CN103792293A - Method of screening target protein ligand - Google Patents
Method of screening target protein ligand Download PDFInfo
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- CN103792293A CN103792293A CN201210422212.XA CN201210422212A CN103792293A CN 103792293 A CN103792293 A CN 103792293A CN 201210422212 A CN201210422212 A CN 201210422212A CN 103792293 A CN103792293 A CN 103792293A
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Abstract
The invention provides a method of screening a micromolecule ligand capable of combining a target protein by utilization of mass spectrometry. The method comprises steps of contacting the target protein with a micromolecule compound library to produce a target protein-micromolecule composite, separating the target protein-micromolecule composite, dissociating the micromolecule in the target protein-micromolecule composite, and detecting the micromolecule dissociated from the composite by utilization of mass spectrometry, thus indirectly screening to obtain the micromolecule having combining functions with the target protein, namely the ligand of the target protein. The method is suitable for high-throughput screening of micromolecule medicines.
Description
Technical field
The present invention relates to the screening of protein ligands, particularly relate to the method for utilizing mass spectrum to screen the little molecule ligand of being combined with target protein, can be used for the discovery to new drug precursor molecule and initiative in new drug development.Background technology
High throughput screening drug is that multiple technologies method is organically combined and the new technical system of formation, it is take the experimental technique of molecular level and cellular level as basis, using microplate format as experimental tool carrier, carry out experimentation with automation operating system, gather experimental data with sensitive detecting instrument fast, data analysis processing experiment being obtained with computing machine.Normally carrying out of it need to have the operating system of the compound library of a high power capacity, robotization, the medicaments sifting model of highly sensitive detection system, high efficiency data handling system and high specific.
High Throughput Screening Assay responds compared with traditional drug screening method that volume is little, automaticity is high, sensitive fast, the feature such as high specificity.Due to the development of combinatorial chemistry, can synthesize hundreds of or thousands of related compounds simultaneously, in the short time, can obtain up to ten thousand compounds.High flux screening has more efficient, quick and economic advantage, participates in more and more widely in drug discovery process, has formed clue molecular screening-the determine technology path of lead compound-lead optimization-pharmaceutical research.High flux molecular medicine screening center is the gordian technique support that joint study institute develops a large amount of new drugs.The drug screening flow process of tentatively setting up at present mainly comprises high flux screening and protein complex crystal structure analysis.
But High Throughput Screening Assay still has some shortcomings at present, these shortcomings comprise: false positive problem; There is not ready-made high flux screening method of testing for some target protein; According to a set of specific high flux screening method of testing of the function development of target protein, the process wasting time and energy often, conventionally need to carry out a lot of changes and optimize just reaching the requirement of setting up high-throughput screening method to the routine biochemistry method of the enzymatic activity of measuring this albumen, once and change target protein to be studied, just need to redesign method; High Throughput Screening Assay conventionally screening to as if the compound monomer of purifying.For example, if the object of tape test is potpourri (different component that natural products rough segmentation obtains), the data representative providing be the whole result of the common interferases activity of polycomponent, singly cannot learn it is which kind of composition in potpourri really plays a role by this result; Protein three-dimensional structure analysis is often subject to the limitation such as difficulty and speed of crystal growth.These factors make existing technology platform in the urgent need to incorporate one (or several) independently module to fill up important technology vacancy, expand and improve the overall route of prodrug screening and optimizing.Mass spectrum relies on the advantage of its numerous uniquenesses and successfully applies precedent, becomes the one preferred technique that meets these large platform fundamental construction needs.
Mass spectrum, due to its high sensitivity and analysis speed fast, can develop into the especially gordian technique means of protein complex of research biomacromolecule.The technology of the profiling protein matter-ligand interaction common with other with the High Throughput Screening Assay of widespread use is compared, and mass spectrophotometry has following outstanding advantage:
1. the combination of utilizing Mass Spectra drug candidate molecule and target protein is a kind of method that universality is very high, studies different target proteins and can adopt essentially identical technology path.
2. without labeled substrate and target protein, reduce the impact that chemical labeling may produce the binding property of albumen.
