CN111298106A

CN111298106A - Conjugate and application thereof in degradation of β -catenin protein

Info

Publication number: CN111298106A
Application number: CN201911166179.7A
Authority: CN
Inventors: 陈晔光; 胡宏岗; 刘磊; 廖洪蔚; 李翔; 赵联正
Original assignee: Tsinghua University
Current assignee: Nanchang Double Angel Biotechnology Development Co ltd
Priority date: 2019-11-25
Filing date: 2019-11-25
Publication date: 2020-06-19
Anticipated expiration: 2039-11-25
Also published as: CN111298106B

Abstract

The invention provides application of E3 ubiquitin ligase VHL in degradation of β -catenin protein or inhibition of Wnt/β -catenin signal path, a conjugate and a preparation method and application thereof, wherein the conjugate comprises a first peptide section, a second peptide section and a connecting arm, wherein the first peptide section is suitable for being combined with β -catenin protein, the second peptide section is suitable for being combined with E3 ubiquitin ligase VHL, and the first peptide section is connected with the second peptide section through the connecting arm.

Description

Conjugate and application thereof in degradation of β -catenin protein

Technical Field

The invention relates to the field of biomedicine, in particular to a conjugate and application thereof in degrading β -catenin protein.

Background

In pathological conditions, once the members of the Wnt/β -catenin signaling pathway are mutated to cause regulatory disorder, excessive β -catenin accumulates in cytoplasm and then enters nucleus to promote cell proliferation, many cancers, especially intestinal cancer, have been proved to be caused by over-activated Wnt/β -catenin signals, therefore, the inhibition of Wnt signals in tumors through the specific degradation of β -catenin is considered to be the most direct and effective anticancer strategy.

In the past, almost all Wnt signal inhibitors taking β -catenin protein as targets are chemical small molecules, and since β -catenin has large binding interfaces with other proteins and many and complex binding proteins, the function of β -catenin is difficult to completely inhibit by the small molecule inhibitors, and no Wnt/β -catenin small molecule inhibitor is approved for clinical use at present.

Therefore, the compounds suitable for degrading β -catenin protein still need to be researched.

Disclosure of Invention

The present invention aims to solve at least to some extent at least one of the technical problems of the prior art.

It should be noted that the present invention has been completed based on the following findings of the inventors:

the inventors of the present invention have performed ubiquitination modification of β -catenin protein using ubiquitin ligase, and the modified β -catenin protein will be degraded by protease, thereby inhibiting Wnt/β -catenin signaling pathway. thus far, nearly hundred types of ubiquitin ligases have been disclosed, each ubiquitin ligase having its own specific substrate, wherein the ubiquitin ligase specifically modifying β 1-catenin protein is mainly β -trcp. e3 ubiquitin ligase VHL, and the substrate is mainly HIF-1 α, and plays a core role in regulating oxygen-induced gene expression.

Therefore, in one aspect of the invention, the invention provides the use of E3 ubiquitin ligase VHL in degrading β -catenin protein or inhibiting Wnt/β -catenin signaling pathway, as mentioned above, almost hundreds of types of ubiquitin ligases have been disclosed so far, each ubiquitin ligase has a specific action substrate, wherein the ubiquitin ligase specifically modifying β -catenin protein mainly comprises β -TrCP. E3 ubiquitin ligase VHL, the action substrate is mainly HIF-1 α, and plays a core role in regulating oxygen-induced gene expression.

According to the embodiment of the invention, the use of the E3 ubiquitin ligase VHL in the degradation of β -catenin protein or the inhibition of Wnt/β -catenin signal pathway can also have the following additional technical characteristics:

according to the embodiment of the invention, the E3 ubiquitin ligase VHL is used for preparing a medicine for treating diseases caused by over-activation of the Wnt/β -catenin signal pathway.

According to an embodiment of the invention, the pharmaceutical composition is for use in the treatment of intestinal cancer.

According to the embodiment of the invention, the method comprises the steps of contacting a candidate drug with a cell, and determining whether β -catenin protein is degraded and/or whether a Wnt/β -catenin signal path is inhibited in the cell before and after the contact, wherein the β -catenin protein is degraded and the Wnt/β -catenin signal path is inhibited after the contact, which are indications that the candidate drug is a target drug containing E3 ubiquitin ligase VHL, so that the drug containing E3 ubiquitin ligase VHL can be effectively obtained by using the method for screening the drug according to the embodiment of the invention, β -catenin is ubiquitinated, so that protease can degrade the Wnt to inhibit the Wnt/β -catenin signal path.

According to the embodiment of the invention, the conjugate can identify and combine β -catenin protein and E3 ubiquitin ligase VHL, so that β -catenin protein can be ubiquinated by the E3 ubiquitin ligase VHL and degraded by protease, the Wnt/β -catenin signal pathway can be inhibited, the effects of inhibiting cancer cell proliferation and tumorigenicity can be achieved, and the conjugate has wide scientific research and clinical application prospects.

According to an embodiment of the invention, the first peptide stretch is selected from one of the following: (1) has the sequence shown in SEQ ID NO: 1 to 7, or a pharmaceutically acceptable salt thereof; (2) a sequence having at least 80% homology to (1).

According to an embodiment of the invention, the second peptide segment is selected from one of the following: (3) has the sequence shown in SEQ ID NO: 8; (4) a sequence having at least 80% homology to (3).

According to an embodiment of the invention, said linking arm is selected from C_1～8Alkyl radical, C_1～8Alkylamino radical, C_1～8Alkoxy radical, C_1～8An alkyl alkenyl group or a polyethylene glycol; wherein, C_1～8Alkyl radical, C_1～8Alkoxy or C_1～8The alkanyl group can be optionally substituted with one or more H, F, Cl, Br, I, hydroxy, alkoxy, amino, carboxy, aryl, or heteroaryl groups.

According to an embodiment of the present invention, the N-terminus of the first peptide fragment is further connected with an acetyl group.

