CN101339167B - Active ingredient high throughput screen method based on target protein and selection - Google Patents

Abstract

The invention relates to the field of medicine, in particular to the field of high-throughput screening, and relates to a high-throughput screening method for active ingredients or active ingredient groups based on target protein affinity selection. The mixed solution obtained from the incubation is loaded on a molecular exclusion chromatography column or an online solid-phase extraction column, eluted with a buffer, the eluate is dissociated, and the dissociated solution is separated by an online solid-phase extraction column, and finally introduced into a high-efficiency The liquid phase-mass spectrometry system is used for analysis to confirm the structure of the compound and perform quantitative analysis to calculate the binding rate of these active ingredients to the target protein to evaluate its activity. The screening method of the invention greatly improves the efficiency of screening active ingredients (groups) from compound groups, and is a high-throughput drug screening method with relatively simple operation and low cost.

Description

A high-throughput screening method for active ingredients based on target protein affinity selection

technical field

The invention relates to the field of high-throughput screening, in particular to a high-throughput screening method for active ingredients or active ingredient groups based on target protein affinity selection, which is a high-affinity screening method for target proteins using multidimensional chromatography-mass spectrometry Active ingredients, especially groups of active ingredients.

Background technique

In the process of innovative drug research, the discovery of biologically active lead compounds (Lead Compounds) is the basis of all research work. At present, the screening of lead compounds mostly adopts screening models at the level of animals, organs (tissues) and cells. Although some effective drugs have been screened from them, this method has high screening costs, requires a large amount of compound samples, and is time-consuming and laborious. The cycle is long, so it is not suitable for the screening of natural products with low content, let alone large-scale and high-throughput screening.

With the advent of the post-gene era, people have a deeper understanding of the disease process, and the key roles of more and more biological macromolecules (such as enzymes, receptors, ion channels, etc.) in physiological and pathological processes have been clarified. The maturity of molecular biology technology provides tools for the expression of protein molecules in vitro. Gene recombination technology is used to express important disease-related enzymes, receptors and other proteins in a suitable expression system, so that molecular-targeted high-throughput and ultra-high-throughput screening models at the molecular or cellular level can be established (see : Broach J.R., Thoner J. High-throughput screening for drug discovery. Nature, 1996, 384: 14; Landro J.A., Taylor I.C., Stirtan W.G, et al.HTS in the new millennium: the role of pharmacology and x flexibility.JPharmacol Methods, 2000, 44: 273-289; Chen Suhong, Wang Hua, Wang Shengqi. Application of ultra-high-throughput screening based on cell function in chemical genomics drug discovery. Foreign Medical Pharmacy, 2002, 29: 345-348, etc.) . Although this high-throughput screening technology has developed into one of the three main technologies in the modern new drug discovery process due to its advantages of rapidity and high efficiency, it has no separation process and compound identification functions, so it is only applicable to single compounds with known structures. The screening of individual compounds (groups), when applied to complex systems such as natural product extracts, especially traditional Chinese medicine extracts, often results in some false negative or false positive results due to the interference between various components (see: Bogustavasky J. HTS assay development: Is smaller really better Drug Discov. Dev., 2004, 7: 37-40).

