CN102095874A - Method for searching small molecular chemical medicament target by using biotin labeling probe and protein chip - Google Patents
Method for searching small molecular chemical medicament target by using biotin labeling probe and protein chip Download PDFInfo
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- CN102095874A CN102095874A CN2011100008137A CN201110000813A CN102095874A CN 102095874 A CN102095874 A CN 102095874A CN 2011100008137 A CN2011100008137 A CN 2011100008137A CN 201110000813 A CN201110000813 A CN 201110000813A CN 102095874 A CN102095874 A CN 102095874A
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- biotin
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Abstract
The invention provides a method for searching a small molecular chemical medicament target by using a biotin labeling probe and a protein chip. The method comprises the following steps of: connecting a biotin and a small molecular chemical medicament to form a biotin and small molecular chemical medicament labeling probe; directly incubating the labeling probe and the protein chip together; and after washing, determining binding sites (namely targets) of specificity between the medicament and the protein fixed on the chip by detecting the biotin labeling probe on the protein chip and BODIPY-FL streptavidin so as to analyze the proteins on the binding sites by using computer software. By the method, the small molecular chemical medicament and the target protein with a medicinal effect on the cell can be searched easily and quickly, and an efficient technical measure can be provided for high-throughout selection of new small molecular chemical medicaments.
Description
Technical field
The invention provides a kind of biotin labeling technology and seek the method for micromolecule chemistry medicine action target spot in conjunction with protein chip technology, belong to material, chemosynthesis, chemical industry, pharmacy and biological crossing domain, be specifically related to a kind of combining and seek the method for micromolecule chemistry medicine action target spot, especially on protein chip, seek the method for the target protein of micromolecule chemistry medicine effect by the biotin labeling particle by biological nano technology and protein chip technology.
Background technology
People studies show that for many years, most drug is to realize that by the function of interfering enzyme in the cell or acceptor it wants drug effect, enzyme or acceptor all are protein, the room that can be occupied by micromolecule is arranged on their surface a lot, when micromolecule was combined in room on enzyme or the acceptor as the key of lock, these micromolecule promptly claimed part.When a kind of medicine and part have same or analogous shape, then this medicine just can be incorporated on enzyme or the acceptor, and bound drug enzyme or acceptor will send a signal to cell, make cell that a series of physiological reaction take place.The enzyme of bound drug or acceptor are pharmaceutically-active direct target spot (target protein).When medicine can in conjunction with target spot when being a plurality of, this medicine may just have spinoff.
Protein chip has the characteristics of high throughput analysis, can show huge superiority aspect the pharmaceutically-active molecule mechanism of announcement.Protein-chip is that protein is fixed on the chip to high-density, but these protein high degree of specificity ground combine with target molecule.When medicine directly with chip on protein have an effect, can reach high flux and analyze pharmaceutically-active target spot apace, this is the most direct method of analysis drug target.For micromolecule chemistry medicine system, can on chip, fix nearly 17,000 human body proteins, hatch with micromolecule chemistry medicine then, thereby tentatively determine micromolecule chemistry medicine action target spot.But when utilizing biochip research mechanism of drug action, what current use was more is genetic chip, as domestic and people adopted mouse 8192 point gene chip researches Chinese medicine compound prescription MRL/pr lupus mouse kidney group is expressed and the Th1/TH2 cell factor than the regulating action of row, experiment adopts organic fluorescent dye Cy3 and Cy5 to detect results of hybridization.Experiment shows that the method for this employing genetic chip research mechanism of drug action all has more bibliographical information at home and abroad, but this method can not the direct acting acceptor of clear and definite medicine.
Though protein-chip be exactly relatively be successfully used to high-throughout drug screening (
China's protein-chip is exactly Be successfully applied to high-flux medicaments sifting .)But do not see the experiment report of biotin labeling technology conjugated protein chip technology being sought micromolecule chemistry medicine target spot is arranged.
Summary of the invention
The objective of the invention is to overcome the deficiencies in the prior art, a kind of short-cut method that micromolecule chemistry medicine has action target spot of seeking is provided.The invention discloses appeal and seek the method for micromolecule chemistry medicine action target spot, especially openly biotin is connected a kind of to form " biotin-micromolecule chemistry medicine " biotinylated probe with micromolecule chemistry medicine, and this probe is used for protein chip to seek the method for drug target.
The present invention by following be exactly that scheme realizes: the present invention is connected the chemical medicine of biotin labeling particle and micromolecule, form a kind of " biotin-micromolecule chemistry medicine " fluorescence probe, then with this fluorescence probe directly and protein chip hatch altogether, after the plain washing of BODIPY-FL strepto-affinity, plain according to the biotinylated probe and the BODIPY-FL strepto-affinity that detect on the protein chip in conjunction with specific binding site between the known protein of determining to fix on medicine and the chip, so pass through these binding sites of computer software analysis albumen.