According to an embodiment of the invention, the C-terminus of the second peptide segment is further linked to an amino group.

According to an embodiment of the invention, the conjugate has the amino acid sequence of SEQ ID NO: 10, or a fragment thereof.

According to the embodiment of the invention, β -catenin protein and E3 ubiquitin ligase VHL can be identified and combined, so that β -catenin protein can be ubiquitinated by the E3 ubiquitin ligase VHL and degraded so as to inhibit a Wnt/β -catenin signal channel, the function of inhibiting cancer cell proliferation and tumorigenicity is achieved, and the pharmaceutical composition has wide scientific research and clinical application prospects.

According to an embodiment of the invention, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient.

According to the embodiment of the invention, the pharmaceutical composition is used for treating diseases caused by over-activation of Wnt/β -catenin signaling pathway.

According to an embodiment of the invention, the pharmaceutical composition is for use in the treatment of colon cancer.

According to an embodiment of the present invention, the pharmaceutical composition is used for inhibiting the proliferation and tumorigenic capacity of intestinal cancer cells.

According to the embodiment of the invention, the method comprises the steps of contacting a candidate drug with a cell, determining whether β -catenin protein is degraded and/or whether a Wnt/β -catenin signal path is inhibited in the cell before and after the contact, wherein the degradation of β -catenin protein and the inhibition of the Wnt/β -catenin signal path are indications that the candidate drug is a target drug, so that the method can screen out drugs capable of identifying and combining β -catenin protein and E3 ubiquitin ligase VHL, and facilitates the ubiquitin VHL of E3 ligase to ubiquitinate β -catenin protein and degrade the same by protease so as to inhibit the Wnt/β -catenin signal path, play a role in inhibiting the proliferation and the oncogenesis capability of cancer cells, and have wide research and clinical application prospects.

According to the embodiment of the invention, the conjugate capable of identifying and combining β -catenin protein and E3 ubiquitin ligase VHL can be obtained, so that the E3 ubiquitin ligase VHL can ubiquitinate β -catenin protein and can be degraded by protease, and the method is simple and convenient to operate, high in yield and purity and suitable for large-scale production.

According to an embodiment of the invention, the protecting group is selected from fluorenylmethoxycarbonyl; the carrier is selected from Rink AmidemBHA amino resin; n, N' -diisopropylcarbodiimide and 2-oxime ethyl cyanoacetate are used as condensation reagents; removing the protecting group on the conjugate in a DMF solution of 8-30 mass% piperidine; performing the olefin metathesis reaction with a Grubbs first generation catalyst; separating the conjugate from the carrier by using a solution containing 70-90 mass% of trifluoroacetic acid, 2-8 mass% of water, 1-5 mass% of 1, 2-ethanedithiol, 2-8 mass% of thioanisole and 5-10 mass% of phenol, and removing a protecting group on the conjugate.

Additional aspects and advantages of the invention will be set forth in part in the description which follows and, in part, will be obvious from the description, or may be learned by practice of the invention.

Drawings

The above and/or additional aspects and advantages of the present invention will become apparent and readily appreciated from the following description of the embodiments, taken in conjunction with the accompanying drawings of which:

FIG. 1 shows a schematic diagram of the analysis of β -catenin protein and Wnt/β -catenin signaling pathway changes according to one embodiment of the present invention;

FIG. 2 is a schematic diagram showing the analysis of the influence of the polypeptide on the tumorigenicity ability of a colon cancer cell line subcutaneously in a nude mouse according to an embodiment of the present invention;

FIG. 3 shows a schematic diagram of an analysis of the effect of a polypeptide according to one embodiment of the present invention on the number of tumors in the intestine of mice.

Detailed Description

The following describes embodiments of the present invention in detail. The following examples are illustrative only and are not to be construed as limiting the invention.

It should be noted that the terms "first" and "second" are used for descriptive purposes only and are not to be construed as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. Further, in the description of the present invention, "a plurality" means two or more unless otherwise specified.

The invention provides application of E3 ubiquitin ligase VHL in degradation of β -catenin protein or inhibition of Wnt/β -catenin signal path, a conjugate and a preparation method thereof, a pharmaceutical composition and a method for screening drugs, which are respectively described in detail below.

Degradation of β -catenin protein by E3 ubiquitin ligase VHL

In one aspect of the invention, the invention provides the use of E3 ubiquitin ligase VHL in degrading β -catenin protein or inhibiting Wnt/β -catenin signal pathway, as mentioned above, almost hundreds of types of ubiquitin ligases have been disclosed so far, each ubiquitin ligase has a specific action substrate, wherein the ubiquitin ligase for specifically modifying and degrading β -catenin protein is mainly β -TrCP. E3 ubiquitin ligase VHL, the action substrate is mainly HIF-1 α, and plays a core role in regulating oxygen-induced gene expression.

According to the embodiment of the invention, the E3 ubiquitin ligase VHL is used for preparing a pharmaceutical composition which is used for treating diseases caused by the over-activation of a Wnt/β -catenin signal channel, and researches show that the over-activation of the Wnt/β -catenin signal channel easily causes the enhancement of the proliferation capacity of cancer cells.

Method for screening drugs

According to the embodiment of the invention, the method comprises the steps of contacting a candidate drug with cells, determining whether β -catenin protein is degraded and/or whether a Wnt/β -catenin signal pathway is inhibited in the cells before and after the contact, wherein the β -catenin protein degradation and the Wnt/β -catenin signal pathway are inhibited after the contact, and the target drug is indicated to be a target drug, and the target drug contains E3 ubiquitin ligase VHL.

As described above, E3 ubiquitin ligase VHL can specifically identify and ubiquitinate β -catenin protein, so that protease can degrade the protein to inhibit Wnt/β -catenin signal pathway, therefore, whether the β -catenin protein is degraded and/or whether the Wnt/β -catenin signal pathway is inhibited before and after the candidate drug contacts cells is determined, and when β -catenin protein and the Wnt/β -catenin signal pathway are inhibited after the candidate drug contacts the cells, the candidate drug is proved to contain E3 ubiquitin ligase VHL, so that the aim of accurately screening the target drug is achieved.

