CN111298106B - Conjugate and application thereof in degradation of beta-catenin protein - Google Patents
Conjugate and application thereof in degradation of beta-catenin protein Download PDFInfo
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/43—Enzymes; Proenzymes; Derivatives thereof
- A61K38/53—Ligases (6)
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
- A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
- A61K47/65—Peptidic linkers, binders or spacers, e.g. peptidic enzyme-labile linkers
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- C12Y—ENZYMES
- C12Y603/00—Ligases forming carbon-nitrogen bonds (6.3)
- C12Y603/02—Acid—amino-acid ligases (peptide synthases)(6.3.2)
- C12Y603/02019—Ubiquitin-protein ligase (6.3.2.19), i.e. ubiquitin-conjugating enzyme
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
Abstract
The invention provides application of E3 ubiquitin ligase VHL in degradation of beta-catenin protein or inhibition of Wnt/beta-catenin signal pathway, a conjugate and a preparation method and application thereof, wherein the conjugate comprises: a first peptide fragment, wherein the first peptide fragment is suitable for being combined with beta-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. The E3 ubiquitin ligase VHL can perform ubiquitination modification on the beta-catenin protein so that the ubiquitinated beta-catenin protein is degraded by protease, thereby inhibiting a Wnt/beta-catenin signal channel, playing a role in inhibiting cancer cell proliferation and tumor formation capability, and having wide scientific research and clinical application prospects.
Description
Technical Field
The invention relates to the field of biomedicine. In particular, the invention relates to a conjugate and application thereof in degrading beta-catenin protein.
Background
The Wnt/beta-catenin signaling pathway plays an important role in controlling the proliferation, differentiation and survival of cells, and the members of the signaling pathway include: wnt ligands secreted outside the cell, transmembrane receptors on the cell membrane, and the degradation complex and beta-catenin inside the cell. As a signal transduction molecule of a classical Wnt signal pathway, the stability of the beta-catenin protein is precisely regulated and controlled. In pathological conditions, once the member of the Wnt/beta-catenin signal pathway is subjected to gene mutation to cause regulatory disorder, excessive beta-catenin accumulates in cytoplasm and then enters nucleus to promote cell proliferation. Many cancers, especially intestinal cancers, have been shown to be due to over-activated Wnt/β -catenin signaling. Therefore, inhibition of Wnt signaling in tumors by specific degradation of β -catenin is considered to be one of the most directly effective anticancer strategies.
In the past, almost all Wnt signal inhibitors taking beta-catenin protein as a target point are chemical small molecules, and because the binding interfaces of the beta-catenin and other proteins are large and the types of the binding proteins are many and complex, the small molecule inhibitors are difficult to completely inhibit the functions of the beta-catenin, and no Wnt/beta-catenin small molecule inhibitor is approved to be clinically used at present.
Therefore, the compounds suitable for degrading beta-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 inventor of the invention utilizes ubiquitin ligase to carry out ubiquitination modification on the beta-catenin protein, and the modified beta-catenin protein is degraded by protease, thereby inhibiting a Wnt/beta-catenin signal channel. Almost one hundred types of ubiquitin ligases have been disclosed so far, each having its own specific substrate, among which the ubiquitin ligase that specifically modifies β -catenin protein is mainly β -TrCP. The substrate of E3 ubiquitin ligase VHL is primarily HIF-1 α, playing a central role in regulating oxygen-induced gene expression. The inventor surprisingly found that E3 ubiquitin ligase VHL can perform ubiquitination modification on the beta-catenin protein, so that the beta-catenin protein is degraded by protease to inhibit Wnt/beta-catenin signal pathway. Based on the situation, the inventor further intensively researches and obtains a conjugate which contains two peptide fragments capable of respectively recognizing and combining beta-catenin protein and E3 ubiquitin ligase VHL, and the conjugate can recognize and combine the beta-catenin protein and the E3 ubiquitin ligase VHL in the environment, so that the E3 ubiquitin ligase VHL is favorable for ubiquitinating the beta-catenin protein to be degraded by protease, and the Wnt/beta-catenin signal pathway is inhibited.