3. mass spectrographic high sensitivity makes the consumption of target protein can be down to Gamma Magnitude.
4. the purity requirement of pair ligand molecular and target protein obviously reduces.Mass spectrum can be distinguished the heterogeneity in mixed substrates, and their binding properties are separately characterized simultaneously.
Target molecule is by the crystal of target proteins matter is contacted with compound molecule mixture alternate sample with the fine three dimensional structure of target protein complex, the X ray diffracting data of the crystal after the crystal of analyzing target proteins matter contacts with target molecule, and then the electron density of the acquisition target molecule of being combined with target proteins.
Existing technical scheme is as follows: the first, choosing of target proteins matter, comprising: the method for the useful X-ray crystallography protein that parses three-dimensional structure can serve as target proteins matter.The second, prepare the crystal of target proteins matter, comprising: use above-mentioned purified target proteins, through the crystal condition of own this target proteins of optimizing, the crystal that growth is in a large number, diffracting power is strong.The 3rd, the preparation of the alternative sample of compound: after natural product extraction, the solvent that extract first uses opposed polarity extracts as ethyl acetate, water after extraction is processed by the method that macroreticular resin separates again, can guarantee to remove like this disturbing factor, can utilize again the difference of two kinds of different separation mechanisms, make little molecule focus on the section that polarity is different, obtain can be used in thus the natural products mixture alternate library of the bio-physical methods such as X-ray crystallography.The 4th, natural products mixture alternate sample is carried out to the mensuration of external activity to target proteins.The 5th, by obtain target proteins with have suppress the target proteins ligand molecular complex that may exist in active sample can stable existence, and have can be for the crystal of the diffracting power of x-ray diffraction experiment.The 6th, carry out the Collection and analysis of X ray diffracting data.The 7th, the identification of compound analysis.
But above method has following shortcoming: one, whole process steps is many, and cost is high, and it is very difficult to obtain the crystal of protein and micromolecular compound.Two, the group that ligand molecular amount is less in the electron density of low resolution, be difficult for crystallization solution in little molecule (as DMSO) etc. itself that exist differentiate, therefore the crystal mass of target proteins is had relatively high expectations, this has limited the range of choice of target protein greatly.Three, under normal circumstances, this technology only has interactional protein for analyzing with single part, requires ligand molecular to reach very high purity.Therefore, the potpourri that forms complexity being gone out by natural products/traditional Chinese medicine extraction for screening, must carry out multistep purifying and obtain being close to single component, could use the method to study seriatim the combination of specific micromolecular compound and target protein.
Summary of the invention
The invention provides the screening technique that a kind of operation is relatively simple, universality is high, analysis speed is fast, for the little molecule ligand of different target proteins and configurations, can adopt the method combination of detector ligand and protein rapidly.
Method of the present invention comprises the following steps: this target protein and micromolecular compound storehouse are contacted to generate target protein-little molecular complex, separate this target protein-little molecular complex, by the little molecular dissociation in this target protein-little molecular complex, then the little molecule that utilizes Mass Spectrometer Method to dissociate from above-mentioned compound, thereby screening obtains having with this target protein the little molecule of combination, the i.e. part of this target protein indirectly.
Particularly, method of the present invention comprises the steps:
1) small-molecule mixture is dissolved in DMSO.
Above-mentioned small-molecule mixture is characterised in that:
In potpourri, the molecular weight of every kind of compound at least differs 3Da; Thereby acid little molecule and alkaline small reach certain balance avoids large pH variation to have impact to protein; Its dissolubility and structure diversity can be used as the feature of prodrug; In potpourri, contained compound number is relevant with its dissolubility, and between each compound without reacting to each other and there is no the problem of overlapping molecular weight.
2) hatch: the target protein of suitable concn is mixed according to the molar ratio of 1: 5～1: 10 with little molecular solution, hatch at ambient temperature 30 minutes.
The protein that purifying is good is concentrated in sort buffer system under normal circumstances: damping fluid is 20mMTris, 100mM sodium chloride, 5mM lime chloride, 0.1mM zinc chloride, 2mM tri-sodium nitrides and 3.5mM DTT, pH=7.0.
