CN103254289B - Tubulin depolymerizing agent polypeptides and application thereof - Google Patents

Abstract

The invention relates to tubulin depolymerizing agent polypeptides 3 and application thereof, particularly polypeptides capable of inhibiting polymerization of tubulin and treating acute lymphatic leukemia. The invention is characterized in that the sequence of the tubulin depolymerizing agent polypeptides 3 is NVTMQLLK disclosed as SEQ NO.1. The invention also relates to application of the tubulin depolymerizing agent polypeptides 3 in preparation of medicines for treating acute lymphatic leukemia. A solid-phase synthesis method is utilized to chemically synthesize the tubulin depolymerizing agent polypeptides 3; and the polypeptides with the bran-new sequence can inhibit tubulin in vitro and treat acute lymphatic leukemia.

Description

A kind of tubulin depolymerizing agent polypeptide and application thereof

Technical field

The present invention relates to tubulin depolymerizing agent polypeptide 3 and application thereof, be specifically related to have the polypeptide that suppresses tubulin polymerization, treatment acute lymphoblastic leukemia.

Background technology

Acute lymphoblastic leukemia (ALL) is carrying out property of one 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, understanding and treatment to acute lymphoblastic leukemia have made great progress in recent years.Wherein, tubulin is being played the part of key player in acute lymphoblastic leukemia.

Microtubule is the chief component of cytoskeleton, by α-tubulin and β-tubulin heterodimer forms, and has the feature of hollow tubular structure.In addition, also has one 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 maintaining in the processes such as cellular form, cell fission, signal transduction and material conveying.Microtubule becomes spindle body in 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, completes cell proliferation.The assembling process of microtubule be α-and 'beta '-tubulin opposite sex dimer between noncovalent interaction closely, this process is driven by GTP hydrolysis.Opposite sex dimer constantly, in the positive pole growth of microtubule, shrinks at negative pole, and maintains dynamic stability.In the time that the dynamic stability of microtubule is destroyed, the mitotic division process of cell is just likely 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 to be divided into two types according to the difference of mechanism of action: the tubulin depolymerizing agent that 1. suppresses tubulin polymerization; 2. promote the tubulin polymerization agent of tubulin polymerization.

The tubulin depolymerizing agent that suppresses tubulin polymerization has the compound such as vinca, colchicine, is used for oncotherapy.This compounds is characterized in the tubulin depolymerizing agent in the vinealeucoblastine(VLB) site that acts on microtubule.Research shows, vinca drug effect, in the mitotic division stage in cell tubulin interference cell cycle, suppresses microtubule polymerization, stops the formation of spindle microtubule, thereby stops tumour cell division propagation.Since this Alkaloid self-discovery has anti-tumor activity, existing vinealeucoblastine(VLB) (vinblastine, 15), vincristine(VCR) (vincristine, 16), vindesine (vindesine, 17) and vinorelbine (vinorelbine, 18) for clinical treatment, and Vinflunine (vinflunine, 19), vinglycinate (vinglycinate, 20) and F 81097 (anhydrovinblastine, 21) be in the clinical study stage.But these have the defect of self, as vinealeucoblastine(VLB) and action target spot avidity little, drug effect is not strong.Its application clinically that vincristine(VCR) has had two important effects limit: (1) vincristine(VCR) neural system toxicity and local irritation are larger; (2) water-soluble relatively low, receptivity is poor.

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

Tubulin depolymerizing agent polypeptide 3 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 depolymerizing agent, has good curative effect to acute lymphoblastic leukemia.

technical scheme

tubulin depolymerizing agent polypeptide 3, is characterized in that its sequence is NVTMQLLK, sees seq NO.1.

The application of tubulin depolymerizing agent polypeptide 3 in preparation treatment acute lymphoblastic leukemia medicine.

beneficial effect

Utilize solid-phase synthesis chemosynthesis tubulin depolymerizing agent polypeptide 3, this polypeptide has brand-new sequence, and this polypeptide can vitro inhibition tubulin, treatment acute lymphoblastic leukemia.In endotoxin shock model experiment, successfully increase the survival rate of mouse.The polypeptide depolymerizing agent that we find can suppress tubulin polymerization 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 mentions that the raw work in polypeptide trust Shanghai is synthetic.

Embodiment 1

The effect of tubulin depolymerizing agent polypeptide 3 to external tubulin polymerization 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 MES damping fluid with every Borneo camphor, at 4 DEG C, use electric homogenizer homogenate; 4 DEG C, the centrifugal 1h of 105000g, gets supernatant, adds isopyknic microtubule polymerization damping fluid, 37 DEG C of water bath heat preservation 30min.26 DEG C, the centrifugal 1h of 105000g, gets precipitation, adds the cold MES damping fluid of approximately 1/10 homogenate volume, stirs gently or precipitation is pulverized with homogenizer; Suspension is put to ice bath 30min, precipitation is dissolved completely.With Lowry ' s method mensuration protein content (SDS polyamide gels electrophoretic method).Tubulin is diluted to 4-5mg/ml with MES damping fluid, puts in liquid nitrogen and preserves.Get freezing tubulin solution, rush its wall by normal-temperature water fast, make it to melt, put into ice bath, be diluted to desired concn (2-3mg/ml) with MES damping fluid, add ATP to 1mmol/l.Set up as " 0 " point at spectrophotometer 350nm taking the tubulin solution taking out from ice bath at once.Then cuvette is measured at 37 DEG C of temperature to the OD value of tubulin 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, increase with tubulin depolymerizing agent polypeptide 3 concentration, polymerization retardation rate declines gradually, illustrates that the tubulin of polymerizing power constantly reduces with the increase of drug level.

Embodiment 2

Growth and the survival IC50 of tubulin depolymerizing agent polypeptide 3 to cultured tumor cells in vitro.

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 tubulin depolymerizing agent polypeptide 3 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, measure absorbance A value at microplate reader 620nm place, 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 8.91 μ M.

Embodiment 3

With vigor in the body of tumor model detection tubulin depolymerizing agent polypeptide 3.

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, tubulin depolymerizing agent polypeptide 3 can be protected small white mouse effectively, improves the survival rate of tumor-bearing mice, and survival rate reaches 39.4%.

SEQUENCE LISTING

Pu Luoda bio tech ltd, <110> Suzhou

<120> tubulin depolymerizing agent polypeptide 3 and application thereof

<130>

<160> 1

<170> PatentIn version 3.3

<210> 1

<211> 8

<212> PRT

<213> artificial sequence

<400> 1

Asn Val Thr Met Gln Leu Leu Lys

1 5

Claims (1)

1. tubulin depolymerizing agent polypeptide 3, is characterized in that its sequence is Seq NO.1.