To this end, in one aspect of the invention, the invention proposes the use of E3 ubiquitin ligase VHL in degrading β -catenin protein or inhibiting Wnt/β -catenin signalling pathway. As described above, almost one hundred types of ubiquitin ligases have been disclosed so far, each having its own specific substrate for action, wherein the ubiquitin ligase that specifically modifies β -catenin protein is mainly β -TrCP. The substrate of E3 ubiquitin ligase VHL is primarily HIF-1 α, playing a central role in regulating oxygen-induced gene expression. The inventor surprisingly finds that E3 ubiquitin ligase VHL can carry out ubiquitination modification on beta-catenin protein, so that protease degrades ubiquitinated beta-catenin protein, thereby inhibiting Wnt/beta-catenin signal pathway, playing a role in inhibiting cancer cell proliferation and tumorigenicity, and having wide scientific research and clinical application prospects.
According to the embodiment of the invention, the use of the E3 ubiquitin ligase VHL in degrading beta-catenin protein or inhibiting Wnt/beta-catenin signaling pathway may further have the following additional technical features:
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/beta-catenin signal pathway.
According to an embodiment of the invention, the pharmaceutical composition is for use in the treatment of intestinal cancer.
In another aspect of the invention, a method of screening for a drug is provided. According to an embodiment of the invention, the method comprises: contacting the candidate agent with the cell; determining whether the beta-catenin protein is degraded and/or whether the Wnt/beta-catenin signal channel is inhibited in the cells before and after the contact; wherein the degradation of the beta-catenin protein and the inhibition of the Wnt/beta-catenin signal pathway after the contact are indications that the candidate drug is a target drug, and the target drug contains E3 ubiquitin ligase VHL. Therefore, the method for screening the medicine can effectively obtain the medicine containing the E3 ubiquitin ligase VHL, so that the beta-catenin is ubiquitinated, the protease degrades the beta-catenin, and the Wnt/beta-catenin signal pathway is inhibited.
In yet another aspect of the invention, the invention features a conjugate. According to an embodiment of the invention, the conjugate comprises: a first peptide fragment, wherein the first peptide fragment is suitable for being combined with beta-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. The conjugate provided by the embodiment of the invention can identify and combine beta-catenin protein and E3 ubiquitin ligase VHL, so that the E3 ubiquitin ligase VHL can ubiquitinate the beta-catenin protein, the beta-catenin protein is degraded by protease, the Wnt/beta-catenin signal pathway can be inhibited, the effects of inhibiting cancer cell proliferation and tumor formation 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 C1～8Alkyl radical, C1～8Alkylamino radical, C1～8Alkoxy radical, C1～8An alkyl alkenyl group or a polyethylene glycol; wherein, C1～8Alkyl radical, C1～8Alkoxy or C1～8The 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.
In yet another aspect of the invention, a pharmaceutical composition is provided. According to an embodiment of the invention, the pharmaceutical composition comprises the conjugate as described above. The pharmaceutical composition provided by the embodiment of the invention can identify and combine beta-catenin protein and E3 ubiquitin ligase VHL, so that the E3 ubiquitin ligase VHL can ubiquitinate the beta-catenin protein, the beta-catenin protein is degraded so as to inhibit a Wnt/beta-catenin signal pathway, the effects of inhibiting cancer cell proliferation and tumor formation can be 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 an embodiment of the invention, the pharmaceutical composition is used for treating diseases caused by over-activation of Wnt/beta-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.
