A kind of anti-adhesion spot kinase polypeptide and application thereof
Technical field
The present invention relates to anti-adhesion spot kinase polypeptide and application thereof, be specifically related to there is anti-adhesion spot kinase activity, treatment acute lymphoblastic leukemia polypeptide.
Background technology
Acute lymphoblastic leukemia (ALL) is a kind of carrying out property malignant disease, it is characterized by a large amount of lymphoblastic neocytes that is similar to.These cells can be found in blood, marrow, lymphoglandula, spleen and other organ.Acute lymphoblastic leukemia accounts for 80% of acute leukemia, and sickness rate peak is between 3 years old to 7 years old.ALL also can betide grownup, accounts for all grownups leukemic 20%.Along with the going deep into of medical research, the understanding of acute lymphoblastic leukemia and treatment are had made great progress in recent years.Wherein, focal adhesion kinase is being played the part of key player in acute lymphoblastic leukemia.
Focal adhesion kinase (focal adhesion kinase, FAK) is the non-receptor protein tyrosine kinase of a kind of kytoplasm, is a member of adhesion plaque mixture family (focal adhesion complex family).There is tyrosine kinase activity, after the stimulation that is subject to Oncoprotein and extracellular matrix-integrin, phosphorylation occurs.In addition, nervelet peptide, endothelin and beta-hypophamine etc. can make its phosphorylation FAK participate in many cell signal paths, and it is the maincenter of born of the same parents' internal/external signal transduction.It can integrate the signal from aspects such as the nutrition outside born of the same parents, pressure, cell adhesions, regulates the activity of downstream molecules, controls metabolism, the propagation of cell, or even the destiny of cell.In recent years research shows growth, grappling that FAK can regulating cell, 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 colorectal carcinoma, mammary cancer, thymic carcinoma, cancer of the stomach and glioblastoma multiforme and melanoma etc.The tumour cell of processing vitro culture with FAK specific inhibitor, can suppress propagation, growth, diffusion and the migration of cell.The FAK of phosphorylation and Src form complex body, can activate or suppress many downstream passages, comprise PI3K/Akt, RIP, p53 and RAS-Erk etc., and initial tumour occurs or cell death inducing.ShRNA technical finesse neuroblastoma cell discovery for Wu etc., FAK can suppress the cell migration of integrin A5B1 induction, and FAK can also be by its kinase domain activation Src.When mammary gland of mouse has lacked after FAK, do not affect the normal development of mammary epithelial cell, but the generation of the mammary cancer that can suppress to be excited by PyMT; Pound out the FAK of the human breast cancer cell of vitro culture, can stop growth, the inducing cell old and feeble (cell senescent) of cancer cells.With the competitive FAK of ATP and PYK2 inhibitor, as PF-562,271, PF-573,228 etc., process the tumour cell of vitro culture, can suppress cell migration.First phase clinical test results shows, FAK inhibitor has obvious therapeutic action to kinds of tumors.FAK is the key protein of the processes such as tumour generation, propagation, migration, and studying new specificity FAK inhibitor will be one of approach of cancer research and treatment.But at present there are no FAK inhibitor for treating acute lymphoblastic leukemia.
The focal adhesion kinase inhibitor that therefore, need to design high specificity is used for the treatment of acute lymphoblastic leukemia.Focal adhesion kinase inhibitor comprises macromole and small molecules.The preparation of macromole inhibitor and use have limited their development, and for example the Half-life in vivo of recombinant human focal adhesion kinase supressor is short.A lot of successfully micromolecular inhibitors, can be in nmole level the kinase whose vigor of anti-adhesion spot, but micromolecular inhibitor lacks specificity, if long-term use lacks specific focal adhesion kinase inhibitor and can produce larger side effect in chronic disease.Thereby from the application point of focal adhesion kinase inhibitor, the acute lymphoblastic leukemia of usining is correct selection as research object.At Acute response stage, body can tolerate inhibition short-term, focal adhesion kinase wide spectrum inhibitor Normal Physiological Function, makes the physiological structure of critical tissue's organ avoid destroying simultaneously, has increased chances of survival.
Anti-adhesion spot kinase polypeptide in this patent has proved in acute lymphoblastic leukemia effective, has the 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 good curative effect to acute lymphoblastic leukemia.
technical scheme
anti-adhesion spot kinase polypeptide, is characterized in that its sequence is ADLIDGYRLVNGTSQRALERLV.
