CN117069745A - Near infrared light activated protein targeted degradation chimeric body and preparation method and application thereof - Google Patents
Near infrared light activated protein targeted degradation chimeric body and preparation method and application thereof Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
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- C07D519/00—Heterocyclic compounds containing more than one system of two or more relevant hetero rings condensed among themselves or condensed with a common carbocyclic ring system not provided for in groups C07D453/00 or C07D455/00
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- A—HUMAN NECESSITIES
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
- A61K41/0057—Photodynamic therapy with a photosensitizer, i.e. agent able to produce reactive oxygen species upon exposure to light or radiation, e.g. UV or visible light; photocleavage of nucleic acids with an agent
- A61K41/0076—PDT with expanded (metallo)porphyrins, i.e. having more than 20 ring atoms, e.g. texaphyrins, sapphyrins, hexaphyrins, pentaphyrins, porphocyanines
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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Abstract
Near infrared light activated protein targeted degradation chimera, and preparation method and application thereof, the structure is as follows:the present invention describes for the first time a Near Infrared (NIR) light activated nano protein targeted degradation chimera (NAP) for in vivo remote controlled proteolysis. NAP is targeted by protein capable of specifically degrading BRD4 protein to degrade chimeric ARV771 through singlet oxygen 1 O 2 ) The cleavable linker is self-assembled by linking to the near infrared photosensitizer. Through regulating and controlling the level of pathogenic protein, the effects of disease research, cancer cell growth inhibition and tumor treatment are achieved. The invention adopts a chemical method to prepare the egg based on singlet oxygen activationThe white targeted degradation medicine is synthesized and applied to the combination of protein in the targeted degradation cells and photodynamic therapy so as to effectively inhibit tumor growth.
Description
Technical Field
The invention belongs to the technical field of protein targeted degradation, and particularly relates to a chimeric for near infrared light activated protein targeted degradation, a preparation method and application thereof.
Background
The proteolytic-targeting chimeric (PROTAC) technology has become a promising way of anticancer, with PROTAC comprising a ligand for the protein of interest and E3 ubiquitin ligase, inducing targeted protein degradation via the ubiquitin-proteasome pathway. Heretofore, PROTAC has been applied to degrade a variety of oncogenic proteins, such as Androgen Receptor (AR), estrogen Receptor (ER), and bromodomain-containing protein 4 (BRD 4), but current research has found that the presence of off-target/targeted toxicity caused by PROTAC activity in normal tissues remains a critical safety issue after systemic administration. Thus, in the course of treatment, tumor-specific therapeutic strategies should be considered, and protein-targeted degradation chimeras are inactive until exposed to the tumor site, and the proteolytic activity is restored by triggering the response group, providing a practical means of manipulating the degradation process. Among them, light has been currently in the optical cage type and optical switch type of PROTAC to temporarily mask proteolytic activity by using photocleavage and photoisomerization groups, respectively, because of its safety and high space-time resolution, and these photoactivated PROTAC show great promise in optical control of targeted protein degradation in living cells. However, their activation depends on uv or visible light irradiation, which greatly hinders in vivo applications. Here we report a Near Infrared (NIR) light activated nano-formulation PROTAC (NAP) for remote controlled proteolysis in tumor cells (scheme 1). The PROTAC was initially proteolytically inactive due to covalent cross-linking. Following systemic administration, NAP accumulates in tumor tissue and enters tumor cells by endocytosis. The released PROTAC then resumes its activity for protein degradation. Meanwhile, the photosensitizer can be used for photodynamic therapy to realize synergistic cancer inhibition.
Disclosure of Invention
The technical problems to be solved are as follows: the invention provides a near infrared light activated protein targeted degradation chimeric body, a preparation method and application thereof, which are suitable for research of activating target proteins and synergizing tumor treatment.
