CN104774904A - Method for screening anti-tumor medicines by using tumor necrosis factor receptor-associated factor 6 - Google Patents
Method for screening anti-tumor medicines by using tumor necrosis factor receptor-associated factor 6 Download PDFInfo
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Abstract
The invention discloses a method for screening anti-tumor medicines by using a tumor necrosis factor receptor-associated factor 6. The method comprises the following steps: by taking a tumor necrosis factor receptor-associated factor 6 shown in SEQ ID NO. 1 as an active pocket of a detection target spot, performing docking on the active pocket and small molecules namely the small molecules of a natural product database or a commercial small-molecule database by using AUTODOCK software, analyzing docking results, predicting the affinity size between the small molecules and the active pocket according to scoring indexes, screening the small molecules with potential anti-tumor activity, and verifying the anti-tumor activity of the small molecules by virtue of experiments. According to the method disclosed by the invention, a key enzyme for activating kinase is selected aiming at kinase which is highly expressed in cancers, action target spots of medicines can be determined, and thus small-molecular anti-tumor medicines with few side effects and high anti-tumor curative effects can be rapidly screened out.
Description
Technical field
The present invention relates to a kind of method of screening antineoplastic drugs, particularly relate to a kind of method of tumor necrosis factor receptor-associated factor screening antineoplastic drugs.
Background technology
Akt (also referred to as protein kinase B, PKB) is the silk Serineprotein kinase of a high conservative on evolving, frequent high level activation in people's cancer.Akt abnormal activation means the tolerance of tumour cell for apoptosis induction, and the exception of cell proliferation, growth, metabolism increases.Akt also exists the situation of excessive activation in most cancer, implies the key player of activation in tumour occurs of Akt.How by the molecular mechanism of raising on cytolemma after being activated about Akt, recently, someone reports tumor necrosis factor receptor-associated factor (TRAF6) is a kind of E3 ubiquitin ligase, can ubiquitination Akt, thus promote that Akt transfers on cytolemma, and be activated.
The generation of tumour, particularly malignant tumour has a strong impact on the health of people, even jeopardizes the life of people, find antitumor drug fast, the antitumor drug of generation and the development, particularly some few side effects of tumour can be reduced as much as possible, need badly at present especially.
Summary of the invention
The object of this invention is to provide a kind of method of tumor necrosis factor receptor-associated factor screening antineoplastic drugs.
Technical scheme of the present invention is summarized as follows:
By the method for tumor necrosis factor receptor-associated factor (TRAF6) screening antineoplastic drugs, comprise the steps: using the TRAF6 shown in SEQ ID NO.1 as the active pocket detecting target spot, with the small molecules in natural products database or commercial small molecules database, utilize AUTODOCK software to carry out active pocket to dock with micromolecular, analyze docking result, according to the avidity size of give a mark index prediction small molecules and active pocket, screening has the small molecules of potential anti-tumor activity, verifies micromolecular anti-tumor activity by experiment.
Described small molecules is quinine, quinine fourth or cinchonine.
Advantage of the present invention is:
For the kinases of high expression level in cancer, select the key enzyme making it activate, determine the action target spot of medicine, rapid screening goes out few side effects, small molecule, anti-tumor drug that antitumor curative effect is high.
Accompanying drawing explanation
Fig. 1 (A) is (1S)-quinolyl-4 (5-vinyl quinine-2-base) molecular structural formula of methyl alcohol (cinchonine) and the binding site with TRAF6 thereof; (B) be (1R)-(6-methoxy quinoline-4-base) ((1S, 4S, 5R)-5-vinyl quinine-2-base) molecular structural formula of methyl alcohol (quinine) and the binding site with TRAF6 thereof; (C) be (S)-(6-methoxy quinoline-4-base) ((1S, 2R, 4S, the 5R)-5-vinyl quinine-2-base) molecular structural formula of methyl alcohol (quinine fourth) and the binding site with TRAF6 thereof.
The growth-inhibiting effect of Fig. 2 cinchonine (in figure A), quinine (in figure B), quinine fourth (in figure C) antithetical phrase Hela cells, after dosing (a) 72 hours (h), (b) 96h, the cell growth condition measured with mtt assay.
Fig. 3 cinchonine (in figure A), quinine (in figure B), quinine fourth (in figure C) are to the growth-inhibiting effect of lung cancer cell line A-549 cell, (a) 72h after dosing, (b) 96h, the cell growth condition measured with mtt assay.
Fig. 4 cultivates Hela cell and adds 48h after different concns compound, with expression (a) cinchonine of flow cytomery AnnexinV and PI, and the effect of (b) quinine and (c) quinine fourth inducing cell early apoptosis.
