CN114181294B

CN114181294B - Blocking polypeptide Q peptide and application thereof

Info

Publication number: CN114181294B
Application number: CN202111516417.XA
Authority: CN
Inventors: 孙建民; 赵晖; 蒋宗英; 郭月
Original assignee: Ningxia Medical University
Current assignee: Ningxia Medical University
Priority date: 2021-12-09
Filing date: 2021-12-09
Publication date: 2023-08-18
Anticipated expiration: 2041-12-09
Also published as: CN114181294A

Abstract

The invention provides a blocking polypeptide Q peptide and application thereof, belonging to the technical field of biological medicines. The amino acid sequence of the blocking polypeptide Q peptide is shown as SEQ ID No.1, and the blocking polypeptide Q peptide can competitively block the combination of PI3K regulatory subunit and catalytic subunit, thereby inhibiting the activation of PI3K molecules and inhibiting the proliferation of tumor cells. The invention provides a new idea for treating tumors.

Description

Blocking polypeptide Q peptide and application thereof

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to a blocking polypeptide Q peptide and application thereof.

Background

PI3ks are involved in a variety of important pathophysiological processes, playing an important role in tumor cell metabolism, growth, proliferation, angiogenesis and tumor metastasis. Excessive activation of PI3K is associated with 30-50% of human tumor cases, and inhibition of PI3K activation can be used as a target for tumor treatment.

The PI3K inhibitor can inhibit the activation of PI3K to inhibit tumor, and the current small molecule inhibitors aiming at PI3K comprise a pan PI3K inhibitor, a PI3K subtype specific inhibitor and a PI3K/mTOR dual inhibitor. The pan-PI 3K inhibitor is an ATP competitive inhibitor, can non-selectively inhibit all subtypes (alpha, beta, delta, gamma) of the I-type PI3K, has poor specificity, and can cause more side reactions, such as fatigue, diarrhea, rash, hyperglycemia and the like, and has dose-dependent toxicity. Thus, many of the pan PI3K inhibitors have terminated in different stages of clinical trials, with only Copanlisib approved for clinical use by the FDA. PI3K subtype specific inhibitors can selectively inhibit different PI3K subtypes, thus resulting in a narrower therapeutic range, but reduced certain toxic effects due to off-target effects of the inhibitor. The side effects of such inhibitors are mainly dependent on their specificity for PI3K isomers, e.g. PI3K alpha inhibitors are mainly responsible for rash and hyperglycemia, PI3K delta inhibitors are mainly gastrointestinal reactions, elevated transaminases and myelosuppression. Among PI3K subtype specific inhibitors, idelalisib (Idelalisib), ubrilisib (Duvelisib), duloxetine (alpeliib) and alpellib (alpeliib) have been approved for clinical use, idelalisib as the first FDA approved PI3K delta inhibitor, and can be used for chronic granulocytic leukemia treatment, but side reactions such as autoimmune reactions, infections, hepatotoxicity and diarrhea due to off-target effects of inhibitors occur to various degrees during the course of treatment. The PI3K/mTOR dual inhibitors have broader toxic effects and no drug has been approved for clinical use by the FDA. Thus, inhibition of PI3K activation by PI3K inhibitors presents a number of problems, and it is necessary to design drugs that inhibit PI3K activation from a new direction.

PI3K molecules can be classified into 3 major classes, type i, type ii, type iii, based on their substrate specificity and structural characteristics. Type i PI3Ks are divided into two subtypes (a and B) according to the mode of regulation, with type IA playing an important role in tumors, type IA PI3Ks consisting of regulatory subunits (p85α, p85β, p55α, p55γ, p50α) and catalytic subunits (p110α, p110β, p110δ) that bind to form dimers being the basis for the structural integrity of type IA PI3K molecules and activation. Currently, there is no report on inhibition of PI3K molecule activation by competitive blocking of PI3K regulatory subunit binding to catalytic subunits.

Disclosure of Invention

In view of the above, the present invention aims to provide a blocking polypeptide Q peptide and application thereof, wherein the blocking polypeptide Q peptide competitively blocks the binding of PI3K regulatory subunit and catalytic subunit, thereby inhibiting the activation of PI3K molecules.

The invention provides a blocking polypeptide Q peptide, the amino acid sequence of which is shown as SEQ ID No. 1.

