Proteolysis targeting chimera for targeted degradation of YY1 transcription factor as well as preparation method and application of proteolysis targeting chimera

Abstract

The invention discloses a proteolysis targeting chimera for targeted degradation of a YY1 transcription factor as well as a preparation method and application of the proteolysis targeting chimera, and relates to the technical field of biology. The proteolysis targeting chimera is a double-stranded DNA molecule and comprises a DNA long chain and a DNA short chain which are complementarily combined, the nucleotide sequence of the DNA long chain is as shown in SEQ ID NO.1, and the 3'end of the DNA long chain is connected with an E3 ubiquitin ligase receptor; the nucleotide sequence of the DNA short chain is as shown in SEQ ID NO. 2. The invention provides the proteolysis targeting chimera aiming at the DNA structure of the YY1 transcription factor, and the proteolysis targeting chimera can accurately degrade the YY1 transcription factor in cells, so that the tumor size is effectively controlled, and the aim of treating cancers is achieved. The proteolysis targeting chimera provided by the invention is stable in structure, simple in preparation method and particularly suitable for clinical popularization and application.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a protein hydrolysis targeted chimera for targeted degradation of YY1 transcription factor, and a preparation method and application thereof. Background Art

[0002] Overexpression of YY1 plays an important role in regulating the sensitivity and tolerance of tumor cells to chemotherapy and immunotherapy, and is involved in the regulation of key genes for cell cycle regulation and programmed cell death. Cell cycle deregulation is an important event in tumorigenesis. Transcription factor YinYang 1 (YY1) belongs to the zinc finger transcription factor family. It is widely involved in various cell biological processes, including cell differentiation, DNA repair, autophagy, apoptosis and proliferation, by activating or inhibiting gene transcription. There is a dynamic interaction between the activity of transcription factor YY1 and different components of the cell cycle and their checkpoints. These interactions often lead to cell cycle disorders and may even lead to tumorigenesis. In addition, overexpression of YY1 plays an important role in regulating the sensitivity and tolerance of tumor cells to chemotherapy and immunotherapy. YY1 is a very promising tumor therapeutic target, but it has the characteristics of (1) a flat and featureless binding site, (2) lack of hydrogen bond donors and acceptors, and (3) lack of a large interaction interface, which makes it currently impossible to target YY1 for clinical tumor therapy inhibitors. Fortunately, PROTAC has shown the ability to target proteins such as transcription factors for degradation and has significant advantages in overcoming drug resistance.

[0003] like Figure 1 It has been shown that protein degradation provides a quality control mechanism during protein folding, which is a normal process within the cell. Most proteins will be degraded by the ubiquitin-proteasome system (UPS), in which proteins are first covalently tagged with ubiquitin, a highly conserved protein containing 76 amino acids (8.6 kDa), and then sent to the 26S proteasome for degradation. Ubiquitination of proteins is a post-translational modification (PTM) process that is carried out in tandem by three enzymes ( Figure 1). In the first step, the E1 ubiquitin activating enzyme activates ubiquitin by using ATP to generate activated ubiquitin-adenosine, which is converted into a thioester intermediate by attachment to the catalytic cysteine ​​in the E1 enzyme. In the second step, ubiquitin is transferred to the catalytic cysteine ​​of the E2 ubiquitin conjugating enzyme via a transthioesterification reaction. Finally, ubiquitin is transferred to the substrate protein via a ternary complex formed between the E2 enzyme, the E3 ubiquitin ligase, and the substrate, resulting in the formation of an isopeptide bond between the carboxyl terminus of ubiquitin and the side chain of a lysine residue on the surface of the target protein. Depending on the type of E3 ligase, ubiquitin can be transferred directly from the E2 to the substrate or passed from the E2 to the E3 and then to the substrate. This cascade of ubiquitination can be repeated to generate a polyubiquitin chain on the substrate that will be recognized and degraded by the 26S proteasome.