3) separate:
Sampling molecular-exclusion chromatography post (for example Sephadex G25) separates target protein-micromolecular compound, and adopts electrophoretic analysis to collect liquid.
Preferably, first use the NH of 50mM
4ac (pH=7) solution for example, soaks thoroughly and rinses molecular-exclusion chromatography post (Sephadex G25), to remove all soluble components that can disturb mass spectrophotometry.Then, the solution of hatching of taking-up 25 μ l is splined in Sephadex G25 post.According to the principle of molecular-exclusion chromatography, what first elute is target protein-micromolecular compound, is not in conjunction with other impurity in excessive little molecule and the solution of target protein and be trapped in eluted afterwards in post.Collect the eluent NH of 25 μ l
4hCO
3, and with SDS-PAGE electrophoretic analysis collect liquid.
4) dissociate: adopt 60% methanol solution that little molecule is dissociated out from target protein-little molecular complex.
5) Spectrometry: get a certain amount of dissociation solution, add the deionized water of equivalent to dilute, take out a part of sample introduction after dilution, carry out chromatography-mass spectroscopy series connection and analyze.
Above-mentioned mass spectrometer is Q-TOF-MS level Four bar-flight time mass spectrum (Waters) preferably.Preferred chromatographic condition is: chromatographic column is selected Waters XTerra MS C18 (2.1mm × 100mm, m), mobile phase A is 3.5 μ: 0.1% aqueous formic acid, and B is 0.1% formic acid-acetonitrile, gradient elution: 20min is from 20to 60%B, 0.2ml/min.Mass spectrum condition: sweep limit m/z is 100-5000, and spray voltage is 3kv, and sheath gas velocity is 100L/h, and acquisition time is spaced apart 1.5s.
6) control experiment
Under identical mass spectrum condition, detect the mass spectrum result in micromolecular compound storehouse of not soaking with target protein, and detect known to target protein have the inhibitor of specific bond effect and target protein soak in conjunction with after mass spectrum result, by gained mass spectrogram under relatively above different situations, know step 5) in the micromolecular information of the specific combination of target protein.
The mass spectrum screening technique expection that the present invention sets up by for the product platform of early stage new drug development provides important technical support, has advantages of as follows:
(1) improving the efficiency of screening active component from compound library, is that a kind of operation is relatively simple, lower-cost high-flux medicaments sifting method;
(2) expand and improve now the elementary screening process of the prodrug molecule extensively adopting, independent operation be expected to overcome the multiple limitation of conventional high flux screening (HTS) based on mass spectrographic screening process, improve the confidence level of result;
(3) can carry out rapid screening to the complex mixture extracting in natural products, directly find effective binding constituents wherein.
Screening technique of the present invention can for systematic Study and the new drug development of the closely-related target proteins of human health, for disease comprise multiple mankind's major diseases such as viral infectious (as SARS and HIV), diabetes, cardiovascular disease, nerve degenerative diseases, cancer, autoimmune disease.Thereby the drug screening technology platform that the present invention sets up, by discovery and the initiative of directly serving for the new drug precursor molecule of these major diseases, produces considerable economic benefit and social benefit.
Accompanying drawing explanation
Fig. 1 illustrates schematic flow sheet of the present invention;
Fig. 2 illustrates the mass spectrum result contrast figure of gained in embodiment.
Embodiment
Be further described with feature to various aspects of the present invention below.
Chinese crude drug obtains extract and extracted component through organic solvent heating and refluxing extraction, liquid-liquid extraction, and each extracted component obtains standard ingredient through normal pressure normal phase silica gel column chromatography partition method again.Analyze the accurate component of mass spectrum preparation spectrometry.Conventional chromatography comprises resin method, preparative liquid chromatography etc.
(1) taking 10mg natural products component (100 kinds) and antipain (the p-tosyl phenylalanyl of N-chloromethyl ketone) is dissolved in respectively in DMSO.
(2) the papain solution of getting 30 μ M mixes according to 1: 10 with natural products, incubated at room 30min.