The invention also provides application of the blocking polypeptide Q peptide in the preparation of the PI3K inhibitor.

Preferably, the PI3K inhibitor comprises a PI3K competitive inhibitor.

The invention also provides application of the blocking polypeptide Q peptide in preparation of antitumor drugs.

Preferably, the anti-tumor comprises inhibiting proliferation of tumor cells.

Preferably, the tumor comprises a gastrointestinal stromal tumor and/or a neuroblastoma.

The invention also provides an anti-tumor drug, which comprises the blocking polypeptide Q peptide and pharmaceutically acceptable auxiliary materials.

Preferably, the dosage form of the medicament comprises an injection or a suppository.

The invention also provides a PI3K inhibitor, which comprises the blocking polypeptide Q peptide and pharmaceutically acceptable auxiliary materials.

The invention provides a blocking polypeptide Q peptide. The blocking polypeptide Q peptide of the invention competitively blocks the combination of PI3K regulatory subunit and catalytic subunit, thereby inhibiting the activation of PI3K molecules and inhibiting the proliferation of tumor cells. The invention provides a new idea for treating tumors.

Drawings

FIG. 1 shows the viability of cells of the human gastrointestinal stromal tumor cell line GIST-T1 after 48h of treatment with the blocking polypeptide;

FIG. 2 shows the viability of cells of the human cell line BE (2) C cells after 48h of treatment with the blocking polypeptide;

FIG. 3 shows the viability of cells after 48h of treatment of human neuroblastoma cell line Kelly with blocking polypeptides.

Detailed Description

The invention provides a blocking polypeptide Q peptide, the amino acid sequence of which is shown as SEQ ID No.1, which comprises the following specific steps: NFAEIATRKMQPPRRRQRRKKRGY.

In the invention, the blocking polypeptide Q peptide comprises a segment of PI3K blocking sequence, as shown in SEQ ID No.2, specifically comprising the following components: NFAEIATRKMQ, PI3K blocking sequences can competitively block the binding of the p85 subunit of the PI3K molecule to the p110 subunit, thereby inhibiting PI3K molecule activation. However, the PI3K blocking sequence cannot enter cells, so that a TAT short peptide sequence derived from HIV is added, as shown in SEQ ID No.3, specifically: PPRRRQRRKKRGY the TAT short peptide sequence derived from HIV assists the blocking of PI3K blocking sequences into cells. The blocking polypeptide Q peptide of the invention competitively blocks the combination of PI3K regulatory subunit and catalytic subunit, thereby inhibiting the activation of PI3K molecules and inhibiting the proliferation of tumor cells.

The preparation method of the blocking polypeptide Q peptide is not particularly limited, and the method is a method for synthesizing a conventional polypeptide in the field. In the specific implementation process of the invention, the blocking polypeptide Q peptide is a fully synthesized full D-type amino acid sequence.

In the present invention, the PI3K inhibitor preferably comprises a PI3K competitive inhibitor.

In the present invention, the antitumor includes inhibiting tumor cell proliferation.

The invention also provides an anti-tumor drug, which comprises the blocking polypeptide Q peptide and pharmaceutically acceptable auxiliary materials. The auxiliary materials are not particularly limited, and conventional auxiliary materials in the field can be adopted.

In the present invention, the dosage form of the drug preferably includes an injection or a suppository.

In the present invention, the tumor preferably comprises a gastrointestinal stromal tumor and/or a neuroblastoma, more preferably comprises a human gastrointestinal stromal tumor or a human neuroblastoma. In the present invention, the human neuroblastoma preferably includes BE (2) C cells or a human neuroblastoma cell line Kelly.

The technical solutions of the present invention will be clearly and completely described in the following in connection with the embodiments of the present invention.

Example 1

1. The blocking polypeptide Q peptide is a fully synthesized full D-type amino acid sequence, the amino acid sequence is shown as SEQ ID No.1, and the blocking polypeptide Q peptide specifically comprises: NFAEIATRKMQPPRRRQRRKKRGY.