[0004] Traditional small molecule inhibitor (SMI) development strategies cannot develop targets that lack suitable binding pockets for directly regulating protein function. However, most proteins will be degraded through the ubiquitin-proteasome system (UPS), and PROTAC technology hijacks the UPS to degrade specific target proteins (POIs). PROTAC consists of a ligand that binds to the POI and another ligand that can recruit the E3 ubiquitin ligase, and the two ligands are connected to each other through a linker. Therefore, PROTACs technology can mark POI ubiquitination and use the UPS system to degrade the target protein, so that non-drugable proteins are degraded.

[0005] There are currently no proteolytic targeting chimeras for the DNA structure of the YY1 transcription factor. Summary of the invention

[0006] The purpose of the present invention is to provide a protein hydrolysis targeting chimera for targeted degradation of YY1 transcription factor and its preparation method and application, so as to solve the problems existing in the above-mentioned prior art. The protein hydrolysis targeting chimera can accurately degrade the YY1 transcription factor in the cell, thereby effectively controlling the tumor size and achieving the purpose of treating cancer.

[0007] To achieve the above object, the present invention provides the following solutions:

[0008] The present invention provides a protein hydrolysis targeting chimera for targeted degradation of YY1 transcription factor. The protein hydrolysis targeting chimera is a double-stranded DNA molecule, comprising a complementary long DNA chain and a short DNA chain; the nucleotide sequence of the long DNA chain is shown in SEQ ID NO.1, and the 3' end of the long DNA chain is connected to an E3 ubiquitin ligase receptor; the nucleotide sequence of the short DNA chain is shown in SEQ ID NO.2.

[0009] Furthermore, the E3 ubiquitin ligase receptor is pomalidomide.

[0010] The present invention also provides a method for preparing the above-mentioned proteolysis targeting chimera, comprising the following steps:

[0011] Connecting the E3 ubiquitin ligase receptor to the 3' end of the DNA long chain with the nucleotide sequence as shown in SEQ ID NO.1 to obtain the DNA long chain connected with the E3 ubiquitin ligase receptor;

[0012] The long-chain DNA connected with the E3 ubiquitin ligase receptor and the short-chain DNA with a nucleotide sequence as shown in SEQ ID NO.2 are subjected to PCR self-assembly reaction to obtain the proteolysis targeting chimera.

[0013] Furthermore, the E3 ubiquitin ligase receptor is connected to an azide group, and the E3 ubiquitin ligase receptor is connected to the 3' end of the DNA long chain through a click chemistry reaction.

[0014] Furthermore, the E3 ubiquitin ligase receptor is pomalidomide.

[0015] Furthermore, the reaction procedure of the PCR self-assembly reaction is: 95°C for 15 min; 4°C for 35 min.

[0016] The present invention also provides the use of the above-mentioned proteolysis targeting chimera in the preparation of a drug for targeted degradation of YY1 transcription factor.

[0017] The present invention also provides the use of the above-mentioned proteolysis targeting chimera in the preparation of a drug for treating tumors, wherein the drug exerts a tumor treatment effect by degrading YY1.

[0018] The present invention also provides a drug for targeted degradation of YY1 or treatment of tumors, comprising the above-mentioned protein hydrolysis targeting chimera.

[0019] Furthermore, the medicine also includes pharmaceutically acceptable excipients.

[0020] The present invention discloses the following technical effects:

[0021] The present invention provides a proteolysis-targeted chimera targeting the DNA structure of the YY1 transcription factor, which consists of two DNA chains, a long chain and a short chain, and the 3' end of the long chain is connected to an E3 ubiquitin ligase receptor. The proteolysis-targeted chimera can accurately degrade the YY1 transcription factor in cells, thereby effectively controlling the size of tumors and achieving the purpose of treating cancer.