Below preparation method of the present invention and application are further specified, specific as follows:
The present invention is connected the chemical medicine of biotin and micromolecule by any one or the arbitrarily several compound actions in covalent bond, coordination bond, hydrogen bond, the Electrostatic Absorption.The product that obtains, is hatched the probe and the protein chip that obtain to remove free impurity component then by separation and purification.
Perhaps adopt biotin changed into and adopt chloro biotin (Biotin-Cl) or bromo biotin (Biotin-Br) molecule to be connected then the probe and the protein chip of acquisition are hatched with micromolecule chemistry medicine.Wherein chloro biotin (Biotin-Cl) of Cai Yonging or bromo biotin (Biotin-Br) molecule will with micromolecule chemistry medicine functional group coupling reaction, functional group comprises a kind of or any several combination in hydroxyl, carboxyl, amino, the shin base.If needed, one section alkane molecule chain can be added between biotin and the micromolecule chemistry medicine, thereby ensures the degree of freedom of micromolecule chemistry medicine.
Above-mentioned connection procedure is to carry out in the mixed liquor of water, organic phase or water and organic solvent.
Described protein-chip, the protein in its point sample district is acceptor, antibody or specific protein, refers in particular to acceptor, antibody or the specific protein relevant with drug mechanism to be measured.
Described micromolecule chemistry medicine is meant the potpourri of compound in any one compound that extracts that the method by chemosynthesis obtains or any two kinds or the state from animal, plant or mineral.These compounds have anticancer or antihepatitic activity, the effect of cardiovascular and cerebrovascular pharmacology, AIDS resisting, anti-ageing, treatment diabetes, treatment prostatic disorders, anti-senile dementia, antibiotic, treatment disease of digestive tract, antirheumatic or comprehensive card of rheumatism, treatment osteoporosis or climacteric active any one or any several effect.
For overcoming the deficiencies in the prior art, the present invention's one biotin labeled molecule is come mark micromolecule chemistry medicine, and then hatches altogether with protein chip, to obtain pharmaceutically-active direct target spot.Biotin is meant Biotin, and biotinylated probe and BODIPY-FL strepto-affinity are plain, and acting force is strong in conjunction with stable, is convenient to detect.In addition, biotin labeling can well keep the activity of micromolecule chemistry medicine.Replace the conventional fluorescent molecule to combine biotin, then can not only significantly improve the stability of hybridization signal, strengthen intensity of hybridization signal, but also can reach the effect of two high throughput testing with protein chip technology.The present invention has important and practical meanings for illustrating micromolecule chemistry medicine action target spot and micromolecule chemistry medicine being screened.
The present invention has actual property characteristics and marked improvement, the superior optical property of biotin labeling molecule has overcome the deficiency of traditional organic fluorescent dye, biotin is connected with micromolecule chemistry medicine, and hatch with protein-chip, can directly obtain the target spot of micromolecule chemistry medicine effect, have broad application prospects aspect the screening of research micromolecule chemistry medicine.
Specific implementation method
Embodiment 1:
Add the biotin molecule of 10mmol and the thionyl chloride of 12mmol in the 50ml dichloromethane solution, stirring reaction obtained the still solution of clear after 2 hours under the room temperature.Evaporate to dryness methylene chloride and thionyl chloride add methylene chloride 50ml again, and 12mmol triethylamine and 10mmol contain amino micromolecule chemistry medicine, and stirring reaction spends the night under the room temperature.The micromolecule of separated free chemistry medicine promptly obtains the molecule of " biotin-micromolecule chemistry medicine " that obtained by covalently bound method.Nuclear magnetic resonance and mass spectrum have been identified molecular structure, and micromolecule chemistry medicine is connected on the biotin molecule.
Embodiment 2:
With micromolecule chemistry medicine biotinylated probe and biotin PBS damping fluid, 1%BSA and 0.1%NonidetP-40 are added on the protein chip.Whole association reaction places the reactor of keeping humidity, at room temperature carries out 1 hour (nothing is rocked).Protein chip is by a large amount of PBS damping fluid (containing 0.1%Nonidet P-40) drip washing afterwards.When albumen is with micromolecular the combination on the detection chip, add BODIPY-FL strepto-affinity element, incubated at room 30 minutes., clean these chips with the PBS damping fluid then, detect by biochip scanner.Found that the plain binding site of 5 BODIPY-FL strepto-affinities is arranged on the chip, illustrate that micromolecule chemistry medicine might act on many human body proteins.
Claims (7)
1. a biotinylated probe is united the method that protein chip is sought micromolecule chemistry medicine action target spot, it is characterized in that adopting following steps: biotin is connected with micromolecule chemistry medicine, form a kind of " biotin-micromolecule chemistry medicine " label probe, then with this label probe directly and protein chip hatch altogether, after the plain washing of BODIPY-FL strepto-affinity, determine specific binding site between the known protein fixing on medicine and the chip according to the biotinylated probe and the plain combination of BODIPY-FL strepto-affinity that detect on the protein chip, and then pass through the albumen of these binding sites of computer software analysis.
