Method for rapidly finding amino acid of target protein combined with protein degradation agent

Abstract

The invention provides a method for rapidly finding amino acid of a target protein combined with a protein degradation agent. According to the method, a mutant type or a wild type of an exogenous expression target protein is provided with a protein tag, an exogenous expression tagged protein band and an endogenous expression label-free protein band can be separated, and a degradation agent can only degrade one of the bands and cannot degrade endogenous and exogenous target protein bands at the same time, so that the degradation efficiency is improved. If the endogenous band is a wild type target protein, the exogenous band is a mutant type target protein, and if the endogenous band is a mutant type target protein, the exogenous band is a wild type target protein. According to the invention, by using a degradation agent DHL-11 of IMPDH2, the key amino acid of DHL-11 binding IMPDH2 protein is successfully identified.

Description

Technical Field

[0001] The present invention belongs to the field of biological and medical technologies, and particularly relates to a method for quickly finding the amino acids of the target protein bound by a protein degrader. Background Art

[0002] Protein degraders overcome the limitations of traditional small molecule inhibitors in drug development by degrading target proteins, and have great application potential. Especially in the field of tumor treatment, multiple protein degrader drugs have entered the clinical trial stage and shown good efficacy. For example, the PROTAC drug ARV-471 targeting the estrogen receptor is conducting a phase III clinical trial for ER+ breast cancer patients and is expected to become an important means for treating this type of breast cancer. Therefore, the development of protein degraders is an emerging treatment strategy that can make up for the deficiencies of traditional drugs and bring new hope for the treatment of various diseases.

[0003] Currently, methods for detecting the binding amino acids of a protein and its degrader include cryo-electron microscopy and X-ray crystallography, mutant library screening, etc. Cryo-electron microscopy and X-ray crystallography require the formation of co-crystals of the protein and the degrader in vitro, and by analyzing the three-dimensional structure of the target protein and its degrader crystal, the possible binding sites and key amino acids on its surface are determined. However, this method has too many limitations. Protein crystallization requires strict experimental conditions, and the crystallization conditions are related to the nature of the protein itself. For the vast majority of proteins, no method for in vitro crystallization has been found, and it is even more difficult to crystallize the protein with its degrader. Currently, only a few co-crystals of proteins and degraders have been discovered. Moreover, the cost of crystallization experiments is very high, and only a few laboratories in the world can conduct them. In addition, crystallization requires a large amount of degrader and a large amount of highly purified protein. Mutant library screening is to construct a mutant library of the amino acids of the target protein, screen the sites sensitive to the degrader, or observe the response of different mutants to the degrader in cell or animal models to inversely locate the key amino acids. However, constructing a mutant library requires a large amount of manpower and financial resources and is difficult to popularize. Therefore, it is very necessary to develop a method that can solve the above technical problems. Summary of the Invention

[0004] The purpose of the present invention is to provide a method for quickly finding the amino acids bound by a protein degrader.

[0005] The purpose of the present invention is achieved as follows. The method for quickly finding the amino acids of the target protein bound by a protein degrader is to exogenously express a mutant or wild-type target protein with a protein tag, and the tag protein is fused with the target protein.

[0006] The present invention uses Western blot technology to detect different bands on the same membrane with target protein antibodies, detect the degradation ability of the degrader on different bands, and quickly find the target protein amino acids bound by the degrader.

[0007] Compared with the prior art, the present invention has the following beneficial effects: quickly finding key amino acids, low cost, requiring very little degrader content, not requiring a large amount of highly purified protein, and saving time, materials, and research costs.

[0008] The abbreviations corresponding to the amino acids involved in the content of the present invention are:

[0009] The IMPDH2 degrader used in the present invention is DHL-11, and its structural formula is as follows:

[0010] The protein + Flag sequence encoded by the human wild-type IMPDH2 expression plasmid used in the present invention is:

[0011] The protein + Flag sequence encoded by the human IMPDH2 mutant 1 (MUT1) expression plasmid used in the present invention is:

[0012] The protein + Flag sequence encoded by the human IMPDH2 mutant 2 (MUT2) expression plasmid used in the present invention is:

[0013] In the above three protein sequences: DYKDHDGDYKDHDIDYKDDDDK is the Flag sequence; the red color indicates the sequence where the amino acid sequence of the corresponding IMPDH2 wild-type protein is mutated to alanine.