Biochromatography is a relatively new method that can investigate the binding of complex systems of compounds (such as Chinese medicine extracts) to enzymes, receptors, cell membranes, etc. This method uses silica gel or gel as a carrier, and coats it Or bonded with active enzymes, receptors or cell membranes to prepare chromatographic stationary phases, and separate different components according to the strength of the interaction between the drug and the enzymes, receptors or cell membranes, but this method exists in practice. Many regrets. For example: 1. The biological characteristics of enzymes, receptors or cell membranes will be affected to a certain extent after they are combined with silica gel and other carriers. 2. This method has strict restrictions on the chromatographic mobile phase, that is, only physiological buffer or very low concentration of organic solvent can be used as the mobile phase, otherwise it will destroy biological macromolecules, so that the chromatography can use different mobile phases to achieve different properties. The separation characteristics of compounds are difficult to play, and it is difficult for compounds to separate from each other on biochromatography. In the literature of "Mao Xiqin et al., The application of three chromatographic modes in the preliminary screening of active ingredients in traditional Chinese medicine, Analytical Chemistry, 2003, 31(8): 992-995", there are Rhizoma Chuanxiong in simulated biofilm chromatographic columns, protein chromatographic From the separation profiles on the column and reversed-phase column, it is obvious that the separation efficiency of biofilm chromatography is not ideal, and it is not as good as the reversed-phase column. 3. The preparation procedure of biochromatography is complex because of the addition of the process of combining silica gel medium with enzymes, receptors or cell membranes, with strict conditions and high cost. For example, a commercially available protein column is as high as tens of thousands of yuan. 4. Because the mobile phase used is a buffer solution, the provided chromatogram has a poor separation effect on the compounds and cannot be combined with LC-MS online, and the structural information of the compounds cannot be given. (See: Kong Liang et al., Molecular Biochromatographic Molecules with Human Serum Albumin as the Stationary Phase Research on Several Active Components of Traditional Chinese Medicine, Chemical Journal of Chinese Universities, 2000, 21:36. He Langchong et al. Enzyme activity and its chromatographic characteristics, Science Bulletin, 1999(6): 633-637. Zhao Xiaojuan et al., Analysis and comparison of active components in the roots and leaves of Huoyanghuo, Analytical Chemistry, 2002, 30(2): 195-197. High Kun et al. Screening and researching the active ingredients in Rhizoma chinensis by cell membrane chromatography, Chinese Journal of Pharmaceutical Sciences, 2003(1): 14-16.)

Contents of the invention

The problem to be solved by the present invention is to establish a method based on target protein affinity selection that can be applied to both monomeric compounds or compound groups with known structures and complex natural product groups (such as traditional Chinese medicine extracts) that lack structural information. High-throughput screening method for active ingredients. The present invention can avoid affecting the accuracy of small molecule compound screening due to the change of the biological characteristics of the target protein due to immobilization. Get active small molecules (groups) and obtain their structural information online, so as to quickly find active lead compounds or active ingredients of traditional Chinese medicines from compounds (groups) or complex systems of traditional Chinese medicines.

The high-throughput screening method of the present invention comprises the following abc three steps successively:

a. Co-incubate the target protein with the test compound or the test compound group in the buffer solution. At this time, the active test compound combines with the target protein to form a target protein-active ingredient complex, and the mixed solution obtained after incubation includes the target protein. -The active ingredient complex and the free small molecular compound that is not bound to the target protein, the mixed solution is loaded on a sizeexclusion chromatography column (sizeexclusion chromatography, SEC) or an online solid-phase extraction column (online solid-phase extraction, SPE), Elute with buffer, and the eluate flows through the UV detector after SEC or the first SPE, and detects at the maximum UV absorption wavelength of the protein at 280nm, and the target protein-active ingredient complex comes out first and is detected as the first A chromatographic peak, the chromatographic peak enters the coil, and the eluent after the chromatographic peak is switched to waste. Since the SPE will be used in the following steps, the SPE in this step is called SPE ₁ , and the SPE used in step b is called SPE ₂ .

The compound to be tested can be a monomeric compound, or a compound group composed of monomeric compounds, or a complex traditional Chinese medicine extract containing unknown components. If the compound to be tested can be combined with the target protein to form a complex, the compound to be tested is an effective drug for the disease related to the target protein, and the specific degree of effectiveness depends on the subsequent method test calculation. If the compound to be tested is a monomeric compound, the complex is a target protein-active ingredient complex, and if the compound to be tested is a compound group, the complex is a target protein-active ingredient group complex, and the present invention is more suitable for compound group For simplicity, high-throughput screening is referred to as the target protein-active ingredient complex.

The above-mentioned target protein is generally consistent with the buffer used for incubation of the test compound and the buffer used for elution of SEC or SPE ₁ , preferably from phosphate buffer at pH 6.0 to 8.0, tris at pH 6.0 to 8.0 Buffer, pH 6.0-8.0 ammonium acetate buffer or pH 6.0-8.0 ammonium bicarbonate buffer.