Conjugates

According to the embodiment of the invention, the conjugate comprises a first peptide segment, a second peptide segment and a connecting arm, wherein the first peptide segment is suitable for being combined with β -catenin protein, the second peptide segment is suitable for being combined with E3 ubiquitin ligase VHL, and the first peptide segment and the second peptide segment are connected through the connecting arm, so that the first peptide segment can specifically recognize and combine β -catenin protein, and the second peptide segment can specifically recruit E3 ubiquitin ligase VHL, thereby facilitating the E3 ubiquitin ligase VHL to ubiquitinate β -catenin protein to be degraded by protease so as to inhibit a Wnt/β -catenin signal path, play a role in inhibiting the proliferation and the tumorigenicity of cancer cells, and have wide scientific research and clinical application prospects.

It should be noted that, the specific amino acid sequences of the first peptide fragment and the second peptide fragment are not strictly limited, as long as the first peptide fragment can be ensured to be capable of specifically recognizing and binding β -catenin protein, and the second peptide fragment can be ensured to be capable of specifically recognizing and binding E3 ubiquitin ligase VHL, and the specific amino acid sequences can be flexibly selected according to the actual situation.

According to the embodiment of the invention, the first peptide segment is selected from one of (1) amino acid sequences shown in SEQ ID NO 1-7 and (2) sequences with at least 80% homology with (1). The inventor finds that the amino acid sequences shown in SEQ ID NO 1-7 can recognize and combine β -catenin protein, and the amino acid sequence is short and convenient to synthesize, wherein the amino acid sequence shown in SEQ ID NO 1 recognizes and combines β -catenin protein, and has strong specificity and good combination capability.

(SEQ ID NO: 1, N terminal → C terminal), the specific amino acid structure is as follows:

(SEQ ID NO: 2, N terminal → C terminal), the specific amino acid structure is as follows:

(SEQ ID NO: 3, N terminal → C terminal), the specific amino acid structure is as follows:

(SEQ ID NO: 4, N terminal → C terminal), the specific amino acid structure is as follows:

(SEQ ID NO: 5, N terminal → C terminal), the specific amino acid structure is as follows:

(SEQ ID NO: 6, N terminal → C terminal), the specific amino acid structure is as follows:

the following steps:

(SEQ ID NO: 7, N terminal → C terminal), the specific amino acid structure is as follows

The first peptide fragment of the present invention may be not only one of the above 7 amino acid sequences, but also one having at least 80%, 85%, 90%, 95% homology with these amino acids, for example, any one of the above amino acid sequences may be subjected to insertion and/or substitution and/or deletion of one or more amino acids, or to insertion position change of an unnatural amino acid (S5), and the present invention is not limited thereto, as long as the homology requirements are satisfied and β -catenin protein can be specifically recognized and bound.

According to an embodiment of the invention, the second peptide segment is selected from one of the following: (3) has the sequence shown in SEQ ID NO: 8; (4) a sequence having at least 80% homology to (3). The inventors found that the sequence of SEQ ID NO: 8 can recognize and combine E3 ubiquitin ligase VHL. And the amino acid sequence is short, so that the synthesis is convenient.

ALAPYIP (SEQ ID NO: 8), the specific amino acid structure is shown as follows:

the second peptide fragment of the present invention may not only be the above amino acid sequence but also have at least 80%, 85%, 90%, 95% homology with these amino acids. For example, any one of the above amino acid sequences may be subjected to insertion and/or substitution and/or deletion of one or more amino acids, and the present invention is not limited thereto, as long as the homology requirement is satisfied and the E3 ubiquitin ligase VHL is specifically recognized and bound thereto.

In the present invention, the amino acid sequence of the linker arm linking the first peptide fragment and the second peptide fragment is not strictly limited, as long as the first peptide fragment and the second peptide fragment can be linked without affecting their respective properties, and the linker arm can be flexibly selected according to actual conditions. According to an embodiment of the invention, the linking arm is selected from C_1～8Alkyl radical, C_1～8Alkylamino radical, C_1～8Alkoxy radical, C_1～8An alkyl alkenyl group or a polyethylene glycol; wherein, C_1～8Alkyl radical, C_1～8Alkylamino radical, C_1～8Alkoxy or C_1～8The alkanyl group can be optionally substituted with one or more H, F, Cl, Br, I, hydroxy, alkoxy, amino, carboxy, aryl, or heteroaryl groups. In some embodiments, the linker arm is selected from C_3～6Alkyl radical, C_3～6Alkylamino radical, C_3～6Alkoxy radical, C_3～6An alkyl alkenyl group or a polyethylene glycol; wherein, C_3～6Alkyl radical, C_3～6Alkylamino radical, C_3～6Alkoxy or C_3～6The alkanyl group can be optionally substituted with one or more H, F, Cl, Br, I, hydroxy, alkoxy, amino, carboxy, aryl, or heteroaryl groups. According to a specific embodiment of the invention, the linker arm is aminocaproic acid having the amino acid sequence of SEQ id no: 9, and (c) 9. Thereby, the synthesis is facilitated without affecting the properties of the first and second peptide fragments.

(SEQ ID NO：9)

n is 1,2 or 3

According to the embodiment of the invention, the N-terminal of the first peptide segment is further connected with acetyl, so that the protease degradation stability of the conjugate is enhanced, and the structure of the conjugate is prevented from being damaged when the protease degrades the ubiquitinated β -catenin protein.

According to an embodiment of the present invention, the C-terminus of the second peptide fragment is further linked with an amino group.

The inventor obtains the sequence shown in SEQ ID NO. 10 through a large number of experiments, and the sequence can specifically identify and firmly combine β -catenin protein and E3 ubiquitin ligase VHL.

(SEQ ID NO：10)

Conjugates according to embodiments of the invention also have at least one of the following advantages:

1. the preparation is convenient: the conjugates have a short sequence length and can be prepared by a mature Fmoc solid phase synthesis method.

