CN103254287A - Tubulin depolymerizing agent polypeptides and application thereof - Google Patents
Tubulin depolymerizing agent polypeptides and application thereof Download PDFInfo
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- CN103254287A CN103254287A CN2013102089369A CN201310208936A CN103254287A CN 103254287 A CN103254287 A CN 103254287A CN 2013102089369 A CN2013102089369 A CN 2013102089369A CN 201310208936 A CN201310208936 A CN 201310208936A CN 103254287 A CN103254287 A CN 103254287A
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Abstract
The invention relates to tubulin depolymerizing agent polypeptides 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 1 is CRALERLV disclosed as SEQ NO.1. The invention also relates to application of the tubulin depolymerizing agent polypeptides 1 in preparation of medicines for treating acute lymphatic leukemia. A solid-phase synthesis method is utilized to chemically synthesize the tubulin depolymerizing agent polypeptides 1; and the polypeptides with the bran-new sequence can inhibit tubulin in vitro and treat acute lymphatic leukemia.
Description
Technical field
The present invention relates to tubulin depolymerizing agent polypeptide 1 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 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.It is leukemic 80% that acute lymphoblastic leukemia accounts for children acute, 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 is formed, 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 polymerization and depolymerization, plays an important role in keeping 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 important role 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.
The tubulin depolymerizing agent that suppresses tubulin polymerization has compounds such as vinca, colchicine, is used for oncotherapy more.This compounds is characterized in acting on the tubulin depolymerizing agent in the vinealeucoblastine(VLB) site of microtubule.Studies show that the vinca drug effect suppresses microtubule polymerization in the mitotic division stage in cell tubulin and interference cell cycle, stops the formation of spindle microtubule, thereby stop tumour cell division propagation.This Alkaloid is spontaneous now to have had since the anti-tumor activity, existing vinealeucoblastine(VLB) (vinblastine,
15), vincristine(VCR) (vincristine,
16), vindesine (vindesine,
17) and vinorelbine (vinorelbine,
18) be used 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 defective of self, and little as vinealeucoblastine(VLB) and action target spot avidity, 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 bigger; (2) water-soluble relatively low, receptivity is poor.
Therefore, need the exploitation good water solubility, the tubulin depolymerizing agent of high specificity is used for the treatment of acute lymphoblastic leukemia.The tubulin depolymerizing agent comprises macromole and small molecules.The preparation of macromole depolymerizing agent and use have limited their development, and for example the transformation period has only 4 minutes in the body of recombinant human tubulin supressor.A lot of successful small molecules depolymerizing agents can suppress the vigor of tubulin in the nmole level, but the small molecules depolymerizing agent lack specificity, if lack specific tubulin depolymerizing agent and can produce side effect greatly medium-term and long-term use of chronic disease.Thereby, from the application point of tubulin depolymerizing agent, be correct selection with acute lymphoblastic leukemia as research object.In the acute reaction phase, body can tolerate short-term, tubulin wide spectrum depolymerizing agent is to the inhibition of normal physiological function, makes the physiological structure of critical tissue's organ avoid destroying simultaneously, increased chances of survival.
Tubulin depolymerizing agent polypeptide 1 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 better curative effect to acute lymphoblastic leukemia.
Technical scheme
Tubulin depolymerizing agent polypeptide 1 is characterized in that its sequence is CRALERLV, sees Seq NO.1.
The application of described tubulin depolymerizing agent polypeptide 1 in preparation treatment acute lymphoblastic leukemia medicine.
Beneficial effect
Utilize solid-phase synthesis chemosynthesis tubulin depolymerizing agent polypeptide 1, this polypeptide has brand-new sequence, but this polypeptide vitro inhibition tubulin, the treatment acute lymphoblastic leukemia.In endotoxin shock model experiment successful increase the survival rate of mouse.The polypeptide depolymerizing agent that we find can suppress 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
It is synthetic that the present invention mentions that polypeptide 1 entrusts Shanghai to give birth to the worker.
Embodiment 1
The effect of 1 pair of external tubulin polymerization of tubulin depolymerizing agent polypeptide and depolymerization.
Get the fresh bovine cerebral tissue, peel off meninx and big 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 cold MES damping fluid of about 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, fast with normal-temperature water towards its wall, make it to melt, put into ice bath, be diluted to desired concn (2-3mg/ml) with the MES damping fluid, adding ATP to 1mmol/l.Setting up at spectrophotometer 350nm with the tubulin solution that takes out from ice bath at once is " 0 " point.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 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 1 concentration, polymerization retardation rate descends gradually, illustrates that the tubulin of polymerizing power constantly reduces with the increase of drug level.