The incubation system should take into account the high activity of the target protein and the solubility of small molecules of the compound. Some compounds, especially those in natural product groups (such as traditional Chinese medicine extracts), have low polarity and are insoluble in the buffer salt system. It is necessary to select a suitable solvent to make the two coexist, such as adding a low concentration of solubilizer, etc. The choice of solubilizer is based on not destroying or substantially not destroying the activity of the target protein. In the actual operation process, the ratio of the solubilizer that the target protein can bear can be checked through experiments. For example, Ellman spectrophotometric analysis proves that the activity of acetylcholinesterase is hardly affected in the buffer salt system containing 5% methanol.

A solubilizing agent is preferably added to the buffer during incubation, and the solubilizing agent is preferably selected from one or a mixed solution of Tween, dimethyl sulfoxide, polyethylene glycol, triton, methanol, and ethanol. The preferred volume ratio of solubilizer to buffer volume is 0.1-5%.

The compound to be tested and the target protein are mixed in the buffer to form a homogeneous mixed system, and different incubation temperatures can be selected according to the stability of the compound components. If the compound to be tested contains more volatile components, an incubation temperature of 4°C is preferred; otherwise, an incubation temperature of 25°C-37°C is selected, and the incubation time is preferably 0.5-1.5 hours.

Size exclusion chromatography is preferably Sephadex G-25, Sephadex G-50 or Sephadex LH-20. The elution flow rate is preferably 0.3-0.5 ml/min.

In size exclusion chromatography, the incubated sample is loaded onto a 3-5cm size exclusion chromatography column (SEC), and eluted with buffer, and the eluate is detected by a UV detector at 280nm after SEC, and the target protein-activity The component complexes are first eluted within 1-4min. Due to the limited pressure that the gel filler can withstand, the elution flow rate is preferably 0.3-0.5ml/min. During the elution process, there are almost no free small molecular compounds around the target protein-active ingredient complex, the dynamic balance between the complex and small molecular compounds is constantly broken, and the small molecules bound to the target protein are continuously dissociated, so the gel The shorter the chromatography process, the better, as long as the protein macromolecules can be separated from the small molecular compounds. In addition, online solid-phase extraction columns (SPE) can also be selected to separate target protein-active ingredient complexes from free small molecule compounds, such as OASIS HLB columns from Waters and other online solid-phase extraction columns with similar principles. This kind of SPE such as HLB uses hydrophilic and lipophilic water-wettable reversed-phase adsorbent as filler, which hardly retains large polar substances such as proteins and buffer salts, and has a good retention effect on polar organic compounds, and Can withstand up to 170bar pressure, high flow rate (2-4ml/min) elution creates turbulence in the column, the target protein-active ingredient complex quickly passes through the HLB and enters the coil in the turbulent flow, greatly reducing the active ingredient bound to the complex early dissociation. At the same time, unbound bulk small molecule compounds are retained on the SPE and do not enter the coil.

b. Mix the eluent and the dissociation solution for dissociation. In step a, the eluent containing the target protein-active ingredient complex is mixed with the dissociation solution to form a dissociation solution, and the active ingredient combined with the target protein in the dissociation solution is dissociated into free compounds, and the dissociation solution is loaded on the coil After the online solid-phase extraction column (SPE ₂ ), and wash the SPE ₂ with a high-flow rate aqueous solution after loading the sample to remove the target protein and buffer salt adsorbed on it, the active components will not be washed out and remain on the column;

After the dissociation solution is loaded on the SPE ₂ column, it is preferably washed with high-flow aqueous solution (2-4ml/min) for 1-3min to remove the target protein and buffer salt. SPE ₂ will form a turbulent chromatogram under the washing of high-flow aqueous solution, which will not retain biological macromolecules such as target proteins, but can well retain small organic molecules.

In the above detection method, the coil can be a 1ml-5ml circular folded coil, which is used for mixing the dissociation solution and the eluent containing the target protein-active ingredient complex. The dissociation solution used for the dissociation of the target protein-active ingredient complex is preferably an aqueous acid solution or a mixed solution of an aqueous acid solution and an organic solvent. The aqueous acid solution is preferably trifluoroacetic acid, formic acid, acetic acid or hydrochloric acid, and the pH is preferably 1-3. The organic solvent is preferably methanol or acetonitrile. The volume ratio of the organic reagent in the mixed solution of the two is preferably 5%-30%. The dissociation solution used in the present invention can effectively dissociate the target protein-active ingredient complex.

