CN101638808A - Method for screening enhanced anti-tumor compounds - Google Patents

Abstract

The invention relates to the field of molecular biology and medicine, in particular to a method for screening enhanced anti-tumor compounds by utilizing two channels. Increase in tumor diseases is nownumber one killer in the world and seriously threatens human survival and health. The prior anti-tumor active compounds screened by aiming at a singe target have the problems of bad patent medicine property or great side effects and the like, and in contrast, medicaments screened through two targets can simultaneously control two transduction pathways. For certain diseases complex in causes, pathological mechanisms and disease development process, multi-target inhibitors are superior to single-target inhibitors in treatment, and multi-target combined blocking-signal transduction is a novel development direction of tumor therapy and medicament development.

Description

The method of screening enhanced anti-tumor compounds

Technical field

The present invention relates to molecular biology and field of medicaments, particularly, the present invention relates to utilize the method for two channels screening enhanced anti-tumor compounds.

Background technology

Tumor disease has now risen to the world No.1 " killer ", serious threat human existence and health.At present, for treatment for cancer is first-selection with chemotherapy and radiotherapy still, though both have obtained suitable curative effect to tumor treatment, but since lack to the specificity of tumour cell thus have bigger toxic side effect and some tumour cell to chemotherapy and radiation handle insensitive, therefore limited their application in clinical to a great extent.The speed of drug research work has been accelerated in the application in antitumor drug research of high flux screening, molecular simulation, drug design method and various biotechnology greatly.Adopt these new technologies, the scientist of Novartis is in the later stage eighties 20th century, expression product with fusion gene Bcr-Abl---Tyrosylprotein kinase is a target, developed new drug Gleevec (STI571) [the Buchdunger E that chronic myeloid leukemia is had better curative effect, Zimmermann J, Mett H, et al.Inhibitionof the abl protein-tyrosine kinase in vitro and in vivo by a 2-phenylaminopyrimidinederivative.Cancer Res, 1996,56:100～104].

Yet the new drug research field still faces many stern challenges at present, and it is still very difficult so far to seek the treatment multigenic disease (as tumour, nerve degenerative diseases, metabolic disease etc.) and the active drug of anti-virus infection (as acquired immune deficiency syndrome (AIDS), hepatitis etc.).Function that major reason is a gene and regulation and control thereof more than people originally imagine more complex.Most of diseases are the results by the common influence of several genes; Exist the dynamic regulation mechanism of idiotype network complexity in the human body.Therefore, at the new drug research thinking and the high flux screening technology of individual molecule target spot, be difficult to comprehensively, intactly reflect the dependency of compound and disease.

Be used for clinical antitumor drug ubiquity poor selectivity now, therefore toxic side effect greatly, easily produces problems such as resistance, continues to seek the selectivity height, anticarcinogen that toxic side effect is little is the emphasis of anti-cancer agent research..Screening anti-tumor medicine is a very important ring in the whole antitumor drug research process, sets up a kind ofly at many target spots, improves selectivity, and the supermatic high-throughput screening method that overcomes the resistance and the new mechanism of action is the task of top priority.

Summary of the invention

The purpose of this invention is to provide a kind of method of utilizing the two channels screening enhanced anti-tumor compounds.

The present invention is a target spot with Aurora B kinases earlier, set up high flux screening model and screened its micromolecular inhibitor, again the micromolecular compound that filters out is carried out vitro kinase and suppress the selectivity experiment, find that it also has the obvious suppression effect to the VEGFR kinase activity. the compound that postsearch screening is obtained carries out testing on the cell levels He in the animal body, find that this compound can not only suppress tumor cell proliferation, the mitotic division of retardance tumour cell, and suppress the tumor neogenetic blood vessels generation, thereby suppress growth of tumor and transfer.