Preparing polypeptide solution: with a commercially available sterile PBS solution (0.0067 mol/L PO ₄ Does not contain calcium and magnesiumThe total synthesized D-type Q peptide and TAT short peptide (PPRRRQRRKKRGY, SEQ ID No.4, TAT short peptide is one amino acid sequence for assisting short peptide to enter cells in the prior art) powder are respectively dissolved by ions with pH of 7.0-7.2 to prepare polypeptide storage solution with the concentration of 5mmol/L, the polypeptide storage solution is preserved at-80 ℃, and the polypeptide storage solution can be temporarily stored at-20 ℃. The volume of sterile PBS solution added was calculated as (1):

wherein V represents the volume (L) of added sterile PBS; m represents the mass (g) of the fully synthesized D-type polypeptide; p represents the purity (%) of the total synthesized D-type polypeptide; mw represents the molecular weight (g/mol) of the total synthesized D-form polypeptide.

2. Through a cell proliferation (MTT) experiment, the Q peptide has a definite effect of inhibiting the proliferation of tumor cells compared with a TAT short peptide in a control group.

The cell proliferation assay procedure was as follows:

(1) Preparing GIST-T1 cells, BE (2) C cells, kelly cells, a pipette, a sterilizing gun head, a sterile 96-well plate, pancreatin, DMEM culture medium, gibco fetal bovine serum, 100 Xgreen streptomycin and the like;

(2) Respectively digesting and centrifuging three tumor cells in a biosafety cabinet to obtain precipitate, adding culture medium to resuspend into cell suspension, counting cells by using a cell counting plate, and diluting the cell suspension to 8×10 ⁴ Uniformly mixing diluted cell suspension, inoculating into 96-well plates, inoculating 36 wells into each tumor cell, and supplementing the volume of the rest empty wells with PBS;

(3) Three cell culture media were prepared: gist-T1 cell culture medium formulation: DMEM medium, 15% fetal bovine serum, 1% penicillin; BE (2) C cell culture Medium formulation: DME/F-12 medium, 10% fetal bovine serum, 1% green streptomycin; kelly cell culture medium formulation: 1640 medium, 10% foetal calf serum, 1% green streptomycin. After 24h, the cells were adherent, and the blocking polypeptide Q peptide and the control polypeptide were diluted with GIST-T1 cell culture medium to final concentrations of 0, 2.5, 5, 10, 20, 40. Mu.M, respectively. Diluting the blocking polypeptide Q peptide and the control group polypeptide with BE (2) C cell culture medium and Kelly cell culture medium respectively to obtain final concentrations of 0, 6.25, 12.5, 25, 50 and 100 μm, sucking out three kinds of tumor cell culture mediums respectively, adding cell culture mediums containing polypeptides with different concentrations respectively, and culturing in a 37 deg.C incubator for 48 hr;

(4) MTT reagent is respectively added into 108 holes of the 2 96-well plates, 50 mu L of each hole is put into a 37 ℃ incubator for 4 hours of incubation;

(5) The liquid in these 108 wells was aspirated, 150 μl of DMSO was added to dissolve the resulting blue-violet pellet, the 96 well plate was gently shaken to dissolve the pellet, the absorbance of the solution at 490nm was measured with a microplate reader and the cell viability was calculated as per (2):

wherein, the liquid crystal display device comprises a liquid crystal display device,refers to the average value of absorbance at 490nm for all wells of the control group with polypeptide concentration 0; n refers to the absorbance at 490nm of experimental wells treated with different concentrations of the polypeptide; k refers to the absorbance value at 490nm of a control well with a polypeptide concentration of 0.

(6) And drawing a survival rate graph according to the calculated cell survival rate.

The experimental results are shown in fig. 1 to 3. As can be seen from fig. 1 to 3, the Q peptide has a clear effect of inhibiting tumor cell proliferation compared with the TAT short peptide of the control group.

Although the foregoing embodiments have been described in some, but not all, embodiments of the invention, according to which one can obtain other embodiments without inventiveness, these embodiments are all within the scope of the invention.

Claims

1. A blocking polypeptide Q peptide has an amino acid sequence of NFAEIATRKMQPPRRRQRRKKRGY.

2. Use of a blocking polypeptide Q peptide according to claim 1 for the preparation of an anti-tumour medicament, wherein the anti-tumour is in the inhibition of gastrointestinal stromal tumour and/or neuroblastoma cell proliferation.

3. An antitumor drug comprising the blocking polypeptide Q peptide of claim 1 and a pharmaceutically acceptable adjuvant, wherein the tumor is gastrointestinal stromal tumor and/or neuroblastoma.

4. A medicament according to claim 3, in the form of an injection or suppository.