(3) papain and little molecular complex are crossed molecular sieve system, first use molecular sieve damping fluid balance nickel ion affinity chromatography post (Amersham Bioscience), then supernatant is splined on to nickel post in 4 ℃, flow velocity 1ml/min, and collect through flow fluid, get 12% separation gel SDS-PAGE electrophoresis for 10 μ l eluents, analyze in eluent whether have papain.
(4) use again 60% methanol-eluted fractions, collect cut and carry out mass spectrophotometry.
(5) natural constituents 0.1mM, sample introduction 1 μ l, mobile phase A is: 0.1% aqueous formic acid, B is 0.1% formic acid-acetonitrile, gradient elution: 30min is from 20 to 60%B, 0.2ml/min.Mass spectrum condition: sweep limit m/z is 100-1000, and spray voltage is 3kv, and acquisition time is spaced apart 1.5s, and result is as shown in figure a; After above-mentioned steps obtains being combined separation with papain, the result of natural products small molecule component is as shown in figure b; Choosing known and papain according to micromolecular architectural feature has and carries out combination in conjunction with the antipain of target spot with papain and test, and obtains the result of the antipain of being combined with papain as schemed as shown in c.
Result as shown in Figure 2, by comparing b figure and c figure, proves that the little molecule in b is specific being attached on target protein.
Although illustrated and described embodiments of the invention, for the ordinary skill in the art, be appreciated that without departing from the principles and spirit of the present invention and can carry out multiple variation, modification, replacement and modification to these embodiment, scope of the present invention is limited by claims and equivalent thereof.
Claims (8)
1. one kind is screened the method for target protein part, it is characterized in that comprising the following steps: this target protein and micromolecular compound storehouse are contacted to generate target protein-little molecular complex, separate this target protein-little molecular complex, by the little molecular dissociation in this target protein-little molecular complex, then the little molecule that utilizes Mass Spectrometer Method to dissociate from above-mentioned compound, thereby screening obtains having with this target protein the little molecule of combination, the i.e. part of this target protein indirectly.
2. according to the method for claim 1, it is characterized in that: described micromolecular compound storehouse has following feature: the molecular weight of every kind of compound at least differs 3Da; Thereby acid little molecule and alkaline small reach certain balance avoids large pH variation to have impact to protein; Micromolecular dissolubility and structure diversity can be served as prodrug; And between each compound, nothing reacts to each other and there is no overlapping molecular weight.
3. according to the method for claim 1 or 2, it is characterized in that comprising the steps:
1) small-molecule mixture is dissolved in DMSO;
2) hatch: target protein is mixed according to the molar ratio of 1: 5～1: 10 with little molecular solution, hatch at ambient temperature 30 minutes;
3) separate: sampling molecular-exclusion chromatography post separates target protein-micromolecular compound, and adopts electrophoretic analysis to collect liquid;
4) dissociate: adopt 60% methanol solution that little molecule is dissociated out from target protein-little molecular complex;
5) Spectrometry: get a certain amount of dissociation solution, add the deionized water of equivalent to dilute, take out a part of sample introduction after dilution, carry out chromatography-mass spectroscopy series connection and analyze;
6) contrast: detect the mass spectrum result in micromolecular compound storehouse of not soaking with target protein under identical mass spectrum condition, and detect known to target protein have the inhibitor of specific bond effect and target protein soak in conjunction with after mass spectrum result, by gained mass spectrogram under relatively above different situations, know step 5) in the micromolecular information of the specific combination of target protein.
4. according to the method for claim 3, it is characterized in that: step 2) in, albumen after purifying is concentrated in sort buffer system: 0.1mM, damping fluid is 20mM Tris, 100mM sodium chloride, 5mM lime chloride, 0.1mM zinc chloride, 2mM tri-sodium nitrides and 3.5mM DTT, pH=7.0.
5. according to the method for claim 3, it is characterized in that: step 3) in first with the NH4Ac solution of 50mM and pH=7, molecular-exclusion chromatography post is soaked thoroughly and is rinsed; Then, that gets 25 μ l hatches solution loading, and according to the principle of molecular-exclusion chromatography, what first elute is target protein-micromolecular compound, and eluted after being trapped in post be not in conjunction with other impurity in excessive little molecule and the solution of target protein; Collect the eluent NH of 25 μ l
4hCO
3, and with SDS-PAGE electrophoretic analysis collect liquid.