AIK (Aurora/Ipl1 Kinase) is present one of known five big modulability kinases families.Up to the present, human Aiks family finds three member: Aik1, Aik2 (Aurora-B) and Aik3 altogether.Wherein, Aik2 mainly works in the cell mitogen phase.It follows albumen to participate in regulating the interaction of kinetochore and spindle microtubule as karyomit(e), this is instantaneous in mid-term to the later stage, Aik2 migrates to cell intermediate zone band [Bischoff, J.R., Anderson by the kinetochore, L., et al. (1998) EMBO J.17,3052-3065], be distributed in cell dish and back mitotic division bridge latter stage, participate in back mitotic division incident, thus with chromosomal separate with division of cytoplasm relevant.The sudden change of Aik2 is suppressed the formation of branch dehiscence furrow, and causes forming syncyte.

VEGFR has 3 kinds to be that VEGFR-1 (Flt-1), VEGFR-2 (KDR/Flk-1), VEGFR-3 (Flt-4) .VEGFR are class Tyrosylprotein kinase transmembrane glycoproteins, the extracellular region that it is made up of 7 class Ig structural domains is striden Tyrosylprotein kinase structural area composition in membrane structure district and the kytoplasm for one.The extracellular fragment of VEGFR is and VEGF bonded zone that both change in conjunction with back VEGFR conformation, cause receptor dimerizationization, its born of the same parents' inner segment tyrosine site generation autophosphorylation, the signal transduction pathway in activation downstream.The nucleus formation of the propagation of VEGF stimulating endothelial cell, the permeability that increases blood vessel and neovascularity is mainly realized by combination and activation VEGFR-2.

The invention provides a kind of method of utilizing the two channels screening enhanced anti-tumor compounds.This method may further comprise the steps successively:

(1) earlier by computer simulation method screening aurora-B kinase inhibitor;

(2) in gained aurora-B kinase inhibitor, screen the kinase whose inhibitor of VEGFR with computer simulation method.

Method of the present invention can also be carried out according to second method:

(1) adopts radio isotope technique screening aurora-B kinase inhibitor;

(2) in the compound that (1) obtains, adopt vitro kinase selectivity experiment screening VEGFR kinase inhibitor.

In the method for utilizing the two channels screening enhanced anti-tumor compounds of the present invention, in second kind of side in the step (1) with in the solution [γ- ³³P]-γ on the ATP- ³³P shifts and is covalently bound to MBP, makes its serine residue phosphorylation, produces to have isotope-labeled substrate ³³P-MBP adds scintillation solution at last and counts by liquid scintillation counter and detect the influence of candidate compound to the aurora-B kinase activity.

In the method for utilizing the two channels screening enhanced anti-tumor compounds of the present invention, at first adopt vitro kinase selectivity experiment screening screening VEGFR kinase inhibitor, in gained VEGFR kinase inhibitor, adopt radio isotope technique screening aurora-B kinase inhibitor then.

Among the present invention, the computer simulation three-dimensional structure of candidate compound can be combined with the computer simulation three-dimensional structure of kinase protein, the computer simulation three-dimensional structure of candidate compound and the reactive site of kinase protein (N end) are combined closely, and this candidate compound is the activeconstituents of mutually deserved kinase inhibitor.

The present invention also provides a kind of test kit that utilizes the two channels screening enhanced anti-tumor compounds, comprises Aurora B, MBP, VEGFR kinases, ATP etc. in this test kit.

Also comprise 33-p-ATP, DMSO etc. in the mentioned reagent box.This test kit can also contain corresponding operation instruction, molecule marker, damping fluid etc.

Among the present invention, kinase inhibitor, promptly described candidate compound can be nucleic acid, amino acid or micromolecular compound.Wherein, nucleic acid comprises DNA, RNA etc., can be siRNA, antisense oligonucleotide, ribozyme or with other nucleic acid molecule of NRN1 nucleotide sequence complementary; Amino acid comprises peptide, antibody, interact protein etc.; Micromolecular compound then comprises natural and the artificial synthetic.

