, in step 5), the fluorescence intensity is detected first, and then the cell viability is detected.

[0026] In a specific implementation, in step 5), the CCK8 reagent is preferably used to detect cell viability.

[0027] Another aspect of the technical solution of the present invention is its use in rapidly screening PROTAC compounds targeting FGFR4 using the screening model described in the first aspect.

[0028] Beneficial effects

[0029] This invention constructs a high-throughput rapid screening model for PROTAC protein degradation drugs. By inserting the target gene into a fusion expression vector tagged with TurboGFP, the target protein is expressed exogenously at high levels in HEK293T cells. Once the target PROTAC compound recognizes and degrades the exogenously expressed target protein, a decrease in its green fluorescence intensity can be detected using a microplate reader. Because this model is based on 96-well plates for cell culture, it achieves high-throughput screening. Furthermore, by observing the decrease in the green fluorescence intensity of the target protein, the formation of the "E3 ligase-PROTAC-target protein" ternary complex and the subsequent target protein-specific degradation can be determined, achieving rapid and high-throughput screening results that cannot be obtained using Western blot techniques.

[0030] Furthermore, the present invention can also screen for the optimal concentration and time for the degradation of target proteins by PROTAC compounds.

[0031] In addition, for the four members of the FGFR family, FGFR1, FGFR2, FGFR3, and FGFR4, the degradation specificity of PROTAC compounds on each member of the FGFR family can be identified by constructing plasmids with TurboGFP tags.

[0032] Furthermore, the core technologies of the high-throughput screening model for PROTAC compounds constructed in this invention—plasmid construction and transient transfection—are fundamental techniques in molecular biology. They are simple, easy to operate, and highly reproducible. The required reagents and consumables are more economical than commercial kits (e.g., Promega). Especially when large-scale screening of PROTAC compounds is required, this invention offers higher cost-effectiveness. Attached Figure Description

[0033] Figure 1 Western blot results of transient transfection of TurboGFP-Vector (blank control) plasmid DNA and TurboGFP-FGFR4 (target protein) plasmid DNA into HEK293T cells.

[0034] Figure 2 The ratio of green fluorescence intensity after culturing HEK293T cells transiently transfected with TurboGFP-FGFR4 for 24 hours and then treating them with different concentrations of PROTAC compound #0334 for 24 hours is shown in the figure. The final concentrations of the detection points shown in the figure are 5 uM, 0.5 uM, 50 nM, 5 nM, and 0.5 nM.

[0035] Figure 3Human gastric cancer cells KATOIII were treated with PROTAC compound #0334 at final concentrations of 10 μM, 1 μM, and 0.5 nM for 24 hours, and the expression level of endogenous FGFR4 protein was detected by Western blot.

[0036] Figure 4 The ratio of green fluorescence intensity after culturing HEK293T cells transiently transfected with TurboGFP-FGFR4 for 24 hours and then treating them with different concentrations of PROTAC compound #0541 for 24 hours is shown in the figure. The final concentrations at the detection points shown in the figure are 5 μM, 0.5 μM, 50 nM, and 5 nM. Detailed Implementation

[0037] The technical solution of the present invention will be described below through specific embodiments. The described embodiments are only some, not all, of the embodiments of the present invention.

[0038] Experimental materials:

[0039] Cell lines: human embryonic kidney cells HEK293T (Pronosai, Cat#CL-0005), human gastric cancer cells KATO III (Pronosai, Cat#CL-0732).

[0040] Cell culture media: DMEM medium (Thermofisher, Cat#11995065), KATO III-specific medium (Pronosai, Cat#CM-0732), OPTI-MEM medium (Thermofisher, Cat#31985070), fetal bovine serum (Avantor, Cat#76294-180), penicillin-streptomycin (Thermofisher, Cat#15140148), PBS for cell culture (Macklin, Cat#P917808), and trypsin (Thermofisher, Cat#25200056).

[0041] Cell culture plates for selection: 96-well plates with black walls and clear bottoms (Thermofisher, Cat#165305).

[0042] Plasmid construction: TurboGFP-Vector(OriGene,Cat#PS100010), TurboGFP-FGFR4(OriGene,Cat#RG204230).

[0043] Plasmid extraction kit: Qiagen's plasmid maxi kit (HiSpeed ​​Plasmid Maxi Kit (10), Cat#12662).

[0044] Transfection reagent: Lipofectamin 2000 (Thermofisher, Cat#11668019).

[0045] PROTAC compounds #0334 and #0541 were provided by the Institute of Materia Medica, Chinese Academy of Medical Sciences.

[0046] Example 1:

[0047] Large-scale amplification of TurboGFP-Vector and TurboGFP-FGFR4 plasmids was performed.

[0048] Remove 50 μL of competent E. coli DH5α cells from -80°C, thaw on ice, add 1 μL of plasmid with a concentration of 100 ng / μL, gently mix, and place on ice for 30 minutes.

[0049] Heat in a 37°C water bath for 1 minute, then immediately place on ice for 2 minutes. Add 1 ml of LB blank culture medium (without antibiotics) and mix well.