In yet another aspect of the invention, a method of screening for a drug is provided. According to an embodiment of the invention, the method comprises: contacting the candidate agent with the cell; determining whether the beta-catenin protein is degraded and/or whether the Wnt/beta-catenin signal channel is inhibited in the cells before and after the contact; wherein the degradation of the beta-catenin protein and the inhibition of the Wnt/beta-catenin signaling pathway after the contact are indications that the candidate drug is a target drug. Therefore, the method provided by the embodiment of the invention can be used for screening out the medicines capable of identifying and combining the beta-catenin protein and the E3 ubiquitin ligase VHL, so that the E3 ubiquitin ligase VHL can ubiquitinate the beta-catenin protein, and the beta-catenin protein is degraded by protease, so that the Wnt/beta-catenin signal channel can be inhibited, the effects of inhibiting the cancer cell proliferation and the tumor formation capability are achieved, and the method has wide scientific research and clinical application prospects.
In yet another aspect of the invention, the invention provides a method of preparing the aforementioned conjugate. According to an embodiment of the invention, the method comprises: synthesizing by solid phase synthesis and subjecting the synthesized product to olefin metathesis to obtain a carrier to which the conjugate is bonded; and separating the conjugate from the carrier and removing the protecting group on the conjugate. The method can obtain a conjugate capable of identifying and combining the beta-catenin protein and the E3 ubiquitin ligase VHL, thereby facilitating the E3 ubiquitin ligase VHL to ubiquitinate the beta-catenin protein so as to be degraded by protease, and facilitating the inhibition of the Wnt/beta-catenin signal pathway. In addition, the method is simple and convenient to operate, high in yield, good in 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 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.
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 the beta-catenin protein and the Wnt/beta-catenin signaling pathway 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 beta-catenin protein or inhibition of Wnt/beta-catenin signal pathway, a conjugate and a preparation method thereof, a pharmaceutical composition and a method for screening drugs, which are respectively described in detail below.
E3 ubiquitin ligase VHL in degradation of beta-catenin protein
In one aspect of the invention, the invention provides the use of E3 ubiquitin ligase VHL in degrading beta-catenin protein or inhibiting Wnt/beta-catenin signaling pathway. As described above, almost one hundred types of ubiquitin ligases have been disclosed so far, each having its own specific substrate for action, wherein the ubiquitin ligase that specifically modifies and degrades β -catenin protein is mainly β -TrCP. The substrate of E3 ubiquitin ligase VHL is primarily HIF-1 α, playing a central role in regulating oxygen-induced gene expression. The inventor surprisingly finds that E3 ubiquitin ligase VHL can also carry out ubiquitination modification on the beta-catenin protein, so that protease degrades the ubiquitinated beta-catenin protein, thereby inhibiting a Wnt/beta-catenin signal pathway, playing a role in inhibiting cancer cell proliferation and tumor formation, and having wide scientific research and clinical application prospects.
The application object of the E3 ubiquitin ligase VHL is not strictly limited, such as reagents, kits, medicaments and the like, and can be flexibly selected according to actual conditions. 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 the Wnt/beta-catenin signal pathway. The research shows that the over-activation of the Wnt/beta-catenin signal channel is easy to cause the enhancement of the proliferation capacity of cancer cells. The beta-catenin protein is ubiquitinated specifically by E3 ubiquitin ligase VHL, so that protease degrades the beta-catenin protein, and the Wnt/beta-catenin signal pathway is inhibited from being over-activated, the proliferation and the tumorigenic capacity of cancer cells are inhibited, the treatment purpose is achieved, and the therapeutic effect is good when the polypeptide is particularly applied to the treatment of intestinal cancer cells, such as intestinal cancer.
Method for screening drugs
In another aspect of the present invention, a method of screening for a drug is provided according to embodiments of the present invention. According to an embodiment of the invention, the method comprises: contacting the candidate agent with the cell; determining whether the beta-catenin protein is degraded and/or whether the Wnt/beta-catenin signal channel is inhibited in the cells before and after the contact; wherein, after the beta-catenin protein degradation and the Wnt/beta-catenin signal channel inhibition are indicated, the candidate drug is the target drug, and the target drug contains E3 ubiquitin ligase VHL.