2. Compared with small molecules, the conjugate provided by the invention has a larger action surface, so that when the conjugate is applied to inhibiting the Wnt/β -catenin signal transduction pathway, the conjugate has the characteristics of high activity and high selectivity, and other signal pathways are not influenced.

3. The polypeptide provided by the invention basically keeps a natural α -spiral structure and has positive charge under physiological conditions, thereby being beneficial to penetrating into cytoplasm and nucleus through endocytosis.

4. The serum stability is high: the conjugate is cyclic peptide, contains unnatural amino acid and is modified by N acetylation, so that the stability of the conjugate is improved.

5. The conjugate provided by the invention can be used for continuously degrading β -catenin in cells so as to inhibit the Wnt signaling pathway for a long time.

6. Low immunogenicity: the peptides provided by the invention are much smaller than proteins and have very low immunogenicity.

Pharmaceutical composition

In yet another aspect of the invention, a pharmaceutical composition is provided. According to an embodiment of the invention, the pharmaceutical composition comprises a conjugate as described above.

As mentioned above, the first peptide segment of the conjugate can specifically recognize and bind β -catenin protein, and the second peptide segment can specifically recruit E3 ubiquitin ligase VHL, so that the E3 ubiquitin ligase VHL can ubiquitinate β -catenin protein and degrade the protein to inhibit Wnt/β -catenin signal path, therefore, whether β -catenin protein is degraded and/or whether Wnt/β -catenin signal path is inhibited before and after the candidate drug contacts cells is determined, and when the β -catenin protein and the Wnt/β -catenin signal path are inhibited after the candidate drug contacts the cell, the candidate drug is proved to contain the conjugate, so that the aim of accurately screening the target drug is achieved, and the conjugate has wide scientific research and clinical application prospects.

According to the embodiment of the invention, the pharmaceutical composition is used for treating diseases caused by over-activation of a Wnt/β -catenin signal pathway, the over-activation of the Wnt/β -catenin signal pathway easily causes the enhancement of the proliferation capacity of cancer cells, and the conjugate can specifically recognize and combine β -catenin protein and E3 ubiquitin ligase VHL, so that β -catenin protein can be conveniently ubiquitinated by the E3 ubiquitin ligase VHL, the protein is degraded by protease, the over-activation of the Wnt/β -catenin signal pathway is inhibited, the proliferation and tumor forming capacity of the cancer cells is inhibited, and the therapeutic purpose is achieved, and particularly the pharmaceutical composition is applied to treating intestinal cancer cells, such as colon cancer.

According to an embodiment of the present invention, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient. The invention does not strictly limit the types of the auxiliary materials and can flexibly select the auxiliary materials according to the conditions. For injectable formulations, pharmaceutically acceptable carriers may include buffers, preservatives, analgesics, solubilizers, isotonic agents (isotonic agents) and stabilizers. For formulations for topical administration, pharmaceutically acceptable carriers may include bases, excipients, lubricants and preservatives. The pharmaceutical composition of the present invention may be prepared in various dosage forms in combination with the above pharmaceutically acceptable carrier. For injectable preparations, the pharmaceutical compositions may be prepared in ampoules, e.g. in single dose dosage form, or in unit dosage forms, e.g. in multidose containers. The pharmaceutical compositions may also be formulated as solutions, suspensions, tablets, pills, capsules and depot preparations.

Among the excipients and diluents suitable for pharmaceutical formulations according to some embodiments of the present invention may be, among others: lactose, glucose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum arabic, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil.

According to other embodiments of the present invention, the adjuvant of the present invention may further include a filler, an anticoagulant, a lubricant, a humectant, a fragrance, and a preservative.

According to the embodiment of the invention, the conjugate and the pharmaceutical composition of the invention can specifically recognize β -catenin protein and E3 ubiquitin ligase VHL, so that β -catenin protein is ubiquitinated, protease is used for degrading the protein, and the Wnt/β -catenin signal pathway is inhibited, and the conjugate and the pharmaceutical composition containing the conjugate are effective in treating or preventing tumors.

The term "administering" as used herein means introducing a predetermined amount of a substance into a patient by some suitable means. The conjugate of the present invention may be administered by any common route as long as it can reach the desired tissue. Various modes of administration are contemplated, including peritoneal, intravenous, intramuscular, subcutaneous, cortical, oral, topical, nasal, pulmonary, and rectal, but the invention is not limited to these exemplified modes of administration. However, since peptides are digested and peptide bonds are cleaved upon oral administration, the active ingredients of orally administered compositions should be coated or formulated to prevent degradation or destruction in the stomach. Preferably, the composition of the present invention can be administered in an injectable formulation. In addition, the pharmaceutical compositions of the present invention may be administered using a specific device that delivers the active ingredient to the target cells.

The administration frequency and dose of the pharmaceutical composition of the present invention can be determined by a number of relevant factors, including the type of disease to be treated, the administration route, the age, sex, body weight and severity of the disease of the patient, and the type of drug as an active ingredient. According to some embodiments of the invention, the daily dose may be divided into 1,2 or more doses in a suitable form for administration 1,2 or more times over the entire period, as long as a therapeutically effective amount is achieved.

The term "therapeutically effective amount" refers to an amount of a compound sufficient to significantly ameliorate some of the symptoms associated with a disease or condition, i.e., to provide a therapeutic effect for a given condition and administration regimen. For example, in the treatment of cancer, a drug or compound that reduces, prevents, retards, inhibits or retards any symptom of a disease or disorder should be therapeutically effective. A therapeutically effective amount of a drug or compound need not cure a disease or condition, but will provide treatment for a disease or condition such that the onset of the disease or condition in an individual is delayed, prevented or prevented, or the symptoms of the disease or condition are alleviated, or the duration of the disease or condition is altered, or the disease or condition becomes less severe, or recovery is accelerated, for example.