The present existing active compound that filters out at single target spot, exist into many problems such as the bad or side effect of the property of medicine is big, by comparison, the medicine that two target spots filter out can be regulated and control two kinds of conduction paths simultaneously, to the very complicated disease of some cause of disease, pathomechanism and progression of disease process, many target spots inhibitor is better than single target spot inhibitor aspect treatment, the conduction of many target spot associating disabling signals is the new developing direction of oncotherapy and drug development.

Embodiment

Embodiment 1 utilizes albumen model configuration SCREENED COMPOUND

The activation loop ring district that the present invention has chosen kinases Aurora B crystalline structure activation conformation (PDB entry code:2BFY) is the screening target spot, adopt Autodock3.0.5 molecular docking software about altogether 70,000 (comprising 50,000 molecules and self-built molecule in the Mybridge database) to be divided into 15 nodes and dock research respectively, each compound produces 30 conformations, according in conjunction with free energy and bunch analytical results, filter out 83 compounds Aurora B is had stronger restraining effect; These are had Aurora B suppresses active compound and adopts that to use the same method with VEGFR2 (PDB entry code:2OH4) be that target spot carries out new round screening.

As a result, obtain to suppress simultaneously 17 of the active compounds of VEGFR2, be mainly compound or its pharmacy acceptable salt with indol-2-one class formation.

Embodiment 2 experiment screening methods

Use escherichia expression system, clone, expression and purifying have obtained people source Aurora-B protein kinase (the NCBI number of landing is NP_004208), are used for the high flux screening screening experiment.Adopt radio isotope technique, AURORAB phosphorylated substrate MBP (UPSTATE company, CAT#13-110, lot#27845), with in the solution [γ- ³³P]-γ on the ATP- ³³P shifts and is covalently bound to substrate, makes its Ser phosphorylation, produces to have isotope-labeled substrate ³³P-MBP.Add scintillation solution at last and count by liquid scintillation counter and detect this enzymic activity, can observe different compounds to its active inhibition situation, with the activity of preliminary assessment compound.

Model system (50uL/ hole)

Aurora?B????????????1uM

ATP?????????????????25uM

MBP?????????????????20uM

33-p-ATP????????????0.4Uci

Compound 2uL

45 minutes reaction times

Solvent: DMSO

The compound of acetonideexample 1 all has some restraining effect to AURORA B, and (3Z)-3-(1H-pyrroles-2-methylene)-1,3-dihydro-2H-indol-2-one, (be S2, (3Z)-3-(1H-pyrrol-2-ylmethylene)-1,3-dihydro-2H-indol-2-one), S4 (1,2-dihydroxy-3-methylanthra-9,10-quinone), S6 ((2Z)-2-(3,4-dihydroxybenzylidene)-1-benzofuran-3 (2H)-one) has remarkable inhibition effect.In order on the compound that filters out utilize invitrogen company " z-lyte " the fluorescence report system is to (the NCBI number of landing: NM_002253) carry out vitro kinase and suppress the selectivity experiment of VEGFR kinases, other compound is compared in discovery, and S2 also has had strong inhibitory effects for the VEGFR kinases.

Wherein analytical procedure is as follows:

1)4X?Test?Compound?Solution???????2.5μL

2)2X?Peptide/Kinase?Mixture???????5μL

3)4X?ATP?Solution?????????????????2.5μL

4) room temperature is one hour

5)Development?Reagent?Solution????5μL

6) room temperature is one hour

7) fluoroscopic examination

The result shows, the above-mentioned screening of process, (3Z)-3-(1H-pyrroles-2-methylene)-1,3-dihydro-2H-indol-2-one, (be S2, (3Z)-3-(1H-pyrrol-2-ylmethylene)-1,3-dihydro-2H-indol-2-one) candidate compound for obtaining.