As previously described, E3 ubiquitin ligase VHL is capable of specifically recognizing and ubiquitinating its β -catenin protein, thereby facilitating protease degradation thereof to inhibit the Wnt/β -catenin signaling pathway. Therefore, whether the beta-catenin protein is degraded and/or whether the Wnt/beta-catenin signal channel is inhibited before and after the candidate drug contacts the cell is determined, and when the beta-catenin protein and the Wnt/beta-catenin signal channel are inhibited after the candidate drug contacts the cell, the candidate drug is proved to contain E3 ubiquitin ligase VHL, so that the aim of accurately screening the target drug is achieved.
Conjugates
In yet another aspect of the invention, the invention features a conjugate. According to an embodiment of the invention, the conjugate comprises: a first peptide fragment, wherein the first peptide fragment is suitable for being combined with beta-catenin protein; a second peptide fragment suitable for binding to E3 ubiquitin ligase VHL; and the first peptide segment is connected with the second peptide segment through the connecting arm. Therefore, the first peptide segment can specifically identify and combine beta-catenin protein, and the second peptide segment can specifically recruit E3 ubiquitin ligase VHL, so that the E3 ubiquitin ligase VHL can ubiquitinate the beta-catenin protein and degrade the beta-catenin protein by protease, a Wnt/beta-catenin signal channel can be inhibited, the effects of inhibiting cancer cell proliferation and tumor formation can be achieved, and the wide scientific research and clinical application prospect can be realized.
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 specifically recognize and bind to the β -catenin protein, and the second peptide fragment can specifically recognize and bind to the E3 ubiquitin ligase VHL, which can be flexibly selected according to the actual situation.
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). The inventors found that the sequence of SEQ ID NO: 1-7 can be identified and combined with beta-catenin protein. And the amino acid sequence is short, so that the synthesis is convenient. Wherein, SEQ ID NO: the amino acid sequence shown in1 has strong specificity and good binding capacity for recognizing and binding the beta-catenin protein.
The first peptide fragment of the present invention may be not only one of the above 7 amino acid sequences, but also have at least 80%, 85%, 90%, 95% homology with these amino acids. For example, any 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 alteration of the position of insertion of an unnatural amino acid (S5), and the present invention is not limited thereto, as long as the above requirement for homology can be satisfied and the 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 C1～8Alkyl radical, C1～8Alkylamino radical, C1～8Alkoxy radical, C1～8An alkyl alkenyl group or a polyethylene glycol; wherein, C1～8Alkyl radical, C1～8Alkylamino radical, C1～8Alkoxy or C1～8The 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 C3～6Alkyl radical, C3～6Alkylamino radical, C3～6Alkoxy radical, C3～6An alkyl alkenyl group or a polyethylene glycol; wherein, C3～6Alkyl radical, C3～6Alkylamino radical, C3～6Alkoxy or C3～6The 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.
According to an embodiment of the present invention, the N-terminus of the first peptide fragment is further connected with an acetyl group. Thereby, 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 beta-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.
According to an embodiment of the invention, the conjugate has the amino acid sequence of SEQ ID NO: 10, or a fragment thereof. The inventor obtains the nucleotide sequence shown in SEQ ID NO: 10, which is capable of specifically recognizing and firmly binding to a beta-catenin protein and an E3 ubiquitin ligase VHL.
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. High activity and high selectivity: compared with small molecules, the conjugate provided by the invention has a larger action surface, so that when the conjugate is applied to inhibiting a Wnt/beta-catenin signal transduction pathway, the conjugate has the characteristics of high activity and high selectivity, and other signal pathways are not influenced.