SPE ₂ plays the role of online enrichment of active components after the coil (see Figure 1). According to the different properties of the compounds to be tested, select the corresponding SPE ₂ , such as the Oasis series of online chromatographic columns of Waters Company or other SPE columns with similar principles.

The concentration of the active ingredient bound to the target protein is so low that the detection limit of mass spectrometry is generally not reached. SPE ₂ can effectively enrich target compounds and remove target proteins, buffer salts and other interfering substances. Select the appropriate SPE ₂ according to the properties of the compounds to be screened. For example, Oasis MCX online chromatographic column and Oasis WCX online chromatographic column of Waters Company should be selected for basic compounds, and Oasis MAX online chromatographic column and Oasis WAX online chromatographic column of Waters Company should be selected for acidic compounds. Chromatographic columns, etc., while the Oasis HLB online chromatographic column of Waters Company can enrich all organic compounds, and other SPEs with similar principles to the above SPE can also be selected to enrich and remove proteins of target compounds.

The compound loaded on SPE ₂ is preferably rinsed with a solution at a high flow rate. According to the nature of the compound, if the compound to be tested is a basic compound, it should be rinsed with alkaline water and alkalized at the same time; if the compound to be tested is an acidic compound, it should be rinsed with acid water and simultaneously For acidification, alkaline water is preferably ammonia solution or diethylamine solution; acid water is preferably trifluoroacetic acid aqueous solution, formic acid aqueous solution, acetic acid aqueous solution or hydrochloric acid aqueous solution. Proteins, buffer salts, etc. are washed to waste, and active ingredients remain on SPE ₂ .

c. Switch the active ingredient on the online solid-phase extraction column (SPE ₂ ) in step b to a high-performance liquid phase-mass spectrometry system (LC-MS) for analysis, confirm the structure of the screened active ingredient, and quantify it Analyze and calculate the binding rate of these active ingredients to the target protein, so as to predict their activity.

After the SPE ₂ is washed to remove the target protein and buffer salt, it is directly switched to LC-MS for analysis, preferably through a six-way switching valve, an eight-way switching valve or a ten-way switching valve. The active ingredients enriched on SPE ₂ are eluted into the C18 reverse-phase chromatographic column by the mobile phase solution (the mobile phase solution preferably contains methanol or acetonitrile aqueous solution) for separation, and then the structural information of the active ingredients is obtained through mass spectrometry detection.

The dissociation solution is loaded on the SPE ₂ downstream, and the active ingredients are concentrated and enriched in the front section of SPE2. After the six-way (or eight-way, ten-way) valve is switched, the mobile phase solution is recoiled on the SPE ₂ , and the active ingredients are recoiled into the recoil. Phase chromatographic analysis column, so that the active components are concentrated into the chromatographic column for separation, under a suitable chromatographic gradient elution, the active component mixture can also obtain good separation effect and chromatographic peak shape through SPE ₂ -LC-MS (see Figure 3).

1. Calculation of the binding rate of the active ingredient to the target protein

By comparing the peak area of small molecular compounds in the sample through SEC/SPE ₁ -SPE ₂ -LC-MS and the peak area of two-way-SPE ₂ -LC-MS, the binding rate of each active ingredient can be calculated. The specific formula is as follows:

Through the screening of compounds in the present invention, possible active ingredients with high affinity to target proteins can be efficiently and quickly screened out from a large number of compounds. Preliminary activity predictions can be made on the screened components through the binding rate.

2. Titration method to calculate the kinetic constant of the compound to be tested

The target protein takes an enzyme as an example. If the test compound competes with a compound with a known enzyme inhibition constant (Ki) for the same site on the enzyme, then the Ki of the test compound can be determined by "titration". The tested compound is used as the titrant and incubated with the enzyme for a period of time, and then titrants of different concentrations are added, that is, compounds with known Ki. As the titrant concentration increases, the titrant is gradually displaced from the active site of the enzyme. The Ki of the tested compound can be calculated from the peak areas of the titrant and the titrant detected by the above screening method. Calculation formula:

$\frac{<span\; class="notranslate">\; [</span>{\mathrm{<span\; class="notranslate">\; ES</span>}}_{\mathrm{<span\; class="notranslate">\; 1</span>}}<span\; class="notranslate">\; ]</span>}{<span\; class="notranslate">\; [</span>{\mathrm{<span\; class="notranslate">\; ES</span>}}_{\mathrm{<span\; class="notranslate">\; 2</span>}}<span\; class="notranslate">\; ]</span>}<span\; class="notranslate">\; =</span>\frac{{<span\; class="notranslate">\; [</span>{\mathrm{<span\; class="notranslate">\; S</span>}}_{\mathrm{<span\; class="notranslate">\; 1</span>}}<span\; class="notranslate">\; ]</span>}_{\mathrm{<span\; class="notranslate">\; 0</span>}}{\mathrm{<span\; class="notranslate">\; Ki</span>}}_{\mathrm{<span\; class="notranslate">\; 2</span>}}}{{<span\; class="notranslate">\; [</span>{\mathrm{<span\; class="notranslate">\; S</span>}}_{\mathrm{<span\; class="notranslate">\; 2</span>}}<span\; class="notranslate">\; ]</span>}_{\mathrm{<span\; class="notranslate">\; 0</span>}}{\mathrm{<span\; class="notranslate">\; <figure-callout\; id="1"\; label="Ki"\; filenames="H2008101246111E00011.png,H2008101246111E00021.png"\; state="\{\{state\}\}">Ki</figure-callout></span>}}_{\mathrm{<span\; class="notranslate">\; 1</span>}}}$

Where [ES ₁ ] is the concentration of the enzyme-titrant complex (in this method, the mass spectrum peak area of the titrant), [ES ₂ ] is the concentration of the enzyme-titrant complex (in this method, the mass spectrum peak area of the titrant). mass spectrum peak area), [S ₁ ] ₀ is the total concentration of the titrant added, [S ₂ ] ₀ is the total concentration of the titrant, Ki ₁ is the Ki of the titrant, and Ki ₂ is the Ki of the titrant (Ki to be found) . From the above, Ki ₂ =[ES ₁ ][S ₂ ] ₀ Ki ₁ /{[ES ₂ ][S ₁ ] ₀ }. (See D.Allen Annis, ^* Naim Nazef, Cheng-Chi Chuang, et al.AGeneral Technique To Rank Protein-Ligand Binding Affinities and Determine Allosteric versus Direct Binding Site Competition in Compound Mixtures.J.AM.CHEM.SOC.2004, 126 , 15495-15503).

Through the molecular weight and fragment information of the compound obtained in step c above or compared with the standard compound, the structure of the screened active ingredient (group) is confirmed. At the same time, a preliminary evaluation of its activity was carried out according to the size of the binding rate calculated above.

In addition to the above steps for compound screening and calculation of its binding rate and kinetic constants, the half maximal inhibitory concentration (IC ₅₀ ) of the screened active compound to the target protein can also be determined by mass spectrometry and calculated by Lineweaver Burk mapping method. The inhibition constant (Ki) of the active compound on the enzyme can be used to better judge the pharmacological activity of the screened compound.

1. Determination of the IC ₅₀ of the active ingredient on the target protein:

Taking enzymes as an example, the specific method is: uniformly mix the substrate of the enzyme with different concentrations of the test compound, add the enzyme solution to initiate the catalytic reaction, and terminate the reaction after a certain period of time. The concentration of the tested compound that can inhibit the activity of the enzyme by 50% is its IC ₅₀ . Using the same conditions and methods to determine the IC ₅₀ of the positive drug, and comparing the IC ₅₀ of the tested compound with the IC ₅₀ of the positive drug, the inhibitory activity of the tested compound on the target enzyme can be obtained.

2. Lineweaver Burk drawing method:

The Lineweaver Burk plot of the enzyme reaction rate and substrate concentration at different substrate concentrations can be used to calculate the Michaelis constant K _m and the maximum reaction velocity V _max of the specific substrate catalyzed by the enzyme. Under different inhibitor concentrations, the type of inhibition of the tested compound on the enzyme can be judged according to the intersection position of the straight lines made by different substrate concentrations, and the tested compound can be calculated by secondary plotting of the corresponding reaction speed formula of the tested compound with different concentrations. The inhibition constant (Ki) of the compound on the enzyme.