[0050] The bacteria were incubated at 37°C on a horizontal shaker at 200 rpm for 1 hour to allow them to recover their growth state and express the plasmid-encoded antibiotic resistance gene AMP. r .

[0051] Spread 100 μL of bacterial culture onto an LB agar plate containing 50 μg / ml ampicillin antibiotic, and incubate upside down at 37°C overnight.

[0052] The next day, the plates were removed, and single colonies with good growth were picked and inoculated into 5 ml of LB liquid medium containing 50 ug / ml ampicillin antibiotic. The culture was carried out at 37°C with a horizontal shaker at 200 rpm for 8 hours. The colonies were then transferred at a ratio of 1:100 to 200 ml of LB liquid medium containing 50 ug / ml ampicillin antibiotic and incubated overnight at 37°C with a horizontal shaker at 200 rpm.

[0053] The following day, plasmids were extracted according to the instructions of Qiagen's plasmid maxi kit (HiSpeed ​​Plasmid Maxi Kit (10), Cat#12662). The DNA concentration and purity were measured, and the plasmids were aliquoted and stored at -20°C to avoid repeated freeze-thaw cycles.

[0054] Example 2:

[0055] Transient plasmid transfection.

[0056] HEK293T cells were revived and passaged for two weeks before use.

[0057] One day before the transfection experiment, 0.32 x 10⁻⁶ cells were inoculated per well.5 HEK293T cells were seeded at a density in 96-well plates with black walls and clear bottoms and cultured overnight until transfection with plasmids the next day.

[0058] (The black-walled / clear-bottomed 96-well plates used for cell culture are suitable for fluorescence signal detection and increase the signal-to-noise ratio.)

[0059] On the day of the experiment, the plasmid and transfection reagent Lipofectamine 2000 were placed on ice. After the plasmid melted, the contents of the plasmid tube and the transfection reagent tube were gently tapped to homogenize them.

[0060] Prepare the mixture required for (n+0.5) samples using a ratio of 0.1 μg plasmid to 0.48 μL Lipofectamine 2000 per well. The specific steps are as follows: ① Dilute 0.1 μg plasmid DNA in 5 μL of OPTI-MEM medium; ② Dilute 0.48 μL Lipofectamine 2000 in 5 μL of OPTI-MEM medium; ③ Gently mix the diluted plasmid DNA and diluted Lipofectamine 2000 transfection reagent using a pipette and incubate at room temperature for 5 minutes.

[0061] To verify that the plasmid-to-transfection reagent transfection ratio used in this invention is optimal, HEK293T cells were transiently transfected at different transfection ratios in preliminary experiments. Western blot analysis was used to detect the expression level of the exogenous FGFR4 protein, ultimately determining the currently used transfection ratio. Figure 1 As shown, the transfection ratio used in this invention can obtain a large amount of exogenous FGFR4 protein expression, and the blank vector did not interfere with the expression of the target gene FGFR4.

[0062] Add the plasmid:transfection reagent mixture to the 96-well plate according to the calculated ratio per well, gently tap the plate wall, and return it to the cell culture incubator to continue culturing for 24 hours.

[0063] Compared to the TurboGFP-FGFR4 working group, a negative control group TurboGFP-Vector and a blank control group (HEK293T cells) were set up.

[0064] Example 3:

[0065] Concentration gradient screening of PROTAC compound #0334.

[0066] The test PROTAC compound #0334 was added to HEK293T cells containing the transient transfection plasmid TurboGFP-FGFR4 from Example 2 and treated for 24 hours.

[0067] To avoid cell death caused by excessively high concentrations of PROTAC compounds, which could affect the fluorescence signal intensity and final results of exogenously expressed FGFR4, the IC50 value of PROTAC compound #0334 was determined through previous cytotoxicity experiments.

[0068] The highest treatment concentration was set at 5 μM drug concentration. The drug was diluted 10 times and the final treatment concentrations were set as follows: 5 μM, 0.5 μM, 50 nM, 5 nM and 0.5 nM, a total of 5 concentration gradients. The blank control DMSO (0 nM) was used as a negative control. Each concentration was set up with 3 replicates.

[0069] Based on the experience gained in this project, the initial screening test time for PROTAC compounds should ideally be 24 hours, but is not limited to 24 hours. The drug detection time can be shortened as the activity of the PROTAC compound increases.

[0070] HEK293T cells transiently transfected with plasmid were treated with PROTAC compound for 24 hours, then removed from the cell culture incubator and placed in a microplate reader. The fluorescence detection wavelength of the microplate reader was set to Ex482 / Em502, and the fluorescence signal of the 96-well plate was read.

[0071] The results are as follows Figure 2 The fluorescence values ​​of exogenously expressed FGFR4 protein are shown in the figure. With the fluorescence value of the blank control DMSO (0 nM) set to 100%, the fluorescence value of PROTAC compound #0334 at the final concentration of 5 μM treatment dropped sharply to 55.56%, and it maintained good protein degradation activity at all five concentration gradients from 5 μM to 0.5 nM. This indicates that PROTAC compound #0334 has a strong ability to degrade FGFR4 protein.

