阅读说明：本技术 一种靶向fgfr1的拮抗短肽 (FGFR 1-targeted antagonistic short peptide ) 是由 吴建章 李物兰 陈玲姿 范蕾 蔡跃飘 于 2019-12-10 设计创作，主要内容包括：本发明属短肽药物学领域,具体涉及FGFR1拮抗短肽的稳定性研究及其抗肿瘤领域的应用,公开了一种FGFR1拮抗短肽,包括短肽化合物P48,短肽化合物P48通过抑制FGFR1通路,抑制细胞增殖和迁移。上述技术方案,本发明人通过不懈努力,获得了短肽化合物P48,并发现其具有稳定的二级结构及相对较长的半衰期。此外,短肽化合物P48能有效靶向抑制FGFR1,在体内外表现出良好的抗肿瘤活性,有望成为癌症治疗的候选肽类抑制剂药物。(The invention belongs to the field of short peptide pharmacology, particularly relates to stability research of FGFR1 antagonistic short peptide and application of the antagonistic short peptide in the field of anti-tumor, and discloses FGFR1 antagonistic short peptide which comprises a short peptide compound P48, wherein the short peptide compound P48 inhibits cell proliferation and migration by inhibiting an FGFR1 channel. The present inventors have diligently made efforts to obtain the short peptide compound P48, and found that it has a stable secondary structure and a relatively long half-life. In addition, the short peptide compound P48 can effectively inhibit FGFR1 in a targeted mode, shows good antitumor activity in vivo and in vitro, and is expected to become a candidate peptide inhibitor drug for cancer treatment.)

1. Antagonistic short targeting FGFR1A peptide characterized by: comprises a short peptide compound P48, wherein the short peptide compound P48 can target and combine with an extramembranous immunoglobulin domain of FGFR1, and the amino acid sequence of the FGFR1 antagonistic short peptide is Ser-Pro-Pro-Arg-Tyr-Pro-Gly-Gly-Ser-NH₂。

2. The antagonistic short peptide targeting FGFR1 according to claim 1, which is characterized in that: FGFR1 antagonizes the use of short peptides as active ingredients in medicaments.

3. The FGFR 1-targeted antagonistic short peptide of claim 1, which is characterized in that: the FGFR1 antagonizes the short peptide, and can be used for treating diseases related to abnormal disorder of FGFR1 by antagonizing an in vivo FGFR1 signal pathway.

4. The FGFR 1-targeted antagonistic short peptide of claim 1, which is characterized in that: aberrant deregulation of FGFR1 includes high FGFR1 expression and mutations.

5. The FGFR 1-targeted antagonistic short peptide of claim 3 or 4, which is characterized in that: the FGFR1 antagonizes the short peptide treatment to tumor.

6. The FGFR 1-targeted antagonistic short peptide of claim 5, which is characterized in that: FGFR1 antagonizes short peptide P48 and inhibits growth and invasion of tumor cells by inhibiting FGFR1 pathway.

7. The antagonistic short peptide targeting FGFR1 according to claim 6, which is characterized in that: the short peptide compound P48 has good anti-tumor activity in vitro and in vivo.

8. The FGFR 1-targeted antagonistic short peptide of claim 7, which is characterized in that: the short peptide compound P48 has stable secondary structure and relatively prolonged half-life.

9. The antagonistic short peptide targeting FGFR1 according to claim 1, which is characterized in that: the FGFR1 antagonistic short peptide can be prepared into medicinal salts, esters or medicinal auxiliary materials.

10. The antagonistic short peptide targeting FGFR1 according to claim 1, which is characterized in that: the FGFR1 antagonistic short peptide drug is prepared into injections, tablets, capsules, aerosols, suppositories, films, controlled release, sustained release agents or nanometer preparations in the form of preparations.

Technical Field

The invention relates to the technical field of short peptide pharmacology, in particular to an antagonistic short peptide targeting FGFR1.

Background

Fibroblast growth factor receptor 1(FGFR1) is involved in the development of various tumors including breast cancer, lung cancer, bladder cancer, ovarian cancer, pancreatic cancer, gastric cancer and the like. FGFR1 is an effective target for cancer therapy. At present, no FGFR1 inhibitor is approved to be on the market, and the search of an effective FGFR1 targeted inhibitor has important research value.

The targeted inhibitor is mainly divided into a small molecule inhibitor, an antibody drug, a peptide inhibitor and derivatives thereof. Compared with small molecule inhibitors, peptide inhibitors have high affinity and specificity and have low adverse reaction. Compared with antibody drugs, peptide inhibitors have low molecular weight and stronger tissue penetration activity. In view of the above-mentioned advantages of peptide inhibitors, it is becoming more and more appreciated by researchers. By 2015, over 60 peptide inhibitors have been approved for marketing globally and over 140 peptide inhibitors are in clinical research. However, studies on FGFR1 antagonist peptides are still stopped in the preclinical phase.