By using the high-throughput screening method of the present invention, all active proteins closely related to diseases from humans and animals can be used as targets for screening compounds. The preferred target protein is from human or animal tissue enzymes or receptors related to Alzheimer's disease, cardiovascular disease or tumor disease. At present, there are a large number of commercially available proteins that can be purchased, and can also be purified and prepared by yourself. The target protein (such as an enzyme or receptor) is generally dissolved in a buffered saline solution at a high concentration, and stored at -20°C to maintain the activity of the target protein. When specifically screening compounds, the appropriate target protein can be selected according to literature information and preliminary experiments. If the literature shows that steroidal alkaloids have cholinesterase inhibitory activity, then cholinesterase can be selected as the target protein for screening steroidal alkaloids. .

Using this screening method, through the separation of SPE ₁ , all target protein-active ingredient (group) complexes enter the coil within 20 seconds (see Figure 2), and can be directly analyzed by LC-MS after 1.5 minutes of online pretreatment. Save more than 2 minutes than the fastest online sample pretreatment currently reported (see Jimmy Flarakos, Kenneth L Morand, and Paul Vouros. High-Throughput Solution-Based Medicinal Library Screening against Human Serum Albumin. Anal. Chem. 2005, 77, 1345-1353.). This greatly improves the efficiency of screening active ingredients (groups) from compounds (groups), and is a high-throughput drug screening method with relatively simple operation and low cost. Using the screening method of the present invention, the contriver has screened out 11 components with high affinity to butyrylcholinesterase (see accompanying drawing 3) from the monomeric compound that is made up of 27 steroidal alkaloids, to These 11 components have carried out further activity test, among them the IC of 4 components is less than 10 _μ M, and positive compound galanthamine is 3.23 _μ M to the IC of BChE under the same condition, this fully demonstrates that the present invention is used for high pass Feasibility of quantitative screening of active compounds.

Description of drawings

是靶蛋白—活性成分复合物，

是靶蛋白，

是游离小分子化合物。Fig. 1 is a diagram of an online screening device for target protein affinity selection of active ingredients. 1. Molecular exclusion chromatographic column/HLB and other solid-phase extraction columns 2. Ultraviolet detector 3. Coil 4. Six-way (or eight-way, ten-way) switching valve 5. C18 reverse-phase chromatographic analysis column 6. Mass spectrometer detector.

is the target protein-active ingredient complex,

is the target protein,

It is a free small molecule compound.

Figure 2 is the 280nm detection spectrum of the target protein-active ingredient complex passing through the first SPE. The SPE of this screening method uses the OASIS HLB online solid-phase extraction column of Waters Company, in the figure 1. Target protein-active ingredient (group) complex 2. Free small molecular compounds that are not bound to the target protein

The total ion chromatogram of the monomeric compound that Fig. 3 (I) 27 steroidal alkaloids forms

(II) (A)-(F) from top to bottom are: ion chromatogram of alkaloid standard extract, ion chromatogram of active ingredient combined with AChE, ion chromatogram of active ingredient combined with BChE . Among them (A)1.ebeienine2.solasodine3.puqiedinone4. Chuanbei ketone5.ebeiedinone(B)6. norpuqiedine8.ebeiedine(C)9. fritillary 10. Pubeidione (D ) 11. Sibecillin 12. Zhebeijingsu 13. Puqiedine base (E) 14. puqiedine7-0115. Zhebeijiasu 16. Isofeibeijiasu (F) 25. Puqiedine glycoside 26. Puqiedine Betone glycosides.

Detailed ways

Example 1

Drug screening for Alzheimer's disease-related targets:

The components with high affinity to acetylcholinesterase (Acetylcholinesterase, AChE) were screened among monomeric compounds composed of 27 steroidal alkaloids.

The monomer compounds to be screened are composed of 27 steroid alkaloids, which are pyrene A, pyrebene B, pyrebene C, pyrebene D, pyrebene E, pyrebene F, fritillin , Sibeisu, Sibeisu glycoside, Pubekone base, Pubekone glycoside, N-norpubekone base, Ibekine glycoside A, Zhebeijiasu, Zhebeijiasu N-oxide, Zhejiang Puqiedinone, Chuanbei ketone, puqiedine-7-01, puqiedinone, puqiedine, puqietinedinone, veratridine, ebeienine, ebeiedinone, ebeiedine, solasodine, isoverticine. It is time-consuming, laborious and inefficient to screen them one by one for their activity. process. The possible active ingredients with high affinity with acetylcholinesterase can be quickly found out by adopting the screening method of the present invention.