The technical scheme is as follows: the structure of the near infrared light activated protein targeted degradation chimera is shown as follows:
the preparation method of the near infrared light activated protein targeted degradation chimera comprises the following steps of: firstly, mixing 1eq mercaptoethanol and 1.2eq potassium fluoride with acetic acid, reacting for 18 hours at 80 ℃, extracting a reaction product by ethyl acetate, and then obtaining a purified product 1 by column chromatography; reacting 1eq of purified product 1 with 2.2eq of acetone and 1eq of trifluoroacetic acid at room temperature for 24 hours, and then carrying out column chromatography to obtain purified product 2;1eq of purified product 2 and 4eq of potassium hydroxide are dissolved in methanol to react for 12 hours at room temperature, after the reaction product is extracted by ethyl acetate, column chromatography is carried out again to obtain purified product 3,1eq of purified product 3 and 4eq of phenyl p-nitrochloroformate, 8eq of TEA are dissolved in methylene dichloride and stirred for 12 hours at room temperature, and column chromatography is carried out to purify intermediate product molecules 4; stirring 1eq of PA and 1.2eq of mono BOC-ethylenediamine in 8eq of TEA for 12h at room temperature, and obtaining PA intermediate 7 through column chromatography; 1eq ARV-771 and 2eq intermediate molecule 4 are stirred for 24 hours at room temperature in 2eq TEA under the protection of nitrogen, 1eq purified product and 1eq PA intermediate 7, 8eq TEA are dissolved in Dichloromethane (DCM) after column chromatography purification, the reaction is carried out for 12 hours at room temperature, and the product molecule NAP is obtained after high performance liquid chromatography purification.
Application of the chimera in preparing medicines for selectively degrading BRD4 in MCF-7 cells.
The chimeric is applied to the preparation of antitumor drugs.
An antitumor drug contains the near infrared light activated protein targeted degradation chimeric.
According to the preparation method of the near infrared activated protein targeted degradation chimera, the protein targeted degradation chimera ARV-771 of the targeted BRD4 protein is synthesized through a standard chemical synthesis flow, the protein targeted degradation chimera ARV-771 is connected with the intermediate product molecule 4 and the PA intermediate 7 of the singlet oxygen response through activated ester, the protein targeted degradation chimera ARV-771 is coupled with the intermediate product molecule 4 and the PA intermediate 7 of the singlet oxygen response to obtain a compound napp, and then the compound NApp is placed in PBS and stands at room temperature to obtain the compound NAP.
The beneficial effects are that: the invention utilizes near infrared light to activate and induce target protein degradation and photodynamic therapy for the first time, and achieves the effects of disease research, cancer cell growth inhibition and tumor therapy through regulating and controlling the level of pathogenic protein. The invention adopts a chemical method to prepare the drug synthesis based on near infrared light activated protein targeted degradation and applies the drug synthesis to the research of protein in targeted degradation cells and photodynamic therapy. (1) Synthesis of near infrared light-activated protein-targeted degradation drugs: the proteolytic-targeting chimeric ARV771 capable of degrading BRD4 protein is selected to be synthesized with photosensitizer PA, and the chemical synthesis is mainly used for preparing near infrared light activated protein targeting degradation drugs through amide condensation reaction. (2) Release of targeted degradation drugs based on near infrared light activated proteins: in the solution, the synthesized near infrared light activated protein targeted degradation drug is irradiated, the release of PROTAC molecule ARV771 is detected through high performance liquid chromatography, and the generation of singlet oxygen is verified in a spectrum experiment. (3) Use of targeted protein degradation in cells based on release of near infrared light activated protein targeted degradation drugs: after co-incubating the near infrared light activated protein targeted degradation drug with tumor cells for illumination, the expression of BRD4 in the cells is detected. And the killing effect of the protein degradation and PDT effect on tumor tissues is verified through in vivo experiments.
Drawings
FIG. 1 is a method for synthesizing a near infrared light-activated protein-targeted degradation drug;
FIG. 2 is a mass spectrometry characterization of near infrared light activated protein targeted degradation drugs;
FIG. 3 is an HPLC chart showing the release of PROTAC in solution after the above drug treatment;
FIG. 4 is a spectrum of singlet oxygen release in solution after the above drug treatment;
FIG. 5 shows BRD4 expression in MCF-7 cells before and after the above drug treatment;
FIG. 6 shows the inhibition of MCF-7 cell proliferation after the above drug treatment;
FIG. 7 shows the growth inhibition of MCF-7 ectopic tumor after the above-mentioned drug treatment;
FIG. 8 shows the expression of BRD4 in MCF-7 ectopic tumor cells after the above drug treatment;
FIG. 9 is a schematic diagram of a method for preparing a protein-targeted degradation compound of the present invention.