Embodiment 2
The growth of 1 pair of cultured tumor cells in vitro of tubulin depolymerizing 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 experiment medicine tubulin depolymerizing agent polypeptide 1 and the positive control medicine vincristine(VCR) of different concns respectively; 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 the experiment medicine is 3.43 μ M.
Embodiment 3
With vigor in the body of tumor model detection tubulin depolymerizing agent polypeptide 1.
Set up the U937 tumor model, positive control medicine vincristine(VCR); 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.The result shows that tubulin depolymerizing agent polypeptide 1 can be protected small white mouse effectively, improves the survival rate of tumor-bearing mice, and survival rate reaches 73.2%.
SEQUENCE LISTING
＜110〉Suzhou Pu Luoda bio tech ltd
＜120〉a kind of tubulin depolymerizing agent polypeptide 1 and application thereof
<130>
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 8
<212> PRT
＜213〉artificial sequence
<400> 1
Cys Arg Ala Leu Glu Arg Leu Val
1 5
Claims (2)
1. tubulin depolymerizing agent polypeptide 1 is characterized in that its aminoacid sequence is Seq NO.1.
2. the application of tubulin depolymerizing agent polypeptide 1 according to claim 1 in preparation treatment acute lymphoblastic leukemia medicine.
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| CN201310208936.9A CN103254287B (en) | 2013-05-30 | 2013-05-30 | A kind of tubulin depolymerizing agent polypeptide and application thereof |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310208936.9A CN103254287B (en) | 2013-05-30 | 2013-05-30 | A kind of tubulin depolymerizing agent polypeptide and application thereof |
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| CN103254287A true CN103254287A (en) | 2013-08-21 |
| CN103254287B CN103254287B (en) | 2015-12-02 |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103923170A (en) * | 2014-01-17 | 2014-07-16 | 南通诚信氨基酸有限公司 | Polyethylene glycol modified tubulin depolymerization agent polypeptide and its application |
| CN104031123A (en) * | 2014-06-27 | 2014-09-10 | 苏州普罗达生物科技有限公司 | Natural killer T cell activator polypeptide and application thereof |
| CN104031124A (en) * | 2014-06-27 | 2014-09-10 | 苏州普罗达生物科技有限公司 | Natural killer T cell activator polypeptide and application thereof |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101812127A (en) * | 2009-02-19 | 2010-08-25 | 中国科学技术大学 | Microtubule-associated protein and coding genes and application thereof |
-
2013
- 2013-05-30 CN CN201310208936.9A patent/CN103254287B/en not_active Expired - Fee Related
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101812127A (en) * | 2009-02-19 | 2010-08-25 | 中国科学技术大学 | Microtubule-associated protein and coding genes and application thereof |
Non-Patent Citations (2)
| Title |
|---|
| 吴作基 等: "微管解聚相关蛋白质Kinesin-13、Stathmin和Katanin", 《生命科学》 * |
| 尚海 等: "微管蛋白抑制剂的研究进展", 《药学学报》 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103923170A (en) * | 2014-01-17 | 2014-07-16 | 南通诚信氨基酸有限公司 | Polyethylene glycol modified tubulin depolymerization agent polypeptide and its application |
| CN104031123A (en) * | 2014-06-27 | 2014-09-10 | 苏州普罗达生物科技有限公司 | Natural killer T cell activator polypeptide and application thereof |
| CN104031124A (en) * | 2014-06-27 | 2014-09-10 | 苏州普罗达生物科技有限公司 | Natural killer T cell activator polypeptide and application thereof |
| CN104031123B (en) * | 2014-06-27 | 2016-06-01 | 青岛大学附属医院 | About natural killer T cells activator polypeptide and application thereof |
| CN104031124B (en) * | 2014-06-27 | 2016-09-21 | 北京中诚华科生物科技有限公司 | A kind of about natural killer T cells activator polypeptide and application thereof |
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| CN103254287B (en) | 2015-12-02 |
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| C41 | Transfer of patent application or patent right or utility model | ||
| CB03 | Change of inventor or designer information |
Inventor after: Kou Jialin Inventor after: Li Hongchen Inventor after: Shen Xiaoyong Inventor after: Li Jiuxia Inventor after: Ge Junnan Inventor before: Luo Ruixue |
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Effective date of registration: 20151021 Address after: 050000, 1, 02, 01 and 04, building 3, A District, Hebei science and Technology Park, Shijiazhuang hi tech Zone, Zhejiang Province Applicant after: HEBEI YOUREN BIOTECHNOLOGY Co.,Ltd. Address before: High tech Zone Suzhou city Jiangsu province 215000 Chuk Yuen Road No. 209 Applicant before: SUZHOU PULUODA BIOLOGICAL SCIENCE AND TECHNOLOGY Co.,Ltd. |
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