[0014] The HEK293T cell line used in the present invention is cultured in DMEM medium containing 10% fetal bovine serum at 37°C, 5% CO2, and 90% humidity.

[0015] After 46 hours of transfection of wild-type and two mutant plasmids of IMPDH2-flag and FANCI plasmid into cells, the cells were treated with a degrader or DMSO for 2 - 10 hours, then the cells were lysed, and Western Blot and CO-IP assays were performed to detect the target proteins. The vector used in this invention is PCDH (No.: VECT231251 of Huayueyang, Beijing), and all IMPDH2-flag type and FANCI overexpression plasmids were constructed on PCDH (constructed by Beijing Tsingke Biotechnology Co., Ltd.).

[0016] The Western Blot experimental protocol is as follows: After cell culture under experimental conditions, the cells were washed with cold phosphate-buffered saline (PBS) and then collected. The cell pellet was dissolved in 2× protein sample buffer (1M Tris-HCl pH = 6.8, 50% glycerol, 10% SDS, 2-mercaptoethanol, 1% bromophenol blue) and boiled at 100°C for 10 minutes. The prepared protein samples were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis and then transferred to a PVDF membrane (Millipore). Blocking was carried out in a 5% skim milk solution for the specific coupling reaction between the proteins on the PVDF membrane and the antibodies. Then the skim milk solution was discarded, and the membrane was washed with PBST (0.5% Tween 20 added to PBS). The coupling reaction between the proteins and the antibodies on the membrane was carried out overnight at 4°C, and then washed with PBST again. Finally, the secondary antibody conjugated with horseradish peroxidase (HRP) (Cell signaling Technology) was diluted in PBST and reacted at room temperature for 2 hours. The protein content on the membrane was measured using Luminata Forte HRP substrate (Millipore).

[0017] The co-IP experimental protocol is as follows: Plate HEK293T cells in a 6-well plate. After adherence, transiently transfect the plasmid using PEI. In separate 1.5 mL EP tubes, add 200 μL of OPTI-MEM. Add 1 μg of the target plasmid to one tube and 4 μL of PEI to the other tube. Let each stand for 5 min. Then add the PEI to the target plasmid, mix well, and continue to stand for 20 min. After that, gently add the mixed OPTI-MEM along the wall of the dish to the 6-well plate. Replace the fresh medium 6 - 8 h later. After 48 h, add DHL-11 or DMSO and continue culturing for 6 h. Add 700 μL of IP lysis buffer and lyse on ice for 30 min. Collect the lysate and place it in a 4°C centrifuge, centrifuge at 12,000 rpm for 10 min. For each treatment group, pipette 30 μL of the lysate into 10 μL of 4×SDS and heat in a 98°C water bath for 10 min as Input. Add 1 - 2 μg of FANCI or IgG antibody to the remaining lysate and incubate overnight on a 4°C shaker. Wash the protein A / G magnetic beads three times in advance with IP lysis buffer. After the incubation, add 30 μL of the protein A / G magnetic beads to each treatment group and continue to incubate on a 4°C shaker for 4 - 6 h. Use a magnetic stand to adsorb the magnetic beads and then aspirate the lysate. Add fresh lysate and shake for 15 min, then aspirate the lysate. Repeat the washing 5 times. After the washing is completed, aspirate the lysate, add 60 μL of 1×SDS and heat in a 98°C water bath for 10 min. Perform Western blot experiments together with the initial Input to observe the results.