The term "treating" is used to refer to obtaining a desired pharmacological and/or physiological effect, such as inhibiting the growth of cancer cells, causing the death of cancer cells, or ameliorating a disease or condition. The effect may be prophylactic in terms of complete or partial prevention of the disease or symptoms thereof, and/or may be therapeutic in terms of a partial or complete cure for the disease and/or adverse effects resulting from the disease. As used herein, "treatment" encompasses treatment of a disease (primarily cancer) in a mammal, particularly a human, including: (a) preventing disease (e.g., preventing cancer) or the occurrence of a disorder in an individual who is susceptible to the disease but has not yet been diagnosed with the disease; (b) inhibiting a disease, e.g., arresting disease progression; or (c) alleviating the disease, e.g., alleviating symptoms associated with the disease. As used herein, "treatment" encompasses any administration of a pharmaceutical composition to an individual to treat, cure, alleviate, ameliorate, reduce, or inhibit a disease in the individual, including, but not limited to, administering a drug comprising a conjugate described herein to an individual in need thereof.

According to embodiments of the invention, the conjugates or pharmaceutical compositions of the invention may be used in conjunction with conventional methods of treatment and/or therapy, or may be used separately from conventional methods of treatment and/or therapy. When the conjugates or pharmaceutical compositions of the invention are administered in combination therapy with other drugs, they may be administered to the individual sequentially or simultaneously. Alternatively, the pharmaceutical compositions of the invention may comprise a combination of a conjugate of the invention, a pharmaceutically acceptable adjuvant or pharmaceutically acceptable excipient, and other therapeutic or prophylactic agents known in the art.

Method for screening drugs

According to the embodiment of the invention, the method comprises the steps of contacting a candidate drug with a cell, determining whether β -catenin protein is degraded and/or whether a Wnt/β -catenin signal channel is inhibited in the cell before and after the contact, wherein β -catenin protein degradation and the Wnt/β -catenin signal channel are inhibited, and the target drug is selected from the pharmaceutical compositions mentioned above, so that the drug capable of identifying and combining β -catenin protein and E3 ubiquitin ligase VHL can be screened by using the method according to the embodiment of the invention, so that the E3 ubiquitin ligase VHL can ubiquitinate β -catenin protein to be degraded by protease, the Wnt/β -catenin signal channel can be inhibited, the effect of inhibiting cancer cell proliferation and tumorigenesis can be achieved, and the method has wide research and clinical application prospects.

Process for preparing conjugates

According to the embodiment of the invention, the method can obtain the conjugate capable of identifying and combining β -catenin protein and E3 ubiquitin ligase VHL, thereby facilitating the ubiquitination of β -catenin protein by the E3 ubiquitin ligase VHL and the degradation of the protein by protease so as to inhibit the Wnt/β -catenin signal path.

According to an embodiment of the invention, the protecting group is selected from fluorenylmethoxycarbonyl; the carrier is selected from Rink Amide MBHA amino resin; n, N' -diisopropylcarbodiimide and 2-oxime ethyl cyanoacetate are used as condensation reagents; removing the protecting group on the conjugate in a DMF solution of 8-30 mass% piperidine; performing the olefin metathesis reaction with a Grubbs first generation catalyst; separating the conjugate from the carrier by using a solution containing 70-90 mass% of trifluoroacetic acid, 2-8 mass% of water, 1-5 mass% of 1, 2-ethanedithiol, 2-8 mass% of thioanisole and 5-10 mass% of phenol, and removing a protecting group on the conjugate. The inventor obtains the better parameters through a large number of experiments, thereby further improving the synthesis efficiency, improving the product yield and purity and reducing the cost.

The scheme of the invention will be explained with reference to the examples. It will be appreciated by those skilled in the art that the following examples are illustrative of the invention only and should not be taken as limiting the scope of the invention. The examples, where specific techniques or conditions are not indicated, are to be construed according to the techniques or conditions described in the literature in the art or according to the product specifications. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.

General synthetic methods

In the following examples, conjugates were prepared with reference to the following methods, unless otherwise specified:

(1) swelling of the resin: 400mg Rink Amide MBHA amino resin is weighed and put into a polypeptide synthesis tube, 5mL DCM is added at room temperature for swelling for more than 20min, and then the solvent is pumped by a water pump.

(2) Washing of the resin: 5mL of DMF was added to the tube, mixed for 1 minute, drained and repeated 3 times. The reaction mixture was washed with DCM, and 5mL of dichloromethane was added to the above synthesis tube, mixed for 1 minute, drained, and repeated 3 times. Finally, the column was washed with DMF, 5mL of DMF was added, mixed for 1 min, aspirated, and repeated 5 times.

(3) Removing Fmoc protecting groups: after the resin was washed, 7mL of 20% piperidine/DMF solution was added to the synthesis tube, the reaction was shaken at 35 ℃ for 10min, and the reaction solution was drained. Then, 7ml of 20% piperidine/DMF solution was added to the synthesis tube, and the mixture was shaken at 35 ℃ for reaction for 10min, and the reaction solution was drained. The resin washing was repeated after each amino acid attachment to remove the Fmoc protecting group, i.e., 5 DMF, 5 DCM, 5 DMF, amounts and methods as described above.

(4) Condensation of amino acids: for natural amino acid (hereinafter abbreviated as AA), Fmoc-AA-OH (4eq.) is weighed and placed in a 10mL centrifuge tube, and then 2-oxime ethyl cyanoacetate (3.6eq.) and N, N' -diisopropylcarbodiimide (8eq.) are added into the centrifuge tube, and 6mL DMF is added and mixed uniformly for activation for three minutes. Adding the activated amino acid into a solid phase synthesis tube, reacting at 35 ℃ for 1h, and washing. For the stapled peptide amino acid- (S) -N-Fmoc-2- (4' -pentenyl) amino acid (hereinafter abbreviated as S)₅Or R₈) For example, Fmoc-R is weighed₅/S₅-OH (2eq.) is placed in a 10mL centrifuge tube, then 2-oxime cyanoethyl acetate (1.8eq.) is added to the centrifuge tube, 6mL of DMF is added, and finally N, N' -diisopropylcarbodiimide (4eq.) is added, mixed evenly and activated for three minutes. Adding the activated amino acid into a synthesis tube, reacting at 35 ℃ for 2h, and washing.