Preparation of AChE: Acetylcholinesterase freeze-dried powder 2000U (Acetylcholinesterase from electric eel, Sigma), fully dissolved with 2ml of ammonium acetate buffer (10mM, pH7.5), prepared to 1000U/ml, placed in -20 ℃ refrigerator use.

Preparation of steroidal alkaloid solution: 27 steroidal alkaloids were dissolved in methanol, the concentration of each steroidal alkaloid was 25 μM, and then diluted with ammonium acetate buffer (10 mM, pH7.5) to a concentration of 2 μM.

Screening: Mix 20μl AChE solution (1000U/ml) and 20μl steroid alkaloid solution (2μM) evenly, and let stand at 25°C for 1h. 40ul incubation solution was loaded on SPE1 (Waters Oasis HLB), ammonium acetate buffer (10mM, pH7.5) was eluted at 2ml/min for 20s, eluent (2ml/min) and dissociation solution (0.2% trifluoroacetic acid aqueous solution Containing 20% acetonitrile, 1ml/min) mixed with the coil (2ml) for 1min dissociation, and the dissociated sample is loaded on SPE2 (Waters Oasis MCX), at this time the six-way switching valve is in the sample loading (LOAD) of Figure 1 position, rinse the SPE with diethylamine aqueous solution (pH10.0) 4ml/min for 21min, then switch the six-way switching valve to the injection analysis (INJECT) position of Figure 1, and the small molecular compounds enriched on SPE2 are eluted into the LC-MS system for separation and analysis. In addition, under the same conditions, the two-pass was used instead of SPE1, and the same sample was separated and analyzed by LC-MS through the above steps.

Chromatography and mass spectrometry analysis conditions: C18 chromatographic column, high performance liquid chromatography-electrospray time-of-flight mass spectrometry (HPLC-ESI/TOFMS); mobile phase: acetonitrile: water (each containing 0.05% diethylamine) gradient elution; ESI ion source ;Positive ion mode, mass range: 400～800m/z; Spray capillary voltage: 3500V; Dry gas (N ₂ ) flow rate: 9.0L/min; Dry gas temperature: 325°C; Atomization pressure: 35psi; Transmission voltage: 150V . The mass-to-charge ratios (m/z) of the [M+H] ⁺ ions of each alkaloid are: Pubeisuin A, 446; Pubeisuin B, 444; Pubeisuin C, 460; Pubeisuin D, 460 ; -norpubetone base, 416; 7-01, 432; puqiedinone, 414; puqiedine, 416; puqietinone, 428; veratridine, 674; ebeienine, 414;

Test results: 9 components with affinity to AChE were screened out from 27 monomeric compounds composed of steroidal alkaloids, see Figure 3. They are veratridine, puqiedine, ebiedine, ebeiedinone, pubeiketone base, pubeiketone base glycoside, pubeidione, puqiedone, pubeisuin B. Their binding rates to 1UAChE were 0.01%, 0.01%, 0.008%, 0.01%, 0.01%, 0.005%, 0.01%, 0.02%, 0.005%, respectively. Among them, the IC50 of Chuanbeidione and ebeiedinone on AChE were 99 μM and 79 μM, respectively. Under the same conditions, the IC ₅₀ of galantamine against AChE is 0.45 μM.

Example 2

Drug screening for Alzheimer's disease-related targets:

The components with high affinity to butyrylcholinesterase (Butyrylcholinesterase, BChE) were screened among monomeric compounds composed of 27 steroidal alkaloids.

The composition of the screened monomer compound is the same as in Example 1.

Preparation of BChE: Butyrylcholinesterase freeze-dried powder 1200U (Butyrylcholinesterase from equine serum, Sigma), fully dissolved with 3ml ammonium acetate buffer (10mM, pH7.5), prepared to 400U/ml, put in -20 ℃ refrigerator For middle equipment.

Preparation of steroidal alkaloid solution: 27 steroidal alkaloids were dissolved in methanol, the concentration of each steroidal alkaloid was 50 μM, and then diluted with ammonium acetate buffer (10 mM, pH7.5) to a concentration of 4 μM.