[0018] Antibodies used in the present invention Description of the drawings

[0019] Figure 1 It is a schematic diagram showing that the IMPDH2 degrader DHL-11 in Example 1 of the present invention can simultaneously degrade the wild-type IMPDH2 protein overexpressed endogenously and exogenously in cells; Figure 2 It is a schematic diagram showing that the IMPDH2 degrader DHL-11 in Example 2 of the present invention can degrade the wild-type IMPDH2 protein expressed endogenously in cells, but cannot degrade the mutant 1 (MUT1) of IMPDH2 expressed exogenously; Figure 3 It is a schematic diagram showing that the IMPDH2 degrader DHL-11 in Example 3 of the present invention can simultaneously degrade the wild-type IMPDH2 protein expressed endogenously in cells and the mutant 2 (MUT2) of IMPDH2 expressed exogenously; Figure 4 It is a schematic diagram showing that DHL-11 in Example 4 of the present invention can disrupt the binding between IMPDH2-Flag and FANCI; Figure 5 Schematic diagram showing that DHL-11 in Embodiment 5 of the present invention does not affect the binding of MUT1 to FANCI; Figure 6 Schematic diagram of the principle of the present invention. Detailed implementation manners

[0020] The present invention will be further described below in conjunction with embodiments, but the present invention is not limited in any way. Any transformation or replacement based on the teachings of the present invention falls within the protection scope of the present invention.

[0021] The method for quickly finding the amino acids of the target protein bound by the protein degrader in the present invention is to exogenously express the mutant or wild-type of the target protein with a protein tag, and the tag protein is fused with the target protein.

[0022] Specifically, the exogenous expression of the mutant or wild-type of the target protein with a protein tag is to transfer the wild-type and two mutant plasmids of IMPDH2-flag and the FANCI plasmid into cells, and after 40-50 h, treat with the degrader or DMSO for 2-10 h, break the cells and perform Western Blot and CO-IP tests to detect the target protein.

[0023] The degrader is a protein, nucleic acid, carbohydrate or lipid.

[0024] The degrader is DHL-11, and its structure is as follows:

[0025] The protein of the mutant or wild-type of the target protein with a protein tag exogenously expressed is one or more.

[0026] The protein bands of the mutant or wild-type of the target protein with a protein tag exogenously expressed can be separated from the endogenous protein bands, and the degrader cannot degrade all types of target proteins; if the endogenous band is the wild-type target protein, the exogenous band is the expressed mutant target protein, and if the endogenous band is the mutant target protein, the exogenous band is the expressed wild-type target protein.

[0027] The present invention will be further described below with specific implementation cases: Example 1 The degrader DHL-11 of IMPDH2 can simultaneously degrade the wild-type IMPDH2 protein overexpressed endogenously and exogenously in cells Figure 1Indicates the protein of wild-type IMPDH2 with an overexpressed exogenous Flag tag (IMPDH2-Flag). The overexpressing cell line was treated with the IMPDH2 degrader DHL-11, and the protein was collected for detection. PCDH is the expressed empty vector as a control. The protein expression of GAPDH was used as an internal reference. From Figure 1 The results show that DHL-11 can degrade the endogenous and exogenous expressed wild-type IMPDH2 protein.

[0028] Example 2 The IMPDH2 degrader DHL-11 can degrade the endogenously expressed wild-type IMPDH2 protein, but cannot degrade the mutant 1 (MUT1) of exogenously expressed IMPDH2. Five target amino acids that may bind DHL-11 in the protein + Flag sequence encoded by the human wild-type IMPDH2 expression plasmid were mutated to A (alanine). Figure 2 Indicates the overexpression of mutant 1 (MUT1) of IMPDH2 with an exogenous Flag tag. The overexpressing cell line was treated with the IMPDH2 degrader DHL-11, and the protein was collected for detection. PCDH is the expressed empty vector as a control. The protein expression of GAPDH was used as an internal reference. From Figure 2 The results show that DHL-11 can degrade the endogenously expressed wild-type IMPDH2 protein, but cannot degrade the exogenously expressed MUT1, proving that DHL-11 and MUT1 cannot bind, indicating that the mutant amino acids of MUT1 affect the binding of IMPDH2 to DHL-11, that is, the target amino acids of MUT1 are the amino acids that bind to the degrader.