There is also no related patent of targeting FGFR1 to tumors by peptide inhibitors. The research lag of the FGFR1 antagonistic peptide is mainly related to the easy degradation and short half-life period of the antagonistic peptide.

Disclosure of Invention

Aiming at the defects in the prior art, the invention aims to provide an antagonistic short peptide targeting FGFR1. The short peptide compound P48 can effectively target and inhibit FGFR1 and shows good antitumor activity in vitro and in vivo.

In order to achieve the purpose, the invention provides the following technical scheme: an antagonistic short peptide targeting FGFR1, comprising a short peptide compound P48, said short peptide compound P48 being targetable to bind to the extracellular immunoglobulin domain of FGFR1, the amino acid sequence of the FGFR1 antagonistic short peptide being:

Ser-Pro-Pro-Arg-Tyr-Pro-Gly-Gly-Gly-Ser-NH₂。

preferably, the FGFR1 antagonizes the short peptide as an active ingredient for use in medicine.

Preferably, the FGFR1 antagonist short peptide is used for treating diseases related to abnormal disorder of FGFR1 by antagonizing an in vivo FGFR1 signal pathway.

Preferably, the aberrant deregulation of FGFR1 comprises high FGFR1 expression and mutation.

Preferably, the FGFR 1-antagonistic short peptide-treated disease is a tumor.

Preferably, FGFR1 antagonizes short peptide P48 and inhibits growth and invasion of tumor cells by inhibiting the FGFR1 pathway.

Preferably, the short peptide compound P48 has a stable secondary structure and a relatively prolonged half-life.

Preferably, the FGFR1 antagonistic short peptide can be prepared into medicinal salts, esters or medicinal auxiliary materials.

Preferably, the FGFR1 antagonistic short peptide drug is prepared into injections, tablets, capsules, aerosols, suppositories, films, controlled release, sustained release agents or nano preparations in the form of preparations.

The invention has the advantages that: compared with the prior art, the inventor has diligently tried to obtain the short peptide compound P48, and finds that the short peptide compound P48 has a stable secondary structure and a relatively long half-life. In addition, the short peptide compound P48 can effectively inhibit FGFR1 in a targeted mode, shows good antitumor activity in vivo and in vitro, and is expected to become a candidate peptide inhibitor drug for cancer treatment.

The invention is further described with reference to the drawings and the specific embodiments in the following description.

Drawings

FIG. 1 is a schematic diagram of the analysis of the structure, stability and binding ability to FGFR1 of short peptide compound P48 according to the example of the present invention;

FIG. 2 is a schematic diagram of the activity of detecting short-peptide compound P48 in HEK-293 highly transformed human embryonic kidney cells and MEF-WT, Balb/c3T3 in inhibiting FGFR1 signaling pathway according to the embodiment of the present invention;

FIG. 3 is a schematic diagram showing the inhibitory activity of a short peptide compound P48 on FGFR1 signaling pathway in various tumor cell lines;

FIG. 4 is a graph showing the in vitro anti-tumor activity of the short peptide compound P48 according to the present invention;

FIG. 5 is a schematic diagram showing the in vivo antitumor activity of short peptide compound P48 according to the present invention.

Detailed Description

The invention is further illustrated in the following examples. These examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

Referring to fig. 1: structure, stability and binding ability to FGFR1 analysis of short-peptide compound P48

(A) The sequences of the precursor peptide P9 and the degraded short peptide P48. (B) a: RMSD of C atoms in the P9 and P48 backbones varied with time as a function of molecular dynamics simulation. b: initial conformations of P9 and P48 and conformational superimpositions at 4ns, 8ns, 12ns, 16ns and 20 ns. (C) Half-life of P9 and P48 in human plasma. (D) a: molecular dynamics simulation and free energy calculation of bFGF-FGFR1 complex. b: key residue analysis to maintain stability of bFGF-FGFR1 complex. (E) Molecular docking of FGFR1 and P48. (F) SPR detects the interaction of FGFR1 and P48.

FIG. 2: the inhibition activity of P48 on FGFR1 signaling pathway is detected in highly transformed human embryonic kidney cell HEK-293 and fibroblast MEF-WT, Balb/c3T 3.

(A) After pretreatment of short-peptide compound P48 for 10 minutes or AZD4547/PD173074 for 2 hours, bFGF was incubated for 15 minutes, inhibition of the drug on FGFR1 and its downstream proteins in HEK-293, MEF-WT, Balb/C3T3 cells was examined, (B) after pretreatment of P48 for 10 minutes or AZD4547/SSR for 2 hours, bFGF stimulation for 15 minutes, inhibition of the drug on PLC γ was examined, (C) flow cytometry analysis of the ability of P48 to bind FGFR1 in MEF-WT and FGFR1-FGFR2-FRS2 α knockout MEF cells, P < 0.05;, P <0.001, vs Control.