Detailed Description
Example 1 Structure and Synthesis method of near-infrared light activated protein-targeted degradation drug
As shown in fig. 1, the standard chemical synthesis procedure synthesizes a near-infrared light activated protein targeting degradation molecular compound napp of the targeting BRD4 protein based on singlet oxygen response, and can self-assemble into NAP; the near infrared light activated protein targeting degradation molecular compound ncpp of the targeting BRD4 protein without singlet oxygen response can be self-assembled into NCP.
The specific method comprises the following steps: firstly, raw materials of mercaptoethanol (5 g) and potassium fluoride (4.6 g) are mixed with acetic acid and reacted for 18 hours at 80 ℃; ethyl acetate extraction three times and petroleum ether ethyl acetate (1:1) column chromatography purification, and acetone (4.0 g), trifluoroacetic acid (140 μl) at room temperature for 24 hours; petroleum ether ethyl acetate (3:1) column chromatography purification product 2g is added with potassium hydroxide (1.8 g), dissolved in methanol and placed for 12 hours at room temperature, ethyl acetate extraction and methylene chloride (30:1) column chromatography purification product (1.0 g), p-nitro phenyl chloroformate (6.17 g) is added, TEA (200 mu L) is dissolved in methylene chloride (10 mL) and reacted for 12 hours at room temperature, methylene chloride is dissolved in methanol (50:1) column chromatography purification to obtain intermediate product molecules 4; further, PA (100 mg) and mono BOC-ethylenediamine (32.43 mg) were stirred in TEA at room temperature for 12h, followed by column chromatography to give PA intermediate 7; ARV-771 (100 mg) and intermediate molecule 4 (213.3 mg) were stirred at room temperature in TEA under nitrogen for 24h, after purification by column chromatography with methylene chloride: methanol (20:1), the product (20 mg) and PA intermediate 7 (11.1 mg), TEA (10. Mu.L) were dissolved in methylene chloride (DCM), reacted at room temperature for 12h, purified by HPLC to give the product molecule NAP, mass spectrum results are shown in FIG. 2, calculated [ M+Na ]] + 1891.7172, actual measurement 1891.8150; calculation result [ M+K]+1907.6861, actual measurement 1907.7853. NCP was obtained under the same conditions.
Example 2 release of near-infrared light activated protein-targeted degradation drug in solution PROTAC
NAP (5. Mu.M) or NCP (5. Mu.M) in PBS was treated with 670nm (300 mW/cm) 2 ) Light irradiation for 5min, standing at room temperature for 12 hr, and high performance liquid chromatography (mobile phase: A: CH) 3 CN，B:H 2 O,0.1% TFA; the flow rate is 1mL/min; elution gradient from 40% A to 95% A) was 0-30 minutes, and the results are shown in FIG. 3, NAP plus light group, with ARV-771 released at 30 minutes, while no release of ARV-771 was observed in the other control groups.
To determine the singlet oxygen production capacity of NAP and NCP, SOSG (10. Mu.M) was added to 5. Mu.M NAP or NCP solution using SOSG as singlet oxygen fluorescent indicator with 670nm (300 mW/cm 2 ) The fluorescence at 520nm was measured after 5min of light irradiation, and the results showed that NAP and NCP were significantly enhanced after light irradiation treatment, indicating singlet oxygen production as shown in FIG. 4.
Example 3 use of protein targeting degradation chimeras to target BRD4 degradation in cells
After incubation for 12h with the addition of different concentrations of the compound NAP, NCP to MCF-7 cells, 670nm (300 mW/cm 2 ) As shown in FIG. 5, BRD4 in the Western blot detection cells is obviously degraded only in NAP light groups after light irradiation for 5min and then incubated for 12h, the degradation effect is enhanced along with the increase of the concentration of the NAP light groups, and the result shows that the compound NAP degrades BRD4 protein in MCF-7 cells under the light condition.
