CN105585618A - Focal adhesion kinase inhibitor polypeptide and application thereof - Google Patents

Abstract

The invention relates to the field of drugs, and in particular relates to a polypeptide with a function for inhibiting the activity of focal adhesion kinase and capable of treating acute lymphocytic leukemia. A sequence of the focal adhesion kinase inhibitor is DLIDGYRLVNGYSQRALERRGD and is a brand new sequence. The focal adhesion kinase inhibitor can in vitro inhibit the activity of the focal adhesion kinase; and in an in-vivo test, the survival rate of tumor-bearing mice is increased, so that the focal adhesion kinase inhibitor has a potential value for developing a new drug.

Description

A kind of focal adhesion kinase inhibitor polypeptide and application thereof

Technical field

The present invention relates to focal adhesion kinase inhibitor polypeptide and application thereof, be specifically related to have anti-adhesion spot kinases and liveProperty, treatment acute lymphatic leukemia polypeptide.

Background technology

Acute lymphatic leukemia (ALL) is carrying out property of one malignant disease, it is characterized by a large amount of pouring that is similar toThe neocyte of bar mother cell. These cells can be found in blood, marrow, lymph node, spleen and other organ. AcuteLymphocytic leukemia accounts for 80% of acute leukemia, and incidence of disease peak is between 3 years old to 7 years old. ALL also can betideAdult, accounts for all adults leukemic 20%. In recent years along with the going deep into of medical research, to the white blood of acute lymphocyticSick understanding and treatment have made great progress. Wherein, focal adhesion kinase is played the part of emphatically in acute lymphatic leukemiaWant role.

Focal adhesion kinase (focaladhesionkinase, FAK) is the non-receptor protein tyrosine-kinase of a kind of kytoplasmEnzyme is a member of adhesion plaque compound family (focaladhesioncomplexfamily). There is EGFR-TK, after the stimulation that is subject to Oncoprotein and extracellular matrix-integrin, there is phosphorylation in activity. In addition, nervelet peptide, inPi Su and vasopressin etc. can make its phosphorylation FAK participate in many cell signal paths, and it is the transduction of born of the same parents' internal/external signalMaincenter. It can integrate the signal from aspects such as the nutrition outside born of the same parents, pressure, cell adhesions, regulates the work of downstream moleculesProperty, metabolism, the propagation of control cell, or even the destiny of cell. In recent years research shows the life that FAK can regulating cellLength, grappling, move, cancerate and the process such as apoptosis, closely related with the generation of tumour.

The tumour that finds that there is at present FAK expression or increased activity has colon cancer, breast cancer, thymic carcinoma, cancer of the stomach and glueMatter blastoma and melanoma etc. The tumour cell of processing in vitro culture with FAK specific inhibitor, can suppress cellPropagation, growth, diffusion and migration. The FAK of phosphorylation and Src form complex, can activate or suppress many downstreams logicalRoad, comprises PI3K/Akt, RIP, p53 and RAS-Erk etc., and initial tumour occurs or cell death inducing. The shRNA such as WuThe discovery of technical finesse neuroblastoma cell, FAK can suppress the cell migration of integrin A5B1 induction, and FAK alsoCan be by its kinase domain activation Src. When mammary gland of mouse has lacked after FAK, do not affect the normal of galactophore epithelial cell and send outEducate, but the generation of the breast cancer that can suppress to be excited by PyMT; Pound out the FAK of the human breast cancer cell of in vitro culture, canTo stop growth, the inducing cell aging (cellsenescent) of cancer cell. With the competitive FAK of ATP and PYK2 inhibitionAgent, as PF-562,271, PF-573,228 etc., process the tumour cell of in vitro culture, can suppress cell migration. OneClinical trial phase result shows, FAK inhibitor has obvious therapeutic action to kinds of tumors. FAK be tumour occur, propagation,The key protein of the processes such as migration, studying new specificity FAK inhibitor will be one of approach of cancer research and treatment. ButAt present there are no FAK inhibitor for treating acute lymphatic leukemia.

The focal adhesion kinase inhibitor that therefore, need to design high specificity is used for the treatment of ALL. StickyAttached spot inhibitors of kinases comprises large molecule and little molecule. Preparation and the use of large molecule inhibitor have limited their development, exampleAs short in the Half-life in vivo of recombined human focal adhesion kinase inhibiting factor. A lot of successfully micromolecular inhibitors, can be at nanomoleThe kinase whose vigor of anti-adhesion spot in level, but micromolecular inhibitor lacks specificity, if used for a long time in chronic diseaseLack specific focal adhesion kinase inhibitor and can produce larger side effect. Thereby, from the application angle of focal adhesion kinase inhibitorDegree is correct selection using ALL as research object. At Acute response stage, body can tolerateInhibition short-term, focal adhesion kinase wide spectrum inhibitor Normal Physiological Function makes the physiological structure of critical tissue's organ simultaneouslyAvoid destroying, increased chances of survival.

Focal adhesion kinase inhibitor polypeptide 1 in this patent has proved in ALL effective, hasThe prospect of developing in other tumor models.

Summary of the invention

Goal of the invention

The invention provides brand-new sequence, this sequence focal adhesion kinase inhibitor, has fine to ALLCurative effect.

Technical scheme

Focal adhesion kinase inhibitor polypeptide, is characterized in that its sequence is DLIDGYRLVNGYSQRALERRGD.

The application of focal adhesion kinase inhibitor polypeptide in preparation treatment ALL medicine.