FIG. 3: the short peptide compound P48 has inhibitory activity on FGFR1 signal channel in various tumor cell lines.

(A) After the short peptide compound P48 is pretreated for 10 minutes, bFGF is incubated for 15 minutes, and the inhibition of the drug on FGFR1 and the downstream protein thereof is detected. (B) After pretreatment of P48 for 10 minutes or AZD4547/SSR for 2 hours, bFGF was incubated for 15 minutes, and the inhibitory activity of the drug on phosphorylated FGFR1 and its downstream proteins in SGC-7901 cells was examined. (C) And (3) stimulating bFGF/aFGF for 15 minutes or stimulating KGF for 20 minutes, and detecting the inhibitory activity of the short-peptide compound P48 on phosphorylated FGFR1 and proteins downstream of the phosphorylated FGFR1 in SGC-7901 cells. (D) EGF was stimulated for 10 minutes and the inhibitory activity of short peptide compound P48 on phosphorylated EGFR and ERK1/2 in PC-9 cells was examined. (E) Flow cytometry was used to examine the binding ability of short peptide compound P48 to FGFR1 in SGC-7901 cells.

FIG. 4: the in vitro antitumor activity of the short peptide compound P48.

(A) The growth inhibition effect of the short peptide compound P48 on cancer cells is detected by an MTT method. (B) The inhibitory activity of the short peptide compound P48 on SGC-7901 cell migration is detected by a cell invasion experiment. (C) The cell cycle arrest effect of the short peptide compound P48 was analyzed by flow cytometry. P <0.001, vs bFGF.

FIG. 5: the in vivo antitumor activity of the short peptide compound P48.

(A) Tumor volume measurements of control and dosing groups. (B) Tumor weight weighing. (C) Inhibition of phosphorylation of FLG and its downstream proteins by short peptide compound P48 in nude mice. (D) Immunohistochemistry detected the inhibitory activity of short peptide compound P48 on the proliferative protein Ki 67.

The invention discloses an antagonistic short peptide targeting FGFR1, which comprises a short peptide compound P48, wherein the short peptide compound P48 can target and combine with an extramembranous immunoglobulin domain of FGFR1, and the amino acid sequence of the antagonistic short peptide of FGFR1 is as follows:

Ser-Pro-Pro-Arg-Tyr-Pro-Gly-Gly-Gly-Ser-NH₂。

preferably, the FGFR1 antagonizes the short peptide as an active ingredient for use in medicine.

Preferably, the FGFR1 antagonist short peptide is used for treating diseases related to abnormal disorder of FGFR1 by antagonizing an in vivo FGFR1 signal pathway.

Preferably, the aberrant deregulation of FGFR1 comprises high FGFR1 expression and mutation.

Preferably, the FGFR 1-antagonistic short peptide-treated disease is a tumor.

Preferably, FGFR1 antagonizes short peptide P48 and inhibits growth and invasion of tumor cells by inhibiting the FGFR1 pathway.

Preferably, the short peptide compound P48 has a stable secondary structure and a relatively prolonged half-life.

Preferably, the FGFR1 antagonistic short peptide can be prepared into medicinal salts, esters or medicinal auxiliary materials.

Preferably, the FGFR1 antagonistic short peptide drug is prepared into injections, tablets, capsules, aerosols, suppositories, films, controlled release, sustained release agents or nano preparations in the form of preparations.

Specifically, the invention obtains the FGFR1 antagonistic short peptide, and the amino acid sequence of the antagonistic short peptide is as follows (example 1):

Ser-Pro-Pro-Arg-Tyr-Pro-Gly-Gly-Gly-Ser-NH₂

the experimental result shows that compared with the precursor peptide P9, the degraded short peptide compound P48 discovered by the invention has stable secondary structure and relatively prolonged half-life. In addition, the molecular docking experiment shows that the short peptide compound P48 and FGFR1 can form stable hydrogen bonds. SPR experiments show that P48 can compete with bFGF in binding FGFR1, and the binding force is stronger at higher concentration (example 1).

Meanwhile, Western blot experiments show that in highly transformed human embryonic kidney cells HEK-293 and fibroblast MEF-WT and Balb/c3T3, a short peptide compound P48 can inhibit the activation of FGFR1 induced by bFGF in a concentration-dependent manner (example 2). furthermore, the inhibition effect of the short peptide compound P48 on FGFR1 signals is similar to that of SSR, the short peptide compound P48 has no obvious inhibition effect on downstream PLC gamma (example 2). wild-type MEF cells and FGFR1-FGFR2-FRS2 α knockout MEF cells are selected, and the detection of a flow cytometer shows that the short peptide compound P48 only has a binding effect on wild-type MEF cells (example 2).