[0022] The protein hydrolysis targeting chimera provided by the present invention has a stable structure and a simple preparation method, and is particularly suitable for clinical promotion and application. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0024] Figure 1 Schematic diagram of the principle of PROTAC targeting transcription factors to degrade proteins;

[0025] Figure 2 The schematic diagram of the structure of the proteolysis targeting chimera prepared by the present invention; wherein 1 is the double-chain part: the binding sequence of the YY1 transcription factor; 2 is the connecting part: the linker of the two ligands, which is 6 A bases; 3 is the E3 ubiquitin ligase receptor: pomalidomide connected with an azide group;

[0026] Figure 3 Schematic diagram of the principle of Protacs-YY1 targeted degradation of YY1;

[0027] Figure 4 The PAGE characterization results of DNA single strand and protein hydrolysis targeting chimera;

[0028] Figure 5 The results of West Blot test of 293T cell experiment; A is the West Blot test result of transfection with different concentrations of Protacs-YY1 solution; B is the West Blot test result of different transfection time;

[0029] Figure 6 The results of West Blot detection of macrophage experiments; A is the West Blot detection result of transfection with different concentrations of Protacs-YY1 solution; B is the West Blot detection result of different transfection time; C is the result of YY1 ubiquitination experiment;

[0030] Figure 7 The observation results of tumors and other organs of mice in each experimental group;

[0031] Figure 8 The synthetic route of pomalidomide with an azide group is shown in FIG. DETAILED DESCRIPTION

[0032] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0033] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0034] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0035] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0036] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0037] The structural formula of pomalidomide (6-azido-N-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)ethyl)hexanamide) with an azide group used in the following examples is as follows:

[0038]

[0039] Its preparation method ( Figure 8 As shown) as follows:

[0040] General Synthesis Procedure of 3-Fluorobenzoic anhydride (25.0 g, 151.00 mmol.) and 3-amino-2,6-piperidone (25.0 g, 151.00 mmol) Weigh in a reaction flask, then add sodium acetate (14.9 g, 181.20 mmol), add 300 mL of glacial acetic acid. The reaction solution temperature rises to 120 ° C, stirs the reaction for 6 h, cools and concentrates in vacuo. Pour water into the reaction flask, precipitate solid, filter to obtain compound 3 (30 g, 72.2%) as a purple solid.

[0041] N-Boc-ethylenediamine (6.8 g, 43.00 mmol.) and DIPEA (15.0 g, 116.00 mmol.) were added to compound 3 (10.7 g, 39.00 mmol. DMSO). The reaction solution was stirred at 60° C. for 2 h. The reactant was dried over anhydrous Na2SO4. After filtration, the solvent was evaporated under reduced pressure and purified by silica gel column chromatography (petroleum ether / ethyl acetate, 1:1, volume / volume) to obtain compound 4 (9.7 g, 59.9%) as a yellow solid.

[0042] Compound 4 (9.7 g, 23.32 mmol) was dissolved in 100 mL DCM and stirred in an ice bath. Trifluoroacetic acid (26.6 g, 20 mmol) was added dropwise to the reaction solution. The reaction was stirred at room temperature for 2 h and concentrated to dryness to obtain 7.2 g of crude yellow solid compound 5.

[0043] Compound 5 (0.50 g, 1.02 mmol), THF and trimethylsilyl acetylene (TMSA, 0.14 g, 1.22 mmol) were added to a flask containing a magnetic stirrer. The mixture was evacuated three times under nitrogen protection. Tetrabutylammonium fluoride (1.53 mL, 1.22 mmol) was then added and stirred at 65 °C for 3 h. The reaction solution was then cooled to room temperature and extracted with EA. The organic layer was separated and concentrated to dryness to obtain compound 6, which is pomalidomide with an azide group.

[0044] Example 1

[0045] In this example, a proteolysis targeting chimera (named Protacs-YY1) was prepared, which consisted of two DNA chains, a long chain (named S7) and a short chain (named S8), as shown in the schematic diagram. Figure 2The 3' end of the long chain is linked to pomalidomide (an E3 ubiquitin ligase receptor) through a click chemistry reaction. The long chain is: 5'-GGCTTCAAAATGGAGACCCTAAAAAA-CRBN-3' (SEQ ID NO.1); the short chain is: 3'-CCGAAGTTTTACCTCTGGGA-5' (SEQ ID NO.2). The schematic diagram of the principle of Protacs-YY1 targeted degradation of YY1 transcription factor is shown in Figure 3 .