Beneficial effect

Utilize solid-phase synthesis chemical synthesis focal adhesion kinase inhibitor polypeptide 1, this polypeptide has brand-new sequence, and this polypeptide canExternal anti-adhesion spot kinases, treatment ALL. In endotoxin shock model experiment, successfully increaseThe survival rate of mouse. The peptide inhibitor that we find is anti-adhesion spot kinases vigor simultaneously, and in test, carries in vivoHigh tumor-bearing mice survival rate, has potential new drug development and is worth.

Detailed description of the invention

Embodiment 1

The effect of focal adhesion kinase inhibitor polypeptide 1 to external focal adhesion kinase activity.

Get fresh cow brain tissue, peel off meninx and large blood vessel, shred, with cold MES buffer solution washing 1-2 time, withEvery Borneo camphor organizes the ratio of 0.5-1ml to add MES buffer solution, at 4 DEG C, uses electric homogenizer homogenate; 4 DEG C,The centrifugal 1h of 105000g, gets supernatant, adds isopyknic microtubule polymerization buffer solution, 37 DEG C of water bath heat preservation 30min. 26 DEG C,The centrifugal 1h of 105000g, gets precipitation, adds the cold MES buffer solution of approximately 1/10 homogenate volume, stirs gently or use homogenizerPrecipitation is pulverized; Suspension is put to ice bath 30min, precipitation is dissolved completely. (SDS is poly-to measure protein content by Lowry ' s methodAcrylamide gel electrophoresis). Focal adhesion kinase is diluted to 4-5mg/ml with MES buffer solution, puts in liquid nitrogen and preserves. Get freezingFocal adhesion kinase solution, rushes its wall by normal-temperature water fast, makes it to melt, and puts into ice bath, is diluted to required dense with MES buffer solutionDegree (2-3mg/ml), adds ATP to 1mmol/l. With the focal adhesion kinase solution that at once takes out from ice bath at spectrophotometricSet up as " 0 " point at meter 350nm place. Then cuvette is measured at 37 DEG C of temperature to the OD value of focal adhesion kinase solution, continuously20-30min, records temperature-light absorption value curve (T-OD curve), in triplicate.

Inhibiting rate calculates: inhibiting rate (%)=(control tube " OD " value-chemical feed pipe " OD " value)/control tube " OD "

Experimental group is established five dosage: 0.75 μ M, 1.5 μ M, 3 μ M, 12 μ M, 24 μ M, positive controls vincristine dosage 4 μ M, skyWhite group adds isopyknic solvent DMSO; Press aforesaid operations and measure light absorption value. As a result, dense with focal adhesion kinase inhibitor polypeptide 1Degree increases, and inhibiting rate raises gradually, illustrates that anti-adhesion spot kinase activity constantly increases with the increase of drug concentration.

Embodiment 2

Growth and the survival IC50 of focal adhesion kinase inhibitor polypeptide 1 to cultured tumor cells in vitro.

Adopt MTT colorimetric method. By the U937 cell of logarithmic growth, add in 96 well culture plates training with 1.0 × 105Support 24h, experimental port, positive drug control wells add respectively Experimental agents focal adhesion kinase inhibitor polypeptide 1 and of variable concentrationsPositive control medicine vincristine; Blank group adds the solvent of same volume. Five multiple holes are established in every hole, cultivate 48h, exist respectively0h, 2h, 8h, 14h, 20h, 24h, 36h,, the every hole of 48h adds MTT, after effect 4h, adds DMSO, hatches 30min, at enzymeMark instrument 620nm place measures absorbance A value, by formula growth of tumour cell inhibiting rate=(1-experimental group light absorption value/control groupLight absorption value) × 100%. The IC50 that calculates Experimental agents is 23.43 μ M.

Embodiment 3

With vigor in the body of tumor model detection focal adhesion kinase inhibitor polypeptide 1.

Set up U937 tumor model, positive control medicine vincristine; Blank group adds the solvent of same volume, experimental groupIf 3 dosage: 0.75,1.5 μ M, 3 μ Mmg/Kg. After 21 days, observe mouse survival quantity, calculate survival rate. Result shows, stickyAttached spot inhibitors of kinases polypeptide 1 can be protected small white mouse effectively, improves the survival rate of tumor-bearing mice, and survival rate reaches 91.31%.

SEQUENCELISTING

<110>Suzhou Pu Luoda bio tech ltd

<120>a kind of medusocongestin polypeptide and preparation and application

<130>

<160>1

<170>PatentInversion3.5

<210>1

<211>26

<212>PRT

<213>artificial sequence

<400>1

IleProAsnArgAlaLysArgSerSerSerGluIleAsnAlaGluIle

151015

AspGlyLeuIleGlnGlnLeuThrThr

2025

<210>2

<211>78

<212>DNA

<213>manually synthetic

<400>2

tatggagttggcttaggagctgtcgcttcattgttatcgtctgtaatagggcttttcgca60

caggatggttttaagaac78

<210>3

<211>21

<212>DNA

<213>artificial sequence

<400>1

cttaggagctgtcgcttcatt21

<210>4

<211>19

<212>DNA

<213>artificial sequence

<400>1

aaaccatcctgtgcgaaaaSEQUENCELISTING

<110>Suzhou Pu Luoda bio tech ltd

<120>a kind of focal adhesion kinase inhibitor polypeptide and application thereof

<130>

<160>1

<170>PatentInversion3.3

<210>1

<211>22

<212>PRT

<213>artificial sequence

<400>1

AspLeuIleAspGlyTyrArgLeuValAsnGlyTyrSerGlnArgAla

151015

LeuGluArgArgGlyAsp

20

19

Claims (2)

1. focal adhesion kinase inhibitor polypeptide, is characterized in that its sequence is DLIDGYRLVNGYSQRALERRGD.

2. focal adhesion kinase inhibitor polypeptide according to claim 1 is at preparation treatment acute lymphatic leukemia medicineApplication in thing.