CN102095875A - Method for searching micro-molecular chemical drug targets by binding click chemical technology and fluorescent dye probe with protein chip - Google Patents
Method for searching micro-molecular chemical drug targets by binding click chemical technology and fluorescent dye probe with protein chip Download PDFInfo
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- CN102095875A CN102095875A CN2011100008141A CN201110000814A CN102095875A CN 102095875 A CN102095875 A CN 102095875A CN 2011100008141 A CN2011100008141 A CN 2011100008141A CN 201110000814 A CN201110000814 A CN 201110000814A CN 102095875 A CN102095875 A CN 102095875A
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Abstract
The invention discloses a method for searching micro-molecular chemical drug targets by binding a click chemical technology and a fluorescent dye probe with a protein chip, which comprises the steps: alkyne is connected with a micro-molecular chemical drug so as to form an alkyne- micro-molecular chemical drug labeled probe; then the labeled probe and a protein chip are directly incubated together, and bound together after reaction with the azide of BODIPY-FL (a fluorescent dye); after washing, the alkyne labeled probe on the detected protein chip is bound with the streptavidin of the BODIPY-FL to determine the specific binding sites (namely the targets) between the drug and the fixed protein on the chip; and further, the protein at the binding sites is analyzed by computer software. By using the method, the micro-molecular chemical drug and target protein having an effect on cells can be searched simply, conveniently and rapidly; and an efficient technical method can be provided for screening new micro-molecular chemical drugs at a high flux.
Description
Technical field
The invention provides a kind of alkynes labelling technique and seek the method for micromolecule chemistry medicine action target spot by the click chemistry technology 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 alkynes label particles 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 successfully applied to high-flux medicaments sifting exactly .)But do not see the experiment report of alkynes labelling technique 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 alkynes is connected a kind of to form " alkynes-micromolecule chemistry medicine " alkynes label 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 alkynes label particles and micromolecule, form a kind of " alkynes-micromolecule chemistry medicine " probe, then with this probe directly and protein chip hatch altogether, through with BODIPY-FL on the nitrine effect, after the washing, specific binding site between the known protein of determining to fix on medicine and the chip according to the BODIPY-FL fluorescence labeling that detects on the protein 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 alkynes 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 alkynes changed into and adopt chloro alkynes or bromo alkynes molecule to be connected then the probe and the protein chip of acquisition are hatched with micromolecule chemistry medicine.Wherein chloro alkynes of Cai Yonging or bromo alkynes 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 alkynes 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 alkynes labeled molecule is come mark micromolecule chemistry medicine, and then hatches altogether with protein chip, to obtain pharmaceutically-active direct target spot.Come mark micromolecule chemistry medicine little with the alkynes labeled molecule for the chemistry and the physical property influence of micromolecule chemistry medicine itself, alkynes label probe and BODIPY-FL nitrine are stable by Huisgen cycloaddition reaction bonded, acting force is strong, is convenient to detect.In addition, the alkynes mark can well keep the activity of micromolecule chemistry medicine.Replace the conventional fluorescent molecule to combine in alkynes, 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 optical property that the alkynes labeled molecule is superior has overcome the deficiency of traditional organic fluorescent dye, alkynes 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 1-bromo-4-hydroxyl butane molecule of 10mmol and the sodium hydride of 11mmol in the 50ml diethyl ether solution, stirring reaction obtained the still solution of clear after 2 hours under the room temperature.Add 10mmol 3-propargyl bromide in this solution, stirring reaction spends the night under the room temperature.Behind the evaporate to dryness diethyl ether solution, add methylene chloride 50ml again, 11mmol 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 " alkynes-micromolecule chemistry medicine " that obtained by covalently bound method.Nuclear magnetic resonance and mass spectrum have been identified molecular structure, and micromolecule chemistry medicine has been connected on the alkynes molecule.
Embodiment 2:
Add the BODIPY-FL carboxylic acid molecules of 10mmol and the borine of 11mmol in the 50ml dichloromethane solution, stirring reaction obtained the still solution of clear after 2 hours under the room temperature.After under ice bath, adding the borine that it is unnecessary that shrend is gone out, after being separated, the dichloromethane solution layer adds 11mmol triethylamine and 10mmolMsCl molecule.Stirring reaction added the 10mmol Sodium azide after 2 hours under the room temperature, and stirring reaction spends the night under the room temperature.After the separation and purification, promptly obtain the BODIPY-FL nitrine molecule that obtains by covalently bound method.Nuclear magnetic resonance and mass spectrum have been identified molecular structure.