2. seek the method for micromolecule chemistry medicine action target spot according to the described a kind of biotinylated probe associating of claim 1 protein chip, it is characterized in that: the chemical medicine of biotin and micromolecule is connected by any one or several arbitrarily compound actions in covalent bond, coordination bond, hydrogen bond, the Electrostatic Absorption; The product that obtains to remove free composition, obtains " biotin-micromolecule chemistry medicine " nanometer label probe by separation and purification, then this probe and protein chip are hatched.
3. seek the method for micromolecule chemistry medicine action target spot according to the described a kind of biotinylated probe associating protein chip of claim 1, it is characterized in that: adopt chloro biotin (Biotin-Cl) or bromo biotin (Biotin-Br) molecule that biotin is connected with micromolecule chemistry medicine, then the probe and the protein chip that obtain are hatched, wherein chloro biotin (Biotin-Cl) of Cai Yonging or bromo biotin (Biotin-Br) molecule will with micromolecule chemistry medicine functional group coupling reaction, functional group comprises hydroxyl, carboxyl, amino, a kind of or any several combination in the shin base.
4. seek the method for micromolecule chemistry medicine action target spot according to a kind of biotinylated probe associating protein chip described in the claim 2-3, it is characterized in that: described connection procedure is to carry out in the mixed liquor of water, organic phase or water and organic solvent.
5. seek the method for micromolecule chemistry medicine action target spot according to the described a kind of biotinylated probe associating protein chip of claim 1-3, it is characterized in that: the protein in its point sample district of described protein-chip is acceptor, antibody or specific protein, refers in particular to acceptor, antibody or the specific protein relevant with drug mechanism to be measured.
6. the combination of label probe according to claim 5 is characterized in that by described any one biotin be nuclear.
7. seek the method for micromolecule chemistry medicine action target spot according to the described a kind of biotinylated probe associating protein chip of claim 1-3, it is characterized in that: described micromolecule chemistry medicine is meant the simplification compound.
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| CN2011100008137A CN102095874A (en) | 2011-01-05 | 2011-01-05 | Method for searching small molecular chemical medicament target by using biotin labeling probe and protein chip |
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| CN2011100008137A CN102095874A (en) | 2011-01-05 | 2011-01-05 | Method for searching small molecular chemical medicament target by using biotin labeling probe and protein chip |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108645830A (en) * | 2018-05-15 | 2018-10-12 | 华中科技大学鄂州工业技术研究院 | A kind of method that namo fluorescence probe combined protein chip finds drug target |
| CN110627852A (en) * | 2019-10-12 | 2019-12-31 | 华中农业大学 | Biotin-labeled naringin, preparation method and application thereof |
| CN114088953A (en) * | 2022-01-19 | 2022-02-25 | 中国中医科学院医学实验中心 | Drug target screening protein chip kit |
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| EP1452868A2 (en) * | 2003-02-27 | 2004-09-01 | Pepscan Systems B.V. | Method for selecting a candidate drug compound |
| CN1719253A (en) * | 2004-07-09 | 2006-01-11 | 中国医学科学院药物研究所 | A kind of high flux screening reverse protein chip, preparation method and detection method thereof |
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2011
- 2011-01-05 CN CN2011100008137A patent/CN102095874A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1452868A2 (en) * | 2003-02-27 | 2004-09-01 | Pepscan Systems B.V. | Method for selecting a candidate drug compound |
| CN1719253A (en) * | 2004-07-09 | 2006-01-11 | 中国医学科学院药物研究所 | A kind of high flux screening reverse protein chip, preparation method and detection method thereof |
Non-Patent Citations (5)
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| AMIR M SADAGHIANI: "Tagging and detection strategies for activity-based proteomics", 《CURRENT OPINION IN CHEMICAL BIOLOGY》, vol. 11, 13 December 2006 (2006-12-13), pages 20 - 28, XP 005881382, DOI: doi:10.1016/j.cbpa.2006.11.030 * |
| LAKHA SLENO: "Proteomic methods for drug target discovery", 《CURRENT OPINION IN CHEMICAL BIOLOGY》, vol. 12, 7 March 2008 (2008-03-07), XP 022547307, DOI: doi:10.1016/j.cbpa.2008.01.022 * |
| RUI CHEN: "Yeast proteomics and protein microarrays", 《JOURNAL OF PROTEOMICS》, vol. 73, 31 October 2010 (2010-10-31), XP 027363344 * |
| 于晓波: "生物素-亲和素偶联探针蛋白芯片检测技术", 《生物工程学报》, vol. 24, no. 3, 25 March 2008 (2008-03-25) * |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108645830A (en) * | 2018-05-15 | 2018-10-12 | 华中科技大学鄂州工业技术研究院 | A kind of method that namo fluorescence probe combined protein chip finds drug target |
| CN110627852A (en) * | 2019-10-12 | 2019-12-31 | 华中农业大学 | Biotin-labeled naringin, preparation method and application thereof |
| CN114088953A (en) * | 2022-01-19 | 2022-02-25 | 中国中医科学院医学实验中心 | Drug target screening protein chip kit |
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Application publication date: 20110615 |