CN103304632A - Tubulin polymerizing agent polypeptides 2 and application thereof - Google Patents

Abstract

The invention relates to the field of medicaments, and in particular relates to polypeptides capable of promoting polymerization of tubulin and treating acute lymphatic leukemia. The sequence of the polypeptides is RQASSIYDDS which is a brand-new sequence. The polypeptides can promote the polymerization of tubulin in vitro at a level of 1 micromole, and can improve the survival rate of tumor-bearing mice in an in vivo test, so that the polypeptides have a potential new medicament development value.

Description

A kind of tubulin polymerization agent polypeptide 2 and application thereof

Technical field

The present invention relates to tubulin polymerization agent polypeptide 2 and application thereof, be specifically related to have the polypeptide that promotes tubulin polymerization, treatment acute lymphoblastic leukemia.

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 the sickness rate peak is between 3 years old to 7 years old.ALL also can betide the grownup, accounts for all grownups leukemic 20%.In recent years along with the going deep into of medical research, understanding and the treatment of acute lymphoblastic leukemia obtained remarkable progress.Wherein, tubulin is being played the part of important role in acute lymphoblastic leukemia.

Microtubule is the chief component of cytoskeleton, by α-tubulin and β-tubulin heterodimer forms, and has the characteristics of hollow tubular structure.In addition, a kind of in addition γTubulin, it is not the moiety of microtubule, but participates in the assembling of microtubule.Microtubule has the dynamics of Aggregation and disaggregation, plays an important role in keeping the processes such as cellular form, cell fission, signal transduction and material conveying.Microtubule becomes spindle body in the prophase of cell division polymerization, moves in two daughter cells to the two poles of the earth and spindle body draws karyomit(e) in mitotic division, finishes cell proliferation.The assembling process of microtubule be α-and 'beta '-tubulin opposite sex dimer between noncovalent interaction closely, this process is driven by the GTP hydrolysis.Opposite sex dimer constantly in the positive pole growth of microtubule, shrinks at negative pole, and keeps dynamic stability.When the dynamic stability of microtubule was destroyed, the mitotic division process of cell just might be prevented from, thereby causes necrocytosis.Because microtubule has extremely important effect in cell fission, now become one of important target spot of antitumor drug research.

Antitubulin has two kinds of sorting techniques.A kind of is that difference according to mechanism of action is divided into two types: the tubulin depolymerizing agent that 1. suppresses tubulin polymerization; 2. promote the tubulin polymerization agent of tubulin polymerization.

Promote the tubulin polymerization agent of tubulin polymerization that the compounds such as taxol are arranged, multiplex in oncotherapy.This compounds is characterized in acting on the tubulin depolymerizing agent in the vinealeucoblastine(VLB) site of microtubule.Studies show that, can rupture by induce dna after taxol and the cells contacting, cause apoptosis.In addition, it can also inducing cell in tumour necrosis factor α(TNF- α) expression of gene, improve protein, comprise the phosphorylation level of MAP kinases and the kinase whose tyrosine residues of Raf-1.In December, 1992, U.S. FDA has been ratified the treatment of taxol for mammary cancer and ovarian cancer.Its application clinically that taxol has had 3 important effects limit: (1) taxol content in several Chinese yew genus plants is very low, and the source is restricted; (2) water-soluble relatively low, receptivity is poor; (3) the cancer cells meeting overexpression P-glycoprotein of crossing with taxol treatment has multidirectional resistance.

Therefore, need the exploitation good water solubility, the little tubulin polymerization agent of side effect is used for the treatment of acute lymphoblastic leukemia.The tubulin polymerization agent comprises macromole and small molecules.The preparation of macromole polymerizing agent and use have limited their development, and for example the recombinant human tubulin promotes the Half-life in vivo of the factor to only have 4 minutes.A lot of successful small molecules polymerizing agents can promote in the nmole level vigor of tubulin, but the small molecules polymerizing agent lack specificity, if use the specific tubulin polymerization agent of shortage can produce larger side effect for a long time in chronic disease.Thereby, from the application point of tubulin polymerization agent, be correct selection with acute lymphoblastic leukemia as research object.At Acute response stage, body can tolerate inhibition short-term, tubulin wide spectrum polymerizing agent Normal Physiological Function, makes simultaneously the physiological structure of critical tissue's organ avoid destroying, and has increased chances of survival.