After incubation for 12h with the addition of various ARV-771, NAP, NCP to A549 cells, 670nm (300 mW/cm 2 ) After 5min of light irradiation and 36h of incubation, as shown in fig. 6, the proliferation rate of CCK-8 detection cells is obviously reduced along with the increase of the incubation concentration, the effect of the NAP light irradiation group is most obvious, and the inhibition of the proliferation of cancer cells by the simultaneous synergistic photodynamic therapy is realized by realizing the protein degradation of the targeted BRD4 after NAP light irradiation.
Example 4 use of protein-targeting degradation chimeras in tumor treatment
Subcutaneous transplantation of 4-6 week old female BALB/c (BALB/c-nude) mice 1X 10 7 MCF-7 cells. When the tumor size reaches about 120mm 3 Every 2 days, let go ofThe same dose (10 mg/kg ARV-771) of PROTAC was administered intravenously. The same dose of control agent (physiological saline) was also injected as a negative control. Tumor size and body weight were measured every 2 days. Mice were sacrificed on day 16 and tumor tissue and major normal tissue were taken for analysis. Tumor inhibition as shown in fig. 7, the tumor growth was the slowest in NAP plus light group, so the near infrared light activated protein targeted degradation chimera could achieve tumor growth inhibition. The BRD4 level in tumor tissues is shown in FIG. 8, and the histone after the independent ARV-771 treatment is degraded, but NAP has the most obvious effect of degrading histone by illumination, and the chimeric is verified to realize the protein degradation of the target BRD4 in vivo.
The above embodiments do not limit the technical solutions of the present invention in any way, and all the technical solutions obtained by adopting equivalent substitution or equivalent transformation fall within the protection scope of the present invention.
Claims (5)
1. The near infrared light activated protein targeted degradation chimera is characterized by comprising the following structure:
2. the method for preparing the near infrared light activated protein targeted degradation chimera according to claim 1, which is characterized by comprising the following steps of: firstly, mixing 1eq mercaptoethanol and 1.2eq potassium fluoride with acetic acid, reacting for 18 hours at 80 ℃, extracting a reaction product by ethyl acetate, and then obtaining a purified product 1 by column chromatography; reacting 1eq of purified product 1 with 2.2eq of acetone and 1eq of trifluoroacetic acid at room temperature for 24 hours, and then carrying out column chromatography to obtain purified product 2;1eq of purified product 2 and 4eq of potassium hydroxide are dissolved in methanol to react for 12 hours at room temperature, after the reaction product is extracted by ethyl acetate, column chromatography is carried out again to obtain purified product 3,1eq of purified product 3 and 4eq of phenyl p-nitrochloroformate, 8eq of TEA are dissolved in methylene dichloride and stirred for 12 hours at room temperature, and column chromatography is carried out to purify intermediate product molecules 4; stirring 1eq of PA and 1.2eq of mono BOC-ethylenediamine in 8eq of TEA for 12h at room temperature, and obtaining PA intermediate 7 through column chromatography; 1eq ARV-771 and 2eq intermediate molecule 4 are stirred for 24 hours at room temperature in 2eq TEA under the protection of nitrogen, 1eq purified product and 1eq PA intermediate 7, 8eq TEA are dissolved in Dichloromethane (DCM) after column chromatography purification, the reaction is carried out for 12 hours at room temperature, and the product molecule NAP is obtained after high performance liquid chromatography purification.
3. Use of the chimera of claim 1 for the preparation of a medicament for the selective degradation of BRD4 in MCF-7 cells.
4. Use of the chimera according to claim 1 for the preparation of an antitumor drug.
5. An antitumor agent comprising the near infrared light-activated protein-targeted degradation chimera according to claim 1.
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| CN202310845309.XA Pending CN117069745A (en) | 2023-07-10 | 2023-07-10 | Near infrared light activated protein targeted degradation chimeric body and preparation method and application thereof |
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