Fig. 5 cultivates the expression of the Bax/Bcl of A549 cell, a () adds quinine 12h, 24h, 48h, 72h and 96h after, b the band in () (a) figure, with after ImageJ software (National Institutes of Health) semiquantitative determination, calculates the ratio histogram graph representation of Bax/ β-actin and Bcl/ β-actin.
Fig. 6 adds 1.5 × 10
-4the quinine of mol/L stimulates 3,6,12 and 24h respectively, after adding the LPS stimulation 3h of 20 μ g/ml again, Western blot method measures the result of phosphorylation Akt (pAkt), and histogram is for A-549 cell (b) is Hela cell with the band (a) of ImageJ software (National Institutes of Health) semiquantitative determination pAkt and β-actin
Fig. 7 cultivates after Hela cell adds compound quinine effect 3,6,12,24h, extracts to detect Akt after protein TRAF6 antibody carries out co-immunoprecipitation and express, and (a) five bands are respectively: 1: blank; 2, add 3h after quinine; 3: add 6h after quinine; 4: add 12h after quinine; 5: add quinine 24h; B () uses the band of ImageJ software (National Institutes of Health) semiquantitative determination Akt and TRAF6, the ratio of histogram graph representation Akt/TRAF6.
Embodiment
Below in conjunction with specific embodiment, the present invention is further illustrated.Embodiments of the invention implement to enable those skilled in the art to, but do not impose any restrictions the present invention.Disclosing of common agents is wherein to make those skilled in the art better implement the present invention, but does not impose any restrictions the present invention.
Embodiment 1
By computer virtual sifting technology, filter out TRAF6 and to be correlated with micromolecular compound:
Protein structural information obtains: download shown in TRAF6 protein structure (PDB ID:3HCT) SEQ ID NO.1 from RCSB Protein Data Bank, using it as the active pocket detecting target spot; By the small molecules in natural products database or commercial small molecules database, the hydrogenation before docking, adds the process such as electric charge; Utilize AUTODOCK software to carry out active pocket to dock with micromolecular, analyze docking result, according to the avidity size of give a mark index prediction small molecules and active pocket, and analyze the mutual relationship of various parameter and the contribution to combination energy, screen small molecules quinine, quinine fourth and the cinchonine (Fig. 1) with potential anti-tumor activity.
TRAF6 be have 523 amino acid structures cell in ubiquitin enzyme, we select TRAF6 to be action target spot, first with computer aided design (CAD) find can with ubiquitin binding enzyme binding site (the i.e. 50-80 of TRAF6,90-110 amino acid) micromolecular compound that combines, synthesis or buy wherein after several compound, tests the molecular mechanism of its anti-tumor activity and effect.
Finding by computer aided design (CAD) can 2 micromolecular compound that with upper amino acid combines amino acid whose with the 50-80 of TRAF6,90-110, sets up compound effects target spot;
Detect the growth-inhibiting effect of several compound to different tumour cell respectively, and bring out apoptotic effect;
Embodiment 2
Compound quinine, quinine fourth and cinchonine that embodiment 1 is screened are on the impact of growth of tumour cell:
Cultivate Hela cell and A-549 cell (purchased from all creation bio tech ltd, Shenyang), be inoculated into 96 orifice plates with the cell density in 4000/hole, after cultivating 24 hours (h), add 1 × 10 respectively
-6, 5 × 10
-5, 1 × 10
-5, 5 × 10
-4, 1 × 10
-4, 2.5 × 10
-4, 5 × 10
-4the quinine of mol/L concentration, quinine fourth or cinchonine, cultivate 48h respectively, cell growth condition is surveyed with mtt assay after 72h and 96h, its result is summarized as Fig. 2 and Fig. 3, Fig. 2 shows the growth that 3 kinds of compounds suppress Hela cell with can having concentration interdependence, and Fig. 3 shows the growth that 3 kinds of compounds suppress A-549 cell with can having concentration interdependence.In figure, A, B, C respectively represent cinchonine (A), quinine (B), quinine fourth (C).
Mtt assay: every hole adds the MTT solution 20 μ l of 5mg/ml, takes out, supernatant discarded in incubator after hatching 4h, every hole adds 150 μ l dmso solution first a ceremonial jade-ladle, used in libation precipitations, measures absorbance by microplate reader under 490nm.
Fig. 2,3 for add after compound 72 and 96h Growth of Cells result statistics and with histogram graph representation, experimental result shows that 3 kinds of compounds can suppress the growth of Hela cell (Fig. 2) and A-549 cell (Fig. 3), shows that 3 kinds of compounds have anti-tumor activity.