Then, Western blot experiments performed on a plurality of tumor cell lines with abnormal FGFR1 disorder were selected, and it was found that the short peptide compound P48 can inhibit the FGFR1 signaling pathway including cervical cancer, melanoma, lung cancer and gastric cancer in a concentration-dependent manner (example 3). In addition, in a gastric cancer cell line, the inhibition effect of the short peptide compound P48 on FGFR1 signal is equivalent to that of SSR, and the activity of the short peptide compound P48 is weaker than that of AZD4547, which is probably related to different binding types of the short peptide compound P48, which is similar to that of SSR, is an extramembranous inhibitor, and AZD4547 is an intramembranous inhibitor. It was further found by flow cytometry experiments that the short peptide compound P48 showed good binding to the FGFR1 site on the cell membrane surface (example 3). Further, it was found by Western blot experiments that the short peptide compound P48 only inhibited FGFR1 activation induced by bFGF, and was ineffective in aFGF, KGF, EGF stimulation (example 3).

In addition, the short peptide compound P48 can obviously inhibit the activity of cancer cells in Hela229, B16-F10, NCI-H460 and SGC-7901 cells. In addition, in the gastric cancer cell SGC-7901, the short peptide compound P48 inhibited cell migration in a concentration-dependent manner as compared with the control group (example 4). The short peptide compound P48 can also effectively block the cell cycle in the G0/G1 phase through flow cycle experiments (example 4).

Finally, in a transplanted tumor mouse model, the short peptide compound P48 can obviously inhibit the growth of gastric cancer cell SGC-7901, and has good inhibition effect on FGFR1 signal pathways (including phosphorylated FLG, FRS2 α and ERK1/2 protein) (example 5). in addition, the short peptide compound P48 can inhibit the expression of Ki67 in a concentration-dependent manner (example 5) compared with a control group through immunohistochemical experiments, and the results show that the short peptide compound P48 can inhibit the cell proliferation by inhibiting the FGFR1 pathway in vivo and has good antitumor activity (example 5).

As described above, our research results show that the short peptide compound P48 has good stability, can exert an antitumor effect by inhibiting the FGFR1 pathway, and has a prospect of being developed as an antitumor drug.

Therefore, the invention provides the application of the high-efficiency and stable FGFR1 antagonistic peptide P48 in preparing the antitumor drug, and the antitumor drug can inhibit cell proliferation and migration by selectively inhibiting an FGFR1 signal channel to play a role in treating tumors.

The invention also provides a pharmaceutical composition for treating tumors, which contains an effective treatment amount of active ingredients and pharmaceutic adjuvants, wherein the active ingredients are the FGFR1 antagonistic short peptide or the pharmaceutically acceptable salts and the pharmaceutic adjuvants thereof. "pharmaceutical excipients" as used herein refers to pharmaceutical carriers which are conventional in the pharmaceutical art, such as: diluents such as starch, sucrose, dextrin, lactose, pregelatinized starch, microcrystalline cellulose, calcium phosphate, and the like; wetting agents such as distilled water, ethanol; binders such as starch slurry, cellulose derivatives, povidone, gelatin, polyethylene glycol, sodium alginate solution, etc.; disintegrating agents such as dry starch, sodium carboxymethyl starch, low-substituted aminopropyl cellulose, effervescent disintegrating agents, etc.; lubricants such as magnesium stearate, aerosil, talc, hydrogenated vegetable oils, polyethylene glycols, sodium lauryl sulfate, etc.; coloring agents such as titanium dioxide, sunset yellow, methylene blue, medicinal iron oxide red, etc.; in addition, other adjuvants such as flavoring agent, sweetener, etc. can also be added into the composition.

Various dosage forms of the pharmaceutical composition of the present invention can be prepared according to conventional production methods in the pharmaceutical field. For example, the active ingredient may be combined with one or more carriers and then formulated into the desired dosage form. The preparation forms of the medicine comprise granules, injections, tablets, capsules, aerosols, suppositories, membranes, dropping pills, ointments, controlled release or sustained release agents or nano preparations. The present invention may be administered in the form of a composition to a patient in need of such treatment by oral, nasal inhalation, rectal or parenteral administration. For oral administration, it can be made into conventional solid preparations such as tablet, powder, granule, capsule, etc., liquid preparations such as aqueous or oil suspension, or other liquid preparations such as syrup, elixir, etc.; for parenteral administration, it can be formulated into solution for injection, aqueous or oily suspension, etc.