Tubulin polymerization agent polypeptide 2 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 tubulin polymerization agent has good curative effect to acute lymphoblastic leukemia.

Technical scheme

Tubulin polymerization agent polypeptide 2 is characterized in that its sequence is RQASSIYDDS.

The application of tubulin polymerization agent polypeptide 2 in preparation treatment acute lymphoblastic leukemia medicine.

Beneficial effect

Utilize solid-phase synthesis chemosynthesis tubulin polymerization agent polypeptide 2, this polypeptide has brand-new sequence, and this polypeptide can external promotion tubulin, the treatment acute lymphoblastic leukemia.In endotoxin shock model experiment successful increase the survival rate of mouse.The tubulin polymerization agent polypeptide 2 that we find can promote the tubulin polymerization vigor simultaneously, and improves the tumor-bearing mice survival rate in the test in vivo, has potential new drug development and is worth.

Embodiment

Embodiment 1

The effect of 2 pairs of external tubulin polymerizations of tubulin polymerization agent polypeptide and depolymerization.

Get fresh cow brain tissue, peel off meninx and large blood vessel, shred, with cold MES damping fluid washing 1-2 time, organize the ratio of 0.5-1ml to add the MES damping fluid with every Borneo camphor, under 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 approximately cold MES damping fluid of 1/10 homogenate volume, stirs gently or with homogenizer precipitation is pulverized; Suspension is put ice bath 30min, precipitation is dissolved fully.Use Lowry ' s method to measure protein content (SDS polyamide gels electrophoretic method).Tubulin is diluted to 4-5mg/ml with the MES damping fluid, puts in the liquid nitrogen and preserves.Get freezing tubulin solution, rush its wall with normal-temperature water fast, make it to melt, put into ice bath, be diluted to desired concn (2-3mg/ml) with the MES damping fluid, add ATP to 1mmol/l.Set up as " 0 " point at spectrophotometer 350nm take the tubulin solution that from ice bath, takes out at once.Then cuvette is measured the OD value of tubulin solution under 37 ℃ of temperature, 20-30min records temperature-light absorption value curve (T-OD curve), 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 dose of paclitaxel 3 μ M, blank group adds isopyknic solvent DMSO; Press aforesaid operations and measure light absorption value.As a result, increase with tubulin polymerization agent polypeptide 2 concentration, polymerization retardation rate raises gradually, illustrates that the tubulin of polymerizing power constantly strengthens with the increase of drug level.

Embodiment 2

The growth of 2 pairs of cultured tumor cells in vitro of tubulin polymerization agent polypeptide and survival IC50.

Adopt the MTT colorimetry.With the U937 cell of logarithmic growth, in 1.0 * 105 addings, 96 well culture plates, cultivate 24h, experimental port, positive drug control wells add respectively Experimental agents tubulin polymerization agent polypeptide 2 and the positive control medicine taxol of different concns; Blank group adds the solvent of equal 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, behind the effect 4h, add DMSO, hatch 30min, measure absorbance A value, by formula growth of tumour cell inhibiting rate=(1-experimental group light absorption value/control group light absorption value) * 100% at microplate reader 620nm place.The IC50 that calculates Experimental agents is 4.34 μ M.

Embodiment 3

With vigor in the body of tumor model detection tubulin polymerization agent polypeptide 2.

Set up the U937 tumor model, positive control medicine taxol; Blank group adds the solvent of equal 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's demonstration, tubulin polymerization agent polypeptide 2 can be protected small white mouse effectively, improves the survival rate of tumor-bearing mice, and survival rate reaches 74.9%.

Sequence table

SEQUENCE LISTING

<110>

＜120〉a kind of tubulin polymerization agent polypeptide 2 and application thereof

<130>

<160> 1

<170> PatentIn version 3.3

<210> 1

<211> 18

<212> PRT

＜213〉artificial sequence

<400> 1

Arg Gln Ala Ser Ser Ile Tyr Asp Asp Ser

1 5

Claims (2)

1. tubulin polymerization agent polypeptide 2 is characterized in that its sequence is RQASSIYDDS.

2. the application of tubulin polymerization agent polypeptide 2 in preparation, treatment acute lymphoblastic leukemia medicine.