[0046] The preparation method of the proteolysis targeting chimera is as follows:

[0047] Two single-stranded DNAs (SEQ ID NO.1 and SEQ ID NO.2) were dissolved in TM buffer (10 mM Tris-base, 50 mM MgCl2, pH 8.0) respectively, with the final concentration of both being 0.1 mM.

[0048] The long-chain DNA dissolved in TM buffer was mixed with pomalidomide linked to an azide group and shaken at 37° C. for 2 h to obtain a solution of long-chain DNA linked to an E3 ubiquitin ligase receptor (named S7-CRBN).

[0049] After the TM buffer solution containing the DNA short strands was mixed with the S7-CRBN solution, a proteolysis-targeted chimera structure was formed by a PCR self-assembly reaction, wherein the reaction procedure of the PCR self-assembly reaction was: 95°C for 15 min; 4°C for 35 min. The two single-stranded DNAs were mixed in equal amounts and concentrations.

[0050] Characterization of proteolysis-targeting chimeras:

[0051] The newly synthesized Protacs-YY1, S7-CRBN, S7 and S8, as well as S7-CRBN stored in a refrigerator at 4°C for one week, were subjected to PAGE electrophoresis. The PAGE electrophoresis was performed by using TBE solution, ddH2O, APS solution and TEMED to prepare a 15% electrophoresis sol, electrophoresing at 120V for 1h, staining with Cyber ​​Green1 solution, and capturing images with a fluorescent imager. The results were obtained. Figure 4 The results shown. The results show that the proteolytic targeting chimera can form a stable structure.

[0052] Example 2

[0053] 1. Methods

[0054] 1.1293T cell experiments

[0055] 1.1.1 293T cells were seeded in 6-well plates (density: 2×10 6cells / well). Use transfection reagent ( 2000DNA Transfection), different concentrations of Protacs-YY1 solution (final concentration of 0.5μM, 1μM) were used to transfect into cells. The blank control was added with an equal amount of PBS buffer solution. The cells were then incubated in an incubator for 24h (37°C, 50% CO2). After incubation, the cells were collected and subjected to West Blot experiments.

[0056] 1.1.2 293T cells were seeded in 6-well plates (density: 2×10 6 cells / well), and Protacs-YY1 was added at a final concentration of 1.0 μM. The cells were incubated in an incubator for 6, 12, and 24 h (37° C., 50% CO 2 ). After incubation, the cells were collected and subjected to West Blot experiments.

[0057] 1.2 Macrophage experiments

[0058] Macrophages were seeded in 6-well plates (density: 2×10 6 cells / well). Use transfection reagent ( 2000DNA Transfection), macrophages were transfected with different concentrations of Protacs-YY1 solution (final concentrations were 0.5μM, 1μM and 1.5μM). The blank control was added with an equal amount of PBS buffer solution. The cells were then incubated in an incubator for 24h (37°C, 50% CO2). After incubation, the cells were collected for subsequent West Blot experiments.

[0059] Macrophages were seeded in 6-well plates (density: 2×10 6 cells / well), and Protacs-YY1 was added at a final concentration of 1.0 μM. The cells were incubated in an incubator for 6, 12, 24, and 36 h (37° C., 50% CO 2 ). After incubation, the cells were collected for subsequent West Blot experiments.

[0060] 1.3West Blot Experiment

[0061] Cells were collected by centrifugation, washed with PBS (4°C × 3 times), and lysed on ice for 50 min with RIPA lysis buffer (RIPA:PMSF=99:1) (Hangzhou Fude Biotechnology). The lysate was centrifuged at 14,847g for 25 minutes at 4°C. The protein concentration of the supernatant was measured using a BCA protein assay kit (Biyuntian Biotechnology (Shanghai, China)). The protein extract was mixed with a dual-color protein loading buffer, denatured in a 100°C bath for 10 minutes, and then loaded onto an SDS-PAGE gel. The protein was then transferred to a nitrocellulose membrane by electrophoresis. The primary antibody used in the present invention was YY1 Monoclonal antibody (Proteintech (Wuhan, China)). The secondary antibody used was HRP-conjugated Affinipure Goat Anti-Rabbit IgG (H+L). Western blot images were captured using an imaging system (Uvitec Ltd., UK). Protein levels were quantified using ImageJ by the grayscale values ​​of the bands in the resulting images.