Embodiment 3 cancer cells suppress experiment

Carry out according to standard MTS experiment, wherein concrete parameter is as follows:

Plant plate: 1000 cells/well

Compound: s2

Incubation time: 96h

Add medicine aftertreatment time: 3h

Compound concentration gradient: unit: uM

Repeated experiments: 6 groups

Data provide form: mean+SD

Cell strain: human liver cancer cell strain (HepG2)

The result shows that compound s 2 of the present invention is about 0.75 μ M to the IC50 that HepG2 suppresses.Specifically see the following form:

Drug level (uM)	??25	??12.5	??6.25	??3.125	??1.5625	??0.78125	??0.390625	??0.1953125	??0
										??s2-h ??epG2	??0.0137± ??0.0131	??0.0142± ??0.00624	??0.0235± ??0.00564	??0.0352± ??0.00868	??0.0578± ??0.0140	??0.061± ??0.0174	??0.208± ??0.0278	??0.355± ??0.009	??0.388± ??0.042

4 couples of rat liver cancer H22 of embodiment tumor killing effect

Experimental animal and strain thereof: Kunming mouse

Source: Shanghai Si Laike laboratory animal responsibility company limited

Production licence number: SCXK (Shanghai) 2003-0003

Occupancy permit number: SYXK (Shanghai) 2004-0015

Body weight: 19-21g

Sex: male

Every treated animal number: 8 (16 of control groups)

Transplanted tumor: rat liver cancer H22

Test method:

Get well-grown rat liver cancer H22 ascites, use physiological saline with 1: 4 dilution (the about 1-2 of cell concn * 10 ⁷Individual/ml), and every the right armpit subcutaneous vaccination of mouse 0.2ml, random packet, establish:

1) blank group

2)s2(12.5mg/kg，ip×7)

3)s2(25mg/kg，ip×7)

Administration is played next day in the inoculation back, and the administration volume is the 0.5ml/20g body weight, and abdominal injection is 7 days continuously.Inoculate back 10 days and take off neck execution animal, dissect and get the knurl piece, claim knurl heavy, the result judges.

The result: behind the inoculation s2, the knurl of mouse is heavy obviously to descend, and the s2 of inoculation is many more, and knurl heavily descends obvious more.

Comparative Examples 1

Method according to embodiment 2, the IC50 value of compound s 2 vitro inhibition Aurora-B kinase activity is about 5.54nm, be about 632nm with the IC50 value that records compound s6 vitro inhibition Aurora-B kinase activity with quadrat method, and compound s 6 to suppress the VEGFR kinase activity optionally the IC50 value also far above S2.

Comparative Examples 2

According to the method for embodiment 3, compound s 2 is about 0.75 μ M to the IC50 that HepG2 suppresses.With record compound s6 with quadrat method the IC50 that sw620 suppresses is about 2 μ M.

Claims (5)

1. a method of utilizing the two channels screening enhanced anti-tumor compounds is characterized in that, this method may further comprise the steps:

(1) earlier by computer simulation method screening aurora-B kinase inhibitor;

(2) in gained aurora-B kinase inhibitor, screen the kinase whose inhibitor of VEGFR with computer simulation method.

2. method according to claim 1 is characterized in that, this method is further comprising the steps of:

(1) adopts radio isotope technique screening aurora-B kinase inhibitor;

(2) in the compound that (1) obtains, adopt vitro kinase selectivity experiment screening VEGFR kinase inhibitor.

3. method according to claim 2 is characterized in that, in the step (1) with in the solution [γ- ³³P]-γ on the ATP- ³³P shifts and is covalently bound to MBP, makes its serine residue phosphorylation, produces to have isotope-labeled substrate ³³P-MBP adds scintillation solution at last and counts by liquid scintillation counter and detect the influence of candidate compound to the aurora-B kinase activity.

4. method according to claim 2 is characterized in that, at first adopts vitro kinase selectivity experiment screening screening VEGFR kinase inhibitor, adopts radio isotope technique screening aurora-B kinase inhibitor then in gained VEGFR kinase inhibitor.

5. a test kit that utilizes the two channels screening enhanced anti-tumor compounds is characterized in that, comprises Aurora B, MBP, VEGFR kinases and ATP in this test kit.