3. Direct penetration of the cell membrane: the polypeptide provided by the invention basically keeps a natural alpha-helical structure, is positively charged under physiological conditions, and is favorable for penetrating a cell membrane 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. Persistent inhibition of the Wnt signaling pathway: the conjugate provided by the invention can continuously degrade beta-catenin in cells so as to inhibit a Wnt signal 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 to the beta-catenin protein, and the second peptide segment can specifically recruit E3 ubiquitin ligase VHL, thereby facilitating the ubiquitination of the beta-catenin protein by the E3 ubiquitin ligase VHL and degrading the beta-catenin protein to inhibit the Wnt/beta-catenin signal pathway. Therefore, whether the beta-catenin protein is degraded and/or whether the Wnt/beta-catenin signal path is inhibited before and after the candidate drug contacts the cell is determined, and when the beta-catenin protein and the Wnt/beta-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 method 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 the Wnt/beta-catenin signal pathway. Over-activation of the Wnt/beta-catenin signaling pathway is likely to result in enhanced proliferation capacity of cancer cells. The conjugate can specifically recognize and combine beta-catenin protein and E3 ubiquitin ligase VHL, so that the E3 ubiquitin ligase VHL can ubiquitinate the beta-catenin protein, the beta-catenin protein is degraded by protease, the over-activation of a Wnt/beta-catenin signal channel is inhibited, the proliferation and tumor forming capacity of cancer cells are inhibited, and the therapeutic purpose is achieved, and the conjugate is particularly applied to the treatment of 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 beta-catenin protein and E3 ubiquitin ligase VHL, thereby facilitating ubiquitination of the beta-catenin protein, degrading the beta-catenin protein by protease, and inhibiting the Wnt/beta-catenin signaling pathway, and are effective in treating or preventing tumors, so that the conjugate and the pharmaceutical composition containing the conjugate can be administered in the treatment of corresponding cancers.
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
In yet another aspect of the invention, a method of screening for a drug is provided. According to an embodiment of the invention, the method comprises: contacting the candidate agent with the cell; determining whether the beta-catenin protein is degraded and/or whether the Wnt/beta-catenin signal channel is inhibited in the cells before and after the contact; wherein the degradation of the beta-catenin protein and the inhibition of the Wnt/beta-catenin signal pathway after the contact are indications that the candidate drug is the target drug, and the target drug is selected from the pharmaceutical compositions. Therefore, the method provided by the embodiment of the invention can be used for screening out the medicines capable of identifying and combining the beta-catenin protein and the E3 ubiquitin ligase VHL, so that the E3 ubiquitin ligase VHL can ubiquitinate the beta-catenin protein, and the beta-catenin protein is degraded by protease, so that the Wnt/beta-catenin signal channel can be inhibited, the effects of inhibiting the cancer cell proliferation and the tumor formation capability are achieved, and the method has wide scientific research and clinical application prospects.
Process for preparing conjugates
In yet another aspect of the invention, the invention provides a method of preparing the aforementioned conjugate. According to an embodiment of the invention, the method comprises: synthesizing by using a solid-phase synthesis method, and subjecting the synthesized product to olefin metathesis reaction so as to obtain a carrier bonded with the conjugate; and separating the conjugate from the carrier and removing the protecting group on the conjugate. The method can obtain a conjugate capable of identifying and combining the beta-catenin protein and the E3 ubiquitin ligase VHL, thereby facilitating the E3 ubiquitin ligase VHL to ubiquitinate the beta-catenin protein so as to be degraded by protease, and facilitating the inhibition of the Wnt/beta-catenin signal pathway. In addition, the method is simple and convenient to operate, high in yield, good in 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 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 "Peptide C18"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 during preparation, freeze drying to obtain target product, and mixingThe pure product is subjected to HPLC analysis and HR-Q-TOF-MS identification.
Example 1 specific degradation of beta-catenin proteins and inhibition of the Wnt/beta-catenin signaling pathway with 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).
As shown in a of fig. 1, when Wnt3a was stimulated, β -catenin protein level was significantly increased, and only conjugate 2 had significant down-regulation effect on β -catenin protein level, while the polypeptide without E3 ubiquitin ligase VHL recognition peptide (experimental group 3) had no significant effect on β -catenin protein level, and in addition, other important components in Wnt/β -catenin signaling pathway, Axin1, Dvl2, GSK3 β and TCF4 were not significantly changed, which indicates that conjugate 2 only acts on β -catenin protein to degrade it.