(5) Detection of condensation reaction: in the condensation reaction, the reaction process is monitored by a Kaiser reagent (ninhydrin qualitative color development), namely a small amount of resin is put into a 2ml centrifuge tube, 3 drops of ninhydrin ethanol solution (5g of ninhydrin dissolved in 100ml of absolute ethanol) and 1 drop of phenol ethanol solution (80g of phenol dissolved in 20ml of ethanol) are added, the mixture is heated at 100 ℃ for 2 minutes, if colorless shows that the amino end condensation is finished, if bluish purple shows that the amino end does not completely react, the condensation is needed to be repeated once. Wherein, the (S) -N-Fmoc-2- (4' -pentenyl) amino acid with naked amino group is colorless in detection.

(6) Olefin metathesis: after receiving the resin grafted with the linear polypeptide, the RCM reaction was performed on the resin. The resin that had been grafted with linear polypeptide was first washed three times with 10mL of DCE in a solid phase synthesis tube. The polypeptide synthesis tube was then drained with an oil pump. 5mL of a DCE solution of Grubbs-generation catalyst (10mmol/L) was added, and the reaction was carried out at 35 ℃ for 2 hours. The RCM reaction was repeated once with suction and finally the resin was washed.

(7) Acetylation reaction: all polypeptides were N-terminally blocked with acetyl groups as follows: fmoc was removed and washed as described above in step (3), and 5mL of acetylating agent (Ac) was added to the synthesis tube₂Pyridine ═ 1:1, v: v) reaction at 35 ℃ for 20 minutes and finally washing again as described above.

(8) Cutting of the resin: the polypeptide was first removed from the resin by oil pumping, and then 15mL of cleavage reagent K (80% TFA, 5% water, 2.5% EDT, 5% thioanisole and 7.5% phenol) was added to the polypeptide synthesis tube and shaken at room temperature for 4 hours. The cleavage reagent was filtered into a 50mL centrifuge tube, the resin rinsed with 5mL K reagent, and the TFA in the centrifuge tube was blown dry with argon. And then adding 35mL of glacial ethyl ether into the centrifugal tube, shaking for precipitation, centrifuging for 3min by using a centrifugal machine with the speed of 3500 rpm, pouring out a supernatant solution, repeating the precipitation process for three times, and air-drying to obtain the crude peptide.

(9) Analysis and purification: the crude peptide was analyzed and purified by shiimazu high performance liquid chromatography. Conditions for HPLC analysis: the analytical column is Grace Vydac "Protein&Peptide C₁₈"(250X 4.6mM,5 μm); the detection wavelength is 214 nm; the mobile phase A is 0.1 percent TFA acetonitrile solution, the mobile phase B is 0.1 percent TFA water solution, and gradient elution is carried out (0-35 min, the mobile phase B is 90-0 percent); the flow rate is 1.0 mL/min; the column temperature was 30 ℃ and the amount of sample was 20. mu.L. Preparing column chromatographic conditions: grace Vydac "Peptidc₁₈"column (250X 10mM, 10 μm); the detection wavelength is 214 nm; the mobile phase A is 0.1 percent TFA acetonitrile solution, the mobile phase B is 0.1 percent TFA water solution, and gradient elution is carried out (0-40 min, the mobile phase B is 90-0 percent); the flow rate was 4.0 mL/min. Collecting main peak in the preparation process, freeze-drying to obtain target product, and performing HPLC analysis and HR-Q-TOF-MS identification on the pure product.

Example 1 specific degradation of β -catenin protein and inhibition of the Wnt/β -catenin signaling pathway Using conjugates

1. In HEK293T cells, 70 μ M of test substance was added to control (unstimulated) and Wnt3a stimulated cells, respectively. Wherein the DMSO group is a control group; the samples to be tested in the control group 1 and the experimental group 1 are SEQ ID NO: 11; the test substance of the control group 2 and the experimental group 2 is conjugate 1(SEQ ID NO: 12); the samples to be tested of the control group 3 and the experimental group 3 are SEQ ID NO: 1; the test substance of the control group 4 and the experimental group 4 was conjugate 2(SEQ ID NO: 10).

(SEQ ID NO：11)

(SEQ ID NO：12)

As shown in A in figure 1, when Wnt3a is stimulated, the protein level of β -catenin is obviously increased, only conjugate 2 has obvious down-regulation effect on the protein level of β -catenin, and the polypeptide without the recognition peptide segment of VHL (experimental group 3) of E3 ubiquitin ligase has no obvious influence on the protein level of β -catenin, and in addition, other important components in the Wnt/β -catenin signal pathway, namely Axin1, Dvl2, GSK3 β and TCF4 have no obvious change, which indicates that conjugate 2 only acts on β -catenin protein to degrade the protein.

2. In HEK293T cells cultured, a Topflash-luciferase reporter gene capable of responding to Wnt/β -catenin signal was transfected with PEI transfection reagent (Polysciences Co.) and Renilla was used as an internal reference, 24 hours after transient expression of the reporter gene, the cells were treated with Wnt3a and the polypeptide for 24 hours, and the cells were harvested for luciferase activity assay to evaluate the degree of activation of Wnt/β -catenin signal.

As shown in B of figure 1, trace amount of Wnt3a can strongly activate Wnt/β -catenin signaling pathway, while experimental groups 1 and 2 still activate Wnt/β -catenin signaling, and experimental group 3 can inhibit Wnt/β -catenin signaling to some extent, while conjugate 2 with the same concentration can more strongly inhibit Wnt/β -catenin signaling pathway.