Embodiment 3
Early stage apoptotic detection-----fluidic cell
In order to study the mechanism of 3 kinds of compound inhibition tumor cells growth, we have inquired into their early stage apoptotic impacts on Hela cell further.Method is as follows:
Cultivate Hela cell (purchased from all creation bio tech ltd, Shenyang), be inoculated in 6cm Tissue Culture Dish, adjustment cell density, cultivates after 24h, add compound be cell density about 40%, add 1 × 10 respectively
-4, 1.5 × 10
-4, 2 × 10
-4the quinine of mol/L concentration, 1 × 10
-4, 1.5 × 10
-4, 1.8 × 10
-4the cinchonine of mol/L concentration, 8 × 10
-5, 1 × 10
-4, 1.2 × 10
-4the quinine fourth of mol/L concentration, uses Flow Cytometry Assay early apoptosis of cells after acting on 48h respectively.
Flow cytometry: in culturing cell and 6cm Tissue Culture Dish, after adding compound stimulation, collecting cell, PBS washes 2 times, 2000 turns of (rpm) centrifugal 5min, supernatant discarded, add Binding Buffer 100 μ l, phosphatidylserine associated proteins (AnnexinV)-FITC 5 μ l and propidium iodide (PI) 5 μ l, after 30 minutes (min) is reacted in room temperature dark place, add the Binding Buffer of 400 μ l, sample detection early apoptosis of cells.
Result shows the expression (Fig. 4) that 3 kinds of compounds all improve AnnexinV and PI of Hela cell, shows that 3 kinds of compounds can promote the early apoptosis of tumour cell.
Embodiment 4
The detection (Bax/Bcl) of apafl
We have also inquired into the expression of compound on intracellular Apoptosis-Related Factors Bax and Bcl.
Cultivate Hela cell (purchased from all creation bio tech ltd, Shenyang), be inoculated in 6cm Tissue Culture Dish, adjustment cell density, cultivates after 24h, and when adding compound, cell density is about 40%.Every ware cell adds 1.5 × 10
-4the quinine of mol/L, stimulates 12h, 24h, 48h, 72h, 96h respectively.Extract total protein of cell, detect the expression of apafl Bax/Bcl by Western blot method.
Western blot: extract total protein of cell, application BCA albuminimetry draws the concentration extracting albumen, by every hole 40 microgram loading.Draw protein sample, add 5 × loading buffer in 1:4 ratio, boil 10min, loading, run SDS electrophoresis, 80V, 20min, 120V, 1h.After electrophoresis terminates, by the half-dried transfer groove that turns in 23V, transfer printing 1h.Take out pvdf membrane, TBST washes 6 times, each 5min, and 2h closed by 5% skim-milk.Proceed in Bax, Bcl or β-actin antibody of 5%BSA dilution, 4 DEG C are shaken and spend the night.Second day, take out pvdf membrane, TBST proceed to after washing film 5% skimmed milk dilution general two anti-in, hatch 40min, TBST washes 50min, adds eECL, and reaction 2min, darkroom exposes.
Result display increases from the expression of Bax after adding quinine 24h, and the expression of Bcl is suppressed, after 48h, this phenomenon more obviously (Fig. 5), and Bax is that apoptosis promotes albumen, and Bcl is IAP, this result supports that quinine facilitates apoptosis further.
Embodiment 5
Akt phosphorylation detects
LPS can promote Akt phosphorylation, in order to inquire into the action pathway of compound, we have studied the phosphorylation that can compound stop Akt.
Cultivate Hela cell (purchased from all creation bio tech ltd, Shenyang), be inoculated in 6cm Tissue Culture Dish, adjustment cell density, when making to add compound, cell density reaches 70%, adds 1.5 × 10
-4the quinine of mol/L stimulates 3,6,12 and 24h respectively.Then, after adding the LPS stimulation 3h of 20 μ g/ml, total protein is extracted.The change of phosphorylation Akt is measured, using β-actin as internal reference albumen by Western blot method.
Western blot method is with mentioned above.
Result shows, the Akt phosphorylation (Fig. 6) that quinine can stop LPS to cause effectively.