2. A Topflash-luciferase reporter gene capable of responding to Wnt/beta-catenin signals was transfected in cultured HEK293T cells using PEI transfection reagent (Polysciences) with a Renilla reference. After 24 hours of transient expression of the reporter gene, cells were treated with Wnt3a and the polypeptide for 24 hours. The harvested cells are tested for luciferase activity to assess the degree of activation of Wnt/beta-catenin signals. The test substances added to each experimental group and control group were the same as those added to the above 1.
As shown in fig. 1B, trace amount of Wnt3a could strongly activate Wnt/β -catenin signaling pathway, and experimental groups 1 and 2 still activated Wnt/β -catenin signaling; experimental group 3 could inhibit Wnt/β -catenin signaling to some extent, while conjugate 2 at the same concentration could inhibit Wnt/β -catenin signaling pathway more strongly.
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 experimentmin/+The mouse, the APC gene is inactivated due to deletion mutation of the APC gene, so that continuous activation of Wnt/beta-catenin signal pathway is caused, and the most obvious phenotype is that a large number of tumors grow in intestinal tracts for weeks after birth. Selecting APC of 11 weeks of agemin/+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 finally all the mice were collected on the 14 th day, and the number of tumors in the intestinal tracts thereof was recorded by dissection. As shown in FIG. 3, the number of intestinal tumors in mice treated with DMSO and experimental groups 2 and 3 was not significantly changed 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 signalingmin/+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 beta-catenin protein
<130> PIDC3194992
<160> 11
<170> PatentIn version 3.5
<210> 1
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<212> PRT
<213> Artificial Sequence
<220>
<223> SEQ ID NO：1
<220>
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<222> (6)..(10)
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Arg Arg Trp Pro Arg S5 Ile Leu Asp S5 His Val Arg Arg Val Trp
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Arg
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<212> PRT
<213> Artificial Sequence
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<221> MISC_FEATURE
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<211> 13
<212> PRT
<213> Artificial Sequence
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<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
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<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
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<210> 6
<211> 16
<212> PRT
<213> Artificial Sequence
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<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
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Arg Arg Trp Pro Gln S5 Ile Leu Asp S5 His Val Arg Arg Val Trp
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Arg
<210> 8
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<213> Artificial Sequence
<220>
<223> SEQ ID NO：8
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Ala Leu Ala Pro Tyr Ile Pro
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<210> 9
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<220>
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<222> (6)..(10)
<223> to each other through an alkyl alkenylalkyl group
<220>
<221> MISC_FEATURE
<222> (17)..(18)
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<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
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<221> MISC_FEATURE
<222> (1)..(1)
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<221> MISC_FEATURE
<222> (3)..(10)
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<400> 10
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1 5 10
<210> 11
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<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)
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<220>
<221> MISC_FEATURE
<222> (21)..(21)
<223> linking amino group
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1 5 10 15
Ala Pro Tyr Ile Pro
20
Claims (7)
1. A conjugate, wherein the conjugate is SEQ ID NO: 10, or a fragment thereof.
2. A pharmaceutical composition comprising the conjugate of claim 1.
3. The pharmaceutical composition of claim 2, further comprising a pharmaceutically acceptable excipient.
4. Use of the pharmaceutical composition of claim 2 or 3 for the preparation of a medicament for the treatment of a disease caused by over-activation of the Wnt/β -catenin signalling pathway.
5. The use according to claim 4, wherein the pharmaceutical composition is for inhibiting the proliferation and tumorigenic capacity of intestinal cancer cells.
6. The use according to claim 4, wherein the pharmaceutical composition is for the treatment of colon cancer.
7. A method of screening for a drug comprising:
contacting the candidate agent with the cell;
determining whether the beta-catenin protein is degraded and/or whether the Wnt/beta-catenin signal channel is inhibited in the cells before and after the contact;
wherein the degradation of the β -catenin protein and the inhibition of the Wnt/β -catenin signaling pathway upon contact is indicative that the candidate drug is a drug of interest selected from the pharmaceutical compositions of claims 2 or 3.
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