Embodiment 3:
Micromolecule chemistry medicine alkynes label probe is 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 drip washing afterwards.When albumen is with micromolecular the combination on the detection chip, add the copper sulphate of BODIPY-FL nitrine molecule and equimolar amounts, sodium ascorbate.In isopyknic water and normal butyl alcohol, reacted 30 minutes under the room temperature., with PBS damping fluid and isopyknic water and clean these chips of normal butyl alcohol, detect then by biochip scanner.Found that 5 protein B ODIPY-FL binding sites are arranged on the chip, illustrate that micromolecule chemistry medicine might act on many human body proteins.
Claims (9)
1. an alkynes label probe is united the method that protein chip is sought micromolecule chemistry medicine action target spot, it is characterized in that adopting following steps: alkynes is connected with micromolecule chemistry medicine, form a kind of " alkynes-micromolecule chemistry medicine " label probe, then with this label probe directly and protein chip hatch altogether, through combining with triazo-compound reaction (click chemistry) back of BODIPY-FL (a kind of fluorescent dye), after washing, determine specific binding site between the known protein fixing on medicine and the chip according to the fluorochrome label probe that detects 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 alkynes label probe associating protein chip of claim 1, it is characterized in that: alkynes is connected with micromolecule chemistry medicine by the covalent bond effect; The product that obtains to remove free composition, obtains " alkynes-micromolecule chemistry medicine " 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 alkynes label probe associating protein chip of claim 1, it is characterized in that: adopt chloro alkynes or bromo alkynes molecule that alkynes is connected with micromolecule chemistry medicine, then the probe and the protein chip that obtain are hatched, wherein chloro alkynes of Cai Yonging or bromo alkynes 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.
4. seek the method for micromolecule chemistry medicine action target spot according to a kind of alkynes label probe associating protein chip described in the claim 1-3, 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 alkynes label 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 alkynes be nuclear.
7. seek the method for micromolecule chemistry medicine action target spot according to the described a kind of alkynes label probe associating protein chip of claim 1-3, it is characterized in that: described micromolecule chemistry medicine is meant the simplification compound.
8. be that carboxyl with BODIPY-FL changes into azide according to the triazo-compound of the described BODIPY-FL of claim 1 (a kind of fluorescent dye).
9. according to described in the claim 1-8, after alkynes mark micromolecule chemistry medicine probe is hatched with protein chip, react by Huisgen cycloaddition with the triazo-compound of BODIPY-FL and to combine.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN112082976A (en) * | 2019-06-14 | 2020-12-15 | 天津方得生物科技有限公司 | In-vitro drug sensitivity detection method based on drug probe and tissue slice |
| CN112147114A (en) * | 2019-06-27 | 2020-12-29 | 成都先导药物开发股份有限公司 | Method for determining interaction of compound and target in living cell by using fluorescence labeling compound |
| CN113189078A (en) * | 2021-03-04 | 2021-07-30 | 吉林大学 | High-throughput screening method for targeted drugs |
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| EP1452868A2 (en) * | 2003-02-27 | 2004-09-01 | Pepscan Systems B.V. | Method for selecting a candidate drug compound |
| CN101881733A (en) * | 2010-07-08 | 2010-11-10 | 浙江工业大学 | method for screening triazole monoamine oxidase inhibitor in high throughput way and compound |
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2011
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| EP1452868A2 (en) * | 2003-02-27 | 2004-09-01 | Pepscan Systems B.V. | Method for selecting a candidate drug compound |
| CN101881733A (en) * | 2010-07-08 | 2010-11-10 | 浙江工业大学 | method for screening triazole monoamine oxidase inhibitor in high throughput way and compound |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112082976A (en) * | 2019-06-14 | 2020-12-15 | 天津方得生物科技有限公司 | In-vitro drug sensitivity detection method based on drug probe and tissue slice |
| CN112147114A (en) * | 2019-06-27 | 2020-12-29 | 成都先导药物开发股份有限公司 | Method for determining interaction of compound and target in living cell by using fluorescence labeling compound |
| CN113189078A (en) * | 2021-03-04 | 2021-07-30 | 吉林大学 | High-throughput screening method for targeted drugs |
| CN113189078B (en) * | 2021-03-04 | 2024-04-16 | 吉林大学 | High-throughput screening method of targeted drugs |
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