Embodiment 6
The relation of TRAF6 and Akt
Cultivate Hela cell (purchased from all creation bio tech ltd, Shenyang), be inoculated in 6cm Tissue Culture Dish, adjustment cell density, when making to add compound, cell density reaches 70%, adds 1.5 × 10
-4the quinine of mol/L stimulates 3 respectively, 6h.The relation of TRAF6 and Akt is detected by co-immunoprecipitation method.Concrete grammar is as follows:
Co-immunoprecipitation:
Peptic cell, obtains cell precipitation, adds cell pyrolysis liquid balance liquid, and 1% proteinase inhibitor, on ice cracking, frequently shake, cracking 30 minutes (min) left and right.12000 turns of (rpm) centrifugal 20min, get supernatant and magnetic bead 4 DEG C hatches 1h.The centrifugal 2min of 800rpm, removes magnetic bead, gets supernatant and the anti-TRAF6 antibody of rabbit (Santa Cruz, USA) 4 DEG C and slowly shakes and spend the night.Second day, get 20 μ l magnetic beads and join in albumen, the anti-TRAF6 mixtures of antibodies of rabbit, 4 DEG C were slowly shaken 6h.Wash 3 times with buffer A, each 2min, 800rpm are centrifugal.In magnetic bead, add 50 μ l 1 × loading buffer, boil, electrophoresis.With mouse-anti TRAF6 (Epitomics, USA) and Akt antibody with Western blot method detect TRAF6 and Akt express, two of TRAF6 anti-selects anti-mouse antibody (Abmart, China), rabbit source antibody used when precipitating TRAF6 albumen to distinguish, other Western blot method method is with described in embodiment 4.
Albumen after result display precipitates with the immunomagnetic beads with TRAF6 antibody, blank group can detect the expression of TRAF6 and Akt simultaneously, and after adding quinine 3h, visible TRAF6 expresses, Akt expresses and obviously reduces, and illustrates that quinine prevents the combination (Fig. 7) of TRAF6 and Akt.
In sum, first we pass through computer simulation design, have found the compound be combined with TRAF6, and shown by above experimental result, quinine, quinine fourth and cinchonine can grow by inhibition tumor cell, promote the early apoptosis of tumour, and enhance the expression promoting apoptosis factor, inhibit the expression of the apoptosis inhibit factor.Further research finds that compound can stop the combination of TRAF6 and Akt albumen, thus inhibit the phosphorylation of Akt, demonstrate and TRAF6 can be used as the target spot of screening anti-tumor medicine, and demonstrate the quinine, quinine fourth and the cinchonine that are combined with TRAF6 there is anti-tumor function, be expected to and apply at anti-tumor aspect from now on.
Experiment main agents:
。
Claims (2)
1. by the method for tumor necrosis factor receptor-associated factor screening antineoplastic drugs, it is characterized in that comprising the steps: using the tumor necrosis factor receptor-associated factor shown in SEQ IDNO.1 as the active pocket detecting target spot, with the small molecules in natural products database or commercial small molecules database, utilize AUTODOCK software to carry out active pocket to dock with micromolecular, analyze docking result, according to the avidity size of give a mark index prediction small molecules and active pocket, screening has the small molecules of potential anti-tumor activity, verify micromolecular anti-tumor activity by experiment.
2. the method for tumor necrosis factor receptor-associated factor screening antineoplastic drugs according to claim 1, is characterized in that described small molecules is quinine, quinine fourth or cinchonine.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105560241A (en) * | 2015-12-11 | 2016-05-11 | 北京夸常生物医疗科技有限公司 | Application and method of quinine dihydrochloride to tumor treatment, and medicine composition |
| CN105687195A (en) * | 2015-12-11 | 2016-06-22 | 北京夸常生物医疗科技有限公司 | Application and method of quinine dihydrochloride to secreting gland disease treatment and medicine composition |
| CN106860457A (en) * | 2015-12-11 | 2017-06-20 | 四川诺邦医疗科技有限公司 | A kind of pharmaceutical composition, preparation method and applications for treating secretion gland disease |
| CN106860458A (en) * | 2015-12-11 | 2017-06-20 | 成都夸常科技有限公司 | Application, method and pharmaceutical composition of the quinine class compound in tumour is treated |
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| CN103294933A (en) * | 2013-05-10 | 2013-09-11 | 司宏宗 | Drug screening method |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105560241A (en) * | 2015-12-11 | 2016-05-11 | 北京夸常生物医疗科技有限公司 | Application and method of quinine dihydrochloride to tumor treatment, and medicine composition |
| CN105687195A (en) * | 2015-12-11 | 2016-06-22 | 北京夸常生物医疗科技有限公司 | Application and method of quinine dihydrochloride to secreting gland disease treatment and medicine composition |
| CN106860457A (en) * | 2015-12-11 | 2017-06-20 | 四川诺邦医疗科技有限公司 | A kind of pharmaceutical composition, preparation method and applications for treating secretion gland disease |
| CN106860458A (en) * | 2015-12-11 | 2017-06-20 | 成都夸常科技有限公司 | Application, method and pharmaceutical composition of the quinine class compound in tumour is treated |
| CN105687195B (en) * | 2015-12-11 | 2021-06-08 | 北京夸常生物医疗科技有限公司 | Application and method of quinine dihydrochloride in treating secretory gland diseases and pharmaceutical composition |
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Application publication date: 20150715 |