[0072] To verify the success of the established model of screening PROTAC compounds by expressing exogenous FGFR4 protein and detecting its fluorescence intensity, the sensitive cell line KATO III was treated with PROTAC compound #0334, and the expression level of endogenous FGFR4 protein was detected by Western blot. The results were as follows: Figure 3 The results are shown. Under the selected drug treatment conditions of 10 μM, 1 μM, and 0.5 nM, with a treatment time of 24 hours, the expression level of endogenous FGFR4 protein was significantly degraded. This demonstrates the success of our established screening model.

[0073] Example 4:

[0074] Concentration gradient screening of PROTAC compound #0541.

[0075] The test PROTAC compound #0541 was added to HEK293T cells containing the transient transfection plasmid TurboGFP-FGFR4 from Example 2 and treated for 24 hours.

[0076] To avoid cell death caused by excessively high concentrations of PROTAC compounds, which could affect the fluorescence signal intensity and final results of exogenously expressed FGFR4, the IC50 value of PROTAC compound #0541 was determined through previous cytotoxicity experiments.

[0077] The highest treatment concentration was set at 5 μM drug concentration. The drug was diluted 10 times and the final treatment concentrations were set at 5 μM, 0.5 μM, 50 nM and 5 nM respectively, forming four concentration gradients. The blank control DMSO (0 nM) was used as a negative control. Three replicates were set for each concentration.

[0078] HEK293T cells transiently transfected with plasmid were treated with PROTAC compound for 24 hours, then removed from the cell culture incubator and placed in a microplate reader. The fluorescence detection wavelength of the microplate reader was set to Ex488 / Em502, and the fluorescence signal of the 96-well plate was read.

[0079] The results are as follows Figure 4 The fluorescence ratios of exogenously expressed FGFR4 protein are shown. With the fluorescence value of DMSO (0 nM) set to 100%, the fluorescence value of PROTAC compound #0541 at a final concentration of 5 μM decreased sharply to 48.19%. Furthermore, compared to PROTAC compound #0334 in Example 3, compound #0541 exhibits a significant dose-dependent degradation of exogenous FGFR4 protein, demonstrating that the established model can respectively exhibit the activity and properties of the tested PROTAC compounds in degrading the target protein.

[0080] In summary, this invention proposes a novel high-throughput rapid screening model for PROTAC protein degradation drugs, which has good screening effect and advantages such as high throughput, specificity, simple operation, strong reproducibility, and cost-effectiveness.

Claims

1. A high-throughput rapid screening model for PROTAC protein degradation drugs, characterized in that... The steps for establishing the screening model are as follows: (1) The target gene was inserted into a blank plasmid vector with a green fluorescent tag, and the plasmid DNA was amplified and extracted in Escherichia coli (E. coli). (2) Seed cells in 96-well plates for cell culture; (3) Transiently transfect plasmids and culture in a cell incubator for 24 hours; (4) Set a concentration gradient according to the IC50 value of the PROTAC protein degradation drug, add it to the corresponding cell culture wells, and continue culturing for 24 hours; (5) Use an enzyme-linked immunosorbent assay (ELISA) reader to detect the intensity of the green fluorescent signal.

2. The high-throughput screening model according to claim 1, wherein, The target gene is human fibroblast growth factor receptor FGFR4.

3. The high-throughput screening model according to claim 1, wherein, The green fluorescent tag blank plasmid vector used was TurboGFP-Vector.

4. The high-throughput screening model according to claim 1, wherein, Escherichia coli (E. coli) competent cells are DH5α.

5. The high-throughput screening model according to claim 1, wherein, The 96-well plates used for cell culture have black walls and a transparent bottom.

6. The high-throughput screening model according to claim 1, wherein, HEK293T cells were used for transient transfection of the plasmid.

7. The high-throughput screening model according to claim 1, wherein, The reagent used for transient transfection was Lipofectamine 2000.

8. The high-throughput screening model according to claim 1, wherein, The wavelengths for detection by the ELISA reader are Ex482nm / Em502nm.

9. The high-throughput screening model according to claim 1, wherein in step (2), the density of HEK293T cells seeded is 0.32 x 10⁻⁶ cells per well in a 96-well plate. 5 indivual.

10. The high-throughput screening model according to claim 1, wherein in step (3), the ratio of plasmid DNA to transfection reagent Lipofectamine 2000 is 0.1ug:0.48ul per well.

11. The high-throughput screening model according to claim 1, wherein in step (4), the final concentration of PROTAC compound #0334 is 5 μM, 0.5 μM, 50 nM, 5 nM, 0.5 nM; and the final concentration of PROTAC compound #0541 is 5 μM, 0.5 μM, 50 nM, 5 nM.

12. Use of the high-throughput screening model according to any one of claims 1-11 in screening PROTAC protein degradation drugs targeting FGFR4.

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