6. according to the method for claim 3 or 5, it is characterized in that: step 3) in molecular-exclusion chromatography post used be Sephadex G25.
7. according to the method for claim 3, it is characterized in that: step 5) chromatography-mass spectroscopy series connection analyze, chromatographic condition: it is the Waters XTerraMS C18 of 2.1mm × 100mm, 3.5 μ m that chromatographic column is selected specification, mobile phase A is: 0.1% aqueous formic acid, B is 0.1% formic acid-acetonitrile, gradient elution: 30min is from 20 to 60%B, 0.2ml/min; Mass spectrum condition: sweep limit m/z is 100-5000, and spray voltage is 3kv, and sheath gas velocity is 100L/h, and acquisition time is spaced apart 1.5s.
8. according to the method for aforementioned any one claim, it is characterized in that: above-mentioned mass spectrum preferably adopts Q-TOF-MS level Four bar-flight time mass spectrum.
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Cited By (7)
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| CN105987888A (en) * | 2015-02-09 | 2016-10-05 | 复旦大学 | Method for fishing screening of active component monomer or active component group from mixture |
| CN107246985A (en) * | 2017-06-10 | 2017-10-13 | 延边大学 | A kind of method that utilization electrophoresis screens target protein natural products multicomponent complex |
| CN107589193A (en) * | 2017-10-25 | 2018-01-16 | 南京工业大学 | A kind of method that protein inhibitor is screened using micro-reaction device |
| CN108896647A (en) * | 2018-07-20 | 2018-11-27 | 遵义医学院 | A kind of method of high-flux fast screening ERp57 inhibitor |
| CN109155148A (en) * | 2015-12-31 | 2019-01-04 | 思科利康有限公司 | The method to identify protein ligand interaction is docked for protein groups |
| CN110018265A (en) * | 2019-01-24 | 2019-07-16 | 河北医科大学第二医院 | A kind of " integration " active ingredient of Chinese herbs screening technique based on target compatibility |
| CN112649517A (en) * | 2019-10-12 | 2021-04-13 | 中国科学院大连化学物理研究所 | Method for screening target protein ligand from organism metabolite |
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Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105987888A (en) * | 2015-02-09 | 2016-10-05 | 复旦大学 | Method for fishing screening of active component monomer or active component group from mixture |
| CN105987888B (en) * | 2015-02-09 | 2019-06-21 | 复旦大学 | A method of it fishes from mixture and screens active constituent monomer or active constituent group |
| CN109155148A (en) * | 2015-12-31 | 2019-01-04 | 思科利康有限公司 | The method to identify protein ligand interaction is docked for protein groups |
| CN109155148B (en) * | 2015-12-31 | 2022-06-24 | 思科利康有限公司 | Methods for proteomic docking to recognize protein-ligand interactions |
| CN107246985A (en) * | 2017-06-10 | 2017-10-13 | 延边大学 | A kind of method that utilization electrophoresis screens target protein natural products multicomponent complex |
| CN107589193A (en) * | 2017-10-25 | 2018-01-16 | 南京工业大学 | A kind of method that protein inhibitor is screened using micro-reaction device |
| CN107589193B (en) * | 2017-10-25 | 2020-05-05 | 南京工业大学 | Method for screening protein inhibitor by using micro-reaction device |
| CN108896647A (en) * | 2018-07-20 | 2018-11-27 | 遵义医学院 | A kind of method of high-flux fast screening ERp57 inhibitor |
| CN110018265A (en) * | 2019-01-24 | 2019-07-16 | 河北医科大学第二医院 | A kind of " integration " active ingredient of Chinese herbs screening technique based on target compatibility |
| CN112649517A (en) * | 2019-10-12 | 2021-04-13 | 中国科学院大连化学物理研究所 | Method for screening target protein ligand from organism metabolite |
| CN112649517B (en) * | 2019-10-12 | 2021-10-15 | 中国科学院大连化学物理研究所 | Method for screening target protein ligand from organism metabolite |
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