[0062] 2. Experimental results

[0063] 2.1293T cell experimental results

[0064] The results of the West Blot assay for 293T cells are shown in Figure 5 ,The results showed that in 293T cells treated with Protacs-YY1, the WB band became lighter and the YY1 content decreased, ,proving that Protacs-YY1 can effectively degrade YY1 in 293T cells.

[0065] 2.2 Macrophage Experimental Results

[0066] The results of the West Blot assay for macrophages are shown in Figure 6 The results showed that the WB bands of macrophages treated with Protacs-YY1 became lighter and the YY1 content decreased. Protacs-YY1 can effectively degrade YY1 in macrophages.

[0067] Example 3

[0068] 1. Methods

[0069] Ten healthy mice (17-21 g) ordered from Qinglongshan Animal Farm in Jiangning District, Nanjing were randomly divided into two groups, 5 mice in each group. RM-1 cells (mouse prostate cancer cells) were injected subcutaneously on the back of each group of mice to implant tumors.

[0070] Protacs-YY1 is mixed with M2-PEP, a vector targeting M2 macrophages, to prepare M2-PEP-YY1-Protacs, which can target M2 macrophages and kill M2 cells by degrading YY1 in M2 cells, preventing the tumor from escaping the immune system and thus treating the tumor.

[0071] After tumor modeling, one group of mice was injected with 250μM M2-PEP-YY1-Protacs solution (200μL, PBS solution) through the tail vein every 24h, and the other group of mice was injected with blank PBS solution of equal volume through the tail vein every 24h. After two weeks of continuous administration, the tumors and organs of the two groups of mice were taken to observe the effects. The results are shown in Figure 7 The results showed that Protacs-YY1 had a good effect in treating tumors. The tumors stopped growing or even shrank within a certain period of time, and there was no harm to other organs.

[0072] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A proteolytic targeting chimera for targeted degradation of YY1 transcription factor, characterized in that: The proteolysis targeting chimera is a double-stranded DNA molecule, including a complementary long DNA chain and a short DNA chain; the nucleotide sequence of the long DNA chain is shown in SEQ ID NO.1, and its 3' end is connected to an E3 ubiquitin ligase receptor; the nucleotide sequence of the short DNA chain is shown in SEQ ID NO.

2.

2. The proteolysis targeting chimera according to claim 1, characterized in that The E3 ubiquitin ligase receptor is pomalidomide.

3. A method for preparing the proteolysis targeting chimera according to claim 1, characterized in that: The following steps are involved: Connecting the E3 ubiquitin ligase receptor to the 3' end of the DNA long chain with the nucleotide sequence as shown in SEQ ID NO.1 to obtain the DNA long chain connected with the E3 ubiquitin ligase receptor; The long-chain DNA connected with the E3 ubiquitin ligase receptor and the short-chain DNA with a nucleotide sequence as shown in SEQ ID NO.2 are subjected to PCR self-assembly reaction to obtain the proteolysis targeting chimera.

4. The preparation method according to claim 3, characterized in that: The E3 ubiquitin ligase receptor is connected with an azide group, and the E3 ubiquitin ligase receptor is connected to the 3' end of the DNA long chain through a click chemistry reaction.

5. The preparation method according to claim 4, characterized in that: The E3 ubiquitin ligase receptor is pomalidomide.

6. The preparation method according to claim 3, characterized in that: The reaction procedure of the PCR self-assembly reaction is: 95° C. for 15 min; 4° C. for 35 min.

7. Use of the proteolysis targeting chimera according to claim 1 or 2 in the preparation of a drug for targeted degradation of YY1 transcription factor.

8. Use of the proteolysis targeting chimera according to claim 1 or 2 in the preparation of a drug for treating tumors, characterized in that: The drug achieves the treatment of tumors by degrading YY1.

9. A drug for targeted degradation of YY1 or treatment of tumors, characterized in that: Comprising the proteolysis targeting chimera of claim 1 or 2.

10. The drug according to claim 9, characterized in that The drug also includes pharmaceutically acceptable excipients.

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