Example 3 the conjugate effectively inhibited the neoplasia of the colon cancer cell line subcutaneously in nude mice

The BALB/C immunodeficient nude mice selected in the experiment are firstly pretreated by DMSO and a sample to be tested on colon cancer cell line LoVo cells, respectively treated with 50 mu M concentration for 24 hours, and then equally pretreated cells (about 3 multiplied by 10) are taken⁶) Injected subcutaneously into nude mice, and after 3 weeks of growth, subcutaneous tumors were removed and weighed.

The results are shown in fig. 2, in which the DMSO group was the control group; the sample of Experimental group 2 was conjugate 1(SEQ ID NO: 12); the test substance of the experimental group 3 is SEQ ID NO: 1; the test substance of test group 4 was conjugate 2(SEQ ID NO: 10), and 10 replicates were tested in each test group.

The subcutaneous tumors of mice treated with experimental groups 2 and 3 both grew to similar sizes compared to the control group, whereas the subcutaneous tumors of mice treated with conjugate 2 grew significantly less than the control group, and therefore it can be considered that conjugate 2 had a significant inhibitory effect on the tumorigenic capacity of colon cancer cells.

Example 4 the conjugate is effective in reducing the number of tumors in the intestinal tract of mice

APC selected for this experiment^min/+The mouse, the deletion mutation of the APC gene leads to the inactivation of the APC gene, thereby causing the continuous activation of the Wnt/β -catenin signal path, the most obvious phenotype is that a large number of tumors can grow in the intestinal tract after several weeks, and the APC of 11 weeks is selected^min/+Mice, divided into 5 groups, one group without treatment (Day0) as a control group, were directly dissected to record the number of tumors in their intestines, and the other four groups were treated with 30mg/kg DMSO, conjugate 1 (experimental group 2), SEQ id no: 1 (Experimental group 3) and conjugate 2 (Experimental group 4) were dissolved in physiological saline to subject the mice to intraperitoneal injection, and the mice were injected 7 times in a 14-day administration period, i.e., every other day, and all the mice were collected on the 14 th day, and their intestines were dissected and recordedAs shown in FIG. 3, the number of intestinal tumors in mice treated with DMSO, experimental groups 2 and 3 did not significantly change compared to the control group without treatment, while the number of intestinal tumors in mice treated with conjugate 2 was significantly reduced compared to the control group, so that it can be considered that the conjugate continuously activates APC due to Wnt/β -catenin signal^min/+The intestinal tumor of the mouse has a healing effect.

In the description herein, references to the description of the term "one embodiment," "some embodiments," "an example," "a specific example," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above are not necessarily intended to refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, various embodiments or examples and features of different embodiments or examples described in this specification can be combined and combined by one skilled in the art without contradiction.

Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention, and that variations, modifications, substitutions and alterations can be made to the above embodiments by those of ordinary skill in the art within the scope of the present invention.

SEQUENCE LISTING

<110> Qinghua university

<120> conjugate and application thereof in degradation of β -catenin protein

<130>PIDC3194992

<160>11

<170>PatentIn version 3.5

<210>1

<211>17

<212>PRT

<213>Artificial Sequence

<220>

<223>SEQ ID NO：1

<220>

<221>MISC_FEATURE

<222>(6)..(10)

<223> to each other through an alkyl alkenylalkyl group

<400>1

Arg Arg Trp Pro Arg S5 Ile Leu Asp S5 His Val Arg Arg Val Trp

1 5 10 15

Arg

<210>2

<211>15

<212>PRT

<213>Artificial Sequence

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<223>SEQ ID NO：2

<220>

<221>MISC_FEATURE

<222>(5)..(9)

<223> to each other through an alkyl alkenylalkyl group

<400>2

Glu Asn Pro Glu S5 Ile Leu Asp S5 His Val Gln Arg Val Met

1 5 10 15

<210>3

<211>13

<212>PRT

<213>Artificial Sequence

<220>

<223>SEQ ID NO：3

<220>

<221>MISC_FEATURE

<222>(3)..(7)

<223> to each other through an alkyl alkenylalkyl group

<400>3

Pro Glu S5 Ile Leu Asp S5 His Val Gln Arg Val Met

1 510

<210>4

<211>14

<212>PRT

<213>Artificial Sequence

<220>

<223>SEQ ID NO：4

<220>

<221>MISC_FEATURE

<222>(3)..(7)

<223> to each other through an alkyl alkenylalkyl group

<400>4

Pro Gln S5 Ile Leu Asp S5 His Val Arg Arg Val Met Arg

1 5 10

<210>5

<211>14

<212>PRT

<213>Artificial Sequence

<220>

<223>SEQ ID NO：5

<220>

<221>MISC_FEATURE

<222>(3)..(7)

<223> to each other through an alkyl alkenylalkyl group

<400>5

Pro Gln S5 Ile Leu Asp S5 His Val Arg Arg Val Trp Arg

1 5 10

<210>6

<211>16

<212>PRT

<213>Artificial Sequence

<220>

<223>SEQ ID NO：6

<220>

<221>MISC_FEATURE

<222>(5)..(9)

<223> to each other through an alkyl alkenylalkyl group

<400>6

Arg Trp Pro Gln S5 Ile Leu Asp S5 His Val Arg Arg Val Trp Arg

1 5 10 15

<210>7

<211>17

<212>PRT

<213>Artificial Sequence

<220>

<223>SEQ ID NO：7

<220>

<221>MISC_FEATURE

<222>(6)..(10)

<223> to each other through an alkyl alkenylalkyl group

<400>7

Arg Arg Trp Pro Gln S5 Ile Leu Asp S5 His Val Arg Arg Val Trp

1 5 10 15

Arg

<210>8

<211>7

<212>PRT

<213>Artificial Sequence

<220>

<223>SEQ ID NO：8

<400>8

Ala Leu Ala Pro Tyr IlePro

1 5

<210>9

<211>24

<212>PRT

<213>Artificial Sequence

<220>

<223>SEQ ID NO：10

<220>

<221>MISC_FEATURE

<222>(1)..(1)