[0029] Example 3 The IMPDH2 degrader DHL-11 can simultaneously degrade the endogenously expressed wild-type IMPDH2 protein and the mutant 2 (MUT2) of exogenously expressed IMPDH2. Four target amino acids that may bind DHL-11 in the protein + Flag sequence encoded by the human wild-type IMPDH2 expression plasmid were mutated to A (alanine). Figure 3 Indicates the overexpression of mutant 2 (MUT2) of IMPDH2 with an exogenous Flag tag. The overexpressing cell line was treated with the IMPDH2 degrader DHL-11, and the protein was collected for detection. PCDH is the expressed empty vector as a control. The protein expression of GAPDH was used as an internal reference. From Figure 3 The results show that DHL-11 can degrade the endogenously expressed wild-type IMPDH2 protein and can also degrade the exogenously expressed MUT2, proving that DHL-11 and MUT2 can bind, indicating that the mutant amino acids of MUT2 do not affect the binding of IMPDH2 to DHL-11.

[0030] Example 4 CO-IP experiment verified that DHL-11 could disrupt the binding between IMPDH2-Flag and FANCI The region where DHL-11 binds to IMPDH2 is also the region where FANCI binds to IMPDH2. Figure 4 It shows that through the CO-IP experiment, it is proved that DHL-11 can disrupt the binding between IMPDH2-Flag and FANCI, resulting in a significant decrease in the amount of FANCI protein bound to IMPDH2, proving that DHL-11 binds to IMPDH2-Flag. FANCI represents overexpression of FANCI protein in cells. Input represents the original protein lysate without immunoprecipitation reaction. IP: Flag means fishing for the protein bound to IMPDH2 with Flag magnetic beads.

[0031] Example 5 CO-IP experiment verified that DHL-11 did not affect the binding between MUT1 and FANCI To prove that MUT1 cannot bind to DHL-11, we detected whether DHL-11 could disrupt the binding between MUT1 and FANCI. Figure 5 It shows that through the CO-IP experiment, it is proved that DHL-11 does not affect the binding between MUT1 and FANCI, and the amount of FANCI protein bound to IMPDH2 does not change, proving that DHL-11 cannot bind to MUT1. FANCI represents overexpression of FANCI protein in cells. Input represents the original protein lysate without immunoprecipitation reaction. IP: Flag means fishing for the protein bound to MUT1 with Flag magnetic beads.

[0032] The above results indicate that through the method of the present invention, it is determined that MUT1 cannot bind to DHL-11, and the CO-IP experiment is carried out for verification. That is, the original unmutated amino acid corresponding to the mutated amino acid of MUT1 is the amino acid to which the IMPDH2 degrader binds.

[0033] The above are the preferred embodiments of the present invention, and are not limitations on the present invention. Any simple modifications, changes and equivalent structural transformations made to the above embodiments according to the technical essence of the present invention still fall within the protection scope of the technical solution of the present invention.

Claims

1. A method for quickly finding the amino acids of the target protein bound by a protein degrader, characterized in that, The method for quickly finding the amino acids of the target protein bound by the protein degrader is to exogenously express the mutant or wild-type target protein with a protein tag, and the tag protein is fused with the target protein.

2. The method according to claim 1, wherein Specifically, for the exogenous expression of the mutant or wild-type target protein with a protein tag, wild-type and mutant plasmids of IMPDH2-flag with different mutation sites are transfected into cells, and then treated with the degrader for 2-10 h. The cells are lysed and Western Blot and CO-IP assays are performed to detect the target protein.

3. The method according to claim 2, wherein The degrader is a small molecule compound, protein, nucleic acid, carbohydrate or lipid.

4. The method according to claim 1, characterized in that, The number of proteins of the mutant or wild-type target protein with a protein tag exogenously expressed is 1 or more.

5. The method according to claim 1, wherein The protein bands of the mutant or wild-type target protein with a protein tag exogenously expressed can be separated from the endogenous protein bands, and different exogenously expressed bands can also be separated. The degrader cannot degrade all types of target proteins; if the endogenous band is the wild-type target protein, the exogenous band is the expressed mutant target protein, and if the endogenous band is the mutant target protein, the exogenous band is the expressed wild-type target protein.

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