The application of anti-adhesion spot kinase polypeptide in treatment acute lymphoblastic leukemia medicine.
beneficial effect
Utilize solid-phase synthesis chemosynthesis anti-adhesion spot kinase polypeptide, this polypeptide has brand-new sequence, and this polypeptide can vitro inhibition focal adhesion kinase, treatment acute lymphoblastic leukemia.In endotoxin shock model experiment, successfully increased the survival rate of mouse.The peptide inhibitor that we find is anti-adhesion spot kinases vigor simultaneously, and in test, improves tumor-bearing mice survival rate in vivo, has potential new drug development and is worth.
Embodiment
The present invention relates to polypeptide synthetic by the biochemical (Shanghai) Co., Ltd. of gill.
Embodiment 1
The effect of anti-adhesion spot kinase polypeptide to external focal adhesion kinase activity.
Get fresh cow brain tissue, peel off meninx and large blood vessel, shred, with cold MES damping fluid washing 1-2 time, with every Borneo camphor, organize the ratio of 0.5-1ml to add MES damping fluid, at 4 ℃, use electric homogenizer homogenate; 4 ℃, the centrifugal 1h of 105000g, gets supernatant, adds isopyknic microtubule polymerization damping fluid, 37 ℃ of water bath heat preservation 30min.26 ℃, the centrifugal 1h of 105000g, gets precipitation, adds the cold MES damping fluid of approximately 1/10 homogenate volume, stirs gently or with homogenizer, precipitation is pulverized; Suspension is put to ice bath 30min, precipitation is dissolved completely.By Lowry ' s method, measure protein content (SDS polyamide gels electrophoretic method).Focal adhesion kinase is diluted to 4-5mg/ml with MES damping fluid, puts in liquid nitrogen and preserves.Get freezing focal adhesion kinase solution, by normal-temperature water, rush its wall fast, make it to melt, put into ice bath, with MES damping fluid, be diluted to desired concn (2-3mg/ml), add ATP to 1mmol/l.The focal adhesion kinase solution at once taking out from ice bath of take is " 0 " point in spectrophotometer 350nm setting.Then cuvette is measured at 37 ℃ of temperature to the OD value of focal adhesion kinase solution, 20-30min, records temperature-light absorption value curve (T-OD curve), in triplicate continuously.
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, and positive controls vincristine(VCR) dosage 3 μ M, blank group adds isopyknic solvent DMSO; Press aforesaid operations and measure light absorption value.As a result, with anti-adhesion spot kinase polypeptide concentration, increase, inhibiting rate raises gradually, illustrates that anti-adhesion spot kinase activity constantly increases with the increase of drug level.
Embodiment 2
Anti-adhesion spot kinase polypeptide is to the growth of cultured tumor cells in vitro and survival IC50.
Adopt MTT colorimetry.By the U937 cell of logarithmic growth, add in 96 well culture plates with 1.0 * 105, cultivate 24h, experimental port, positive drug control wells add respectively Experimental agents anti-adhesion spot kinase polypeptide and the positive control medicine vincristine(VCR) of different concns; Blank group adds the solvent of same volume.Five multiple holes are established in every hole, cultivate 48h, respectively 0h, 2h, 8h, 14h, 20h, 24h, 36h,, the every hole of 48h adds MTT, after effect 4h, add DMSO, hatch 30min, at microplate reader 620nm place, measure absorbance A value, by formula growth of tumour cell inhibiting rate=(1-experimental group light absorption value/control group light absorption value) * 100%.The IC50 that calculates Experimental agents is 33.43 μ M.
Embodiment 3
With vigor in the body of tumor model detection anti-adhesion spot kinase polypeptide.
Set up U937 tumor model, positive control medicine vincristine(VCR); Blank group adds the solvent of same volume, and experimental group is established 3 dosage: 0.75,1.5 μ M, 3 μ M mg/Kg.After 21 days, observe mouse survival quantity, calculate survival rate.Result demonstration, anti-adhesion spot kinase polypeptide can be protected small white mouse effectively, improves the survival rate of tumor-bearing mice, and survival rate reaches 83.31%.
SEQUENCE LISTING
<110> timely snow, sieve
<120> anti-adhesion spot kinase polypeptide and application thereof
<130>
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 22
<212> PRT
<213> artificial sequence
<400> 1
Ala Asp Leu Ile Asp Gly Tyr Arg Leu Val Asn Gly Thr Ser Gln Arg
1 5 10 15
Ala Leu Glu Arg Leu Val
20