<223> attachment of acetyl group

<220>

<221>MISC_FEATURE

<222>(6)..(10)

<223> to each other through an alkyl alkenylalkyl group

<220>

<221>MISC_FEATURE

<222>(17)..(18)

<223> attachment of aminocaproic acid

<220>

<221>MISC_FEATURE

<222>(24)..(24)

<223> linking amino group

<400>9

Arg Arg Trp Pro Arg S5 Ile Leu Asp S5 His Val Arg Arg Val Trp

1 5 10 15

Arg Ala Leu Ala Pro Tyr Ile Pro

20

<210>10

<211>14

<212>PRT

<213>Artificial Sequence

<220>

<223>SEQ ID NO：11

<220>

<221>MISC_FEATURE

<222>(1)..(1)

<223> attachment of acetyl group

<220>

<221>MISC_FEATURE

<222>(3)..(10)

<223> to each other through an alkyl alkenylalkyl group

<400>10

Pro Gln R8 Ile Leu Asp Gln His Val S5 Arg Val Met Lys

1 5 10

<210>11

<211>21

<212>PRT

<213>Artificial Sequence

<220>

<223>SEQ ID NO：12

<220>

<221>MISC_FEATURE

<222>(3)..(10)

<223> to each other through an alkyl alkenylalkyl group

<220>

<221>MISC_FEATURE

<222>(14)..(15)

<223> are connected through vigabatrin

<220>

<221>MISC_FEATURE

<222>(21)..(21)

<223> linking amino group

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Pro Gln R8 Ile Leu Asp Gln His Val S5 Arg Val Met Lys Ala Leu

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Ala Pro Tyr Ile Pro

20

Claims

Use of E3 ubiquitin ligase VHL in the degradation of β -catenin protein or in the inhibition of Wnt/β -catenin signalling pathway.
2. The use according to claim 1, wherein the E3 ubiquitin ligase VHL is used for the preparation of a pharmaceutical composition for the treatment of a disease caused by overactivation of the Wnt/β -catenin signaling pathway;

optionally, the medicament is used for inhibiting the proliferation and tumorigenic capacity of intestinal cancer cells;

optionally, the medicament is for the treatment of colon cancer.
3. A method of screening for a drug comprising:

contacting the candidate agent with the cell;

determining whether β -catenin protein is degraded and/or whether Wnt/β -catenin signaling pathway is inhibited in cells before and after the contact;

wherein, the β -catenin protein degradation and the inhibition of the Wnt/β -catenin signal pathway after the contact are indications that the candidate drug is the target drug, and the target drug contains E3 ubiquitin ligase VHL.
4. A conjugate, comprising:

a first peptide fragment, wherein the first peptide fragment is suitable for being combined with β -catenin protein;

a second peptide stretch adapted to bind to E3 ubiquitin ligase VHL;

and the first peptide segment and the second peptide segment are connected through the connecting arm.
5. The conjugate of claim 4, wherein the first peptidyl fragment is selected from one of the following:

(1) has the sequence shown in SEQ ID NO: 1 to 7, or a pharmaceutically acceptable salt thereof;

(2) a sequence having at least 80% homology to (1);

optionally, the second peptide segment is selected from one of:

(3) has the sequence shown in SEQ ID NO: 8;

(4) a sequence having at least 80% homology to (3);

optionally, the linker arm is selected from C_1～8Alkyl radical, C_1～8Alkylamino radical, C_1～8Alkoxy radical, C_1～8An alkyl alkenyl group or a polyethylene glycol; wherein, C_1～8Alkyl radical, C_1～8Alkylamino radical, C_1～8Alkoxy or C_1～8The alkanyl group can be optionally substituted with one or more H, F, Cl, Br, I, hydroxy, alkoxy, amino, carboxy, aryl, or heteroaryl groups;

optionally, the N-terminal of the first peptide fragment is further connected with acetyl;

optionally, the C-terminus of the second peptide segment is further linked with an amino group;

optionally, a polypeptide having the sequence of SEQ ID NO: 10, or a fragment thereof.
6. A pharmaceutical composition comprising the conjugate of claim 4 or 5.
7. The pharmaceutical composition of claim 6, further comprising a pharmaceutically acceptable excipient;

optionally, the pharmaceutical composition is used for treating diseases caused by over-activation of Wnt/β -catenin signal pathway;

optionally, the pharmaceutical composition is used for inhibiting the proliferation and tumorigenic capacity of intestinal cancer cells;

optionally, the pharmaceutical composition is for use in the treatment of colon cancer.
8. A method of screening for a drug comprising:

contacting the candidate agent with the cell;

determining whether β -catenin protein is degraded and/or whether Wnt/β -catenin signaling pathway is inhibited in cells before and after the contact;

wherein the β -catenin protein degradation and inhibition of the Wnt/β -catenin signaling pathway following contact are indicative that the drug candidate is a drug of interest selected from the pharmaceutical composition of claim 6 or 7.
9. A method of preparing the conjugate of claim 4 or 5, comprising:

providing a carrier and a synthetic substrate, synthesizing by using a solid-phase synthesis method, and carrying out olefin double decomposition reaction on a synthetic product so as to obtain the carrier bonded with the conjugate; and

separating the conjugate from the carrier and removing the protecting group on the conjugate.
10. The process according to claim 9, characterized in that the protecting group is selected from fluorenylmethoxycarbonyl;

the carrier is selected from Rink Amide MBHA amino resin;

n, N' -diisopropylcarbodiimide and 2-oxime ethyl cyanoacetate are used as condensation reagents;

removing the protecting group on the conjugate in a DMF solution of 8-30 mass% piperidine;

performing the olefin metathesis reaction with a Grubbs first generation catalyst;

separating the conjugate from the carrier by using a solution containing 70-90 mass% of trifluoroacetic acid, 2-8 mass% of water, 1-5 mass% of 1, 2-ethanedithiol, 2-8 mass% of thioanisole and 5-10 mass% of phenol, and removing a protecting group on the conjugate.