Screening: Mix 30μl BChE solution (400U/ml) with 10μl steroidal alkaloid solution (4μM) evenly, and let stand at 25°C for 1h. 40ul of the incubation solution was injected into SPE1, and the rest of the steps were the same as in Example 1.

Chromatography and mass spectrometry conditions are the same as in Example 1.

Test results: 11 components that bind to AChE with high affinity were screened out from 27 monomeric compounds composed of steroidal alkaloids (see Figure 3). They are veratridine, puqiedine, ebiedine, ebeiedinone, pubeiketone base, pubeiketone base glycoside, pubeidiketone, Chuanbei ketone, iso-Zhebeijiasu, zhebeibeijingsu, and ibeibeiline glycoside A. Their binding rates to BChE were 0.82%, 0.80%, 2.18%, 1.84%, 0.39%, 0.19%, 0.24%, 0.89%, 0.44%, 0.39%, 0.17%, respectively. Among them, the IC ₅₀ of four compounds against BChE is less than 10 μm, namely ebeiedinone9.07μM, ebeiedine4.48μM, puqiedine8.49μM, and trabeinone 9.09μM. Under the same conditions, the IC ₅₀ of galantamine against BChE was 3.23 μM. It can be known that this type of steroidal alkaloids has better inhibitory activity on butyrylcholinesterase.

Example 3

Drug screening for cardiovascular disease-related targets

To screen the components with high affinity for angiotensin converting enzyme (Angiotensin Converting Enzyme, ACE, sigma) among the monomeric compounds composed of 17 fritillaria steroidal alkaloids.

The screened monomeric compounds are composed of 17 steroidal alkaloids, which are Pubeisuin E, Pubeisuin A, Pubeisuin B, Pubeisuin C, Pubeisuin D, puqiedine, Pubeisuanine, Pubeisu Bequinone glycoside, plebetin, ebeidinone, ebeidine, zhebeijiasu, zhebeijingsu, sibeisu, sibeisu glycoside, fritillary glycoside, ibetinine glycoside A.

Preparation of ACE: Angiotensin Converting Enzyme freeze-dried powder 6U (Angiotensin Converting Enzyme from rabbitlung, Sigma), fully dissolved with 200ul ammonium acetate buffer (10mM, PH7.5), prepared to 30U/ml, placed in a -20°C refrigerator For middle equipment.

Preparation of steroidal alkaloid solution: 17 Fritillaria steroidal alkaloids were dissolved with methanol, the concentration of each steroidal alkaloid was 25 μM, and then diluted to the concentration with ammonium acetate buffer (10mM, PH7.5) 2 μM.

Screening: 20μl ACE solution (30U/ml) was evenly mixed with 20μl Fritillaria steroidal alkaloid solution (2μM), and allowed to stand at 25°C for 1h. 40ul incubation solution was injected into SPE1. All the other steps are the same as in Example 1.

Chromatography and mass spectrometry conditions are the same as in Example 1.

Test results: From 17 monomeric compounds (groups) composed of Fritillaria steroidal alkaloids, 3 components that bind to AChE with high affinity were preliminarily screened out. They are pyrethine E, pyrethine A, and pyrethine B, and the binding rates are 0.4%, 0.01%, and 0005%, respectively.

Example 4

Drug screening for tumor disease-related targets

Screen for components with high affinity to histone deacetylase 6 (Histone deacetylase6, HDAC6).

Compounds to be screened: myostatin chloride, Trapoxin (TPX), FK228, trichostatin A.

Preparation of HDAC6: 50ug of HDAC6 freeze-dried powder was fully dissolved in 200ul of ammonium acetate buffer (10mM, pH7.5), prepared to 250ug/ml, and placed in a -20°C refrigerator for use.

The configuration of the screened compound solution: myostatin, Trapoxin (TPX), FK228, and trichostatin A were dissolved in ammonium acetate buffer (10mM PH7.5), and the concentration of each compound was 2 μM.

Screening: Mix 20 μl HDAC solution (250ug/ml) with 20 μl mixed solution of the compound to be screened (2 μM) evenly, and let stand at 25°C for 1 hour. 40ul incubation solution was injected into SPE1. All the other steps are the same as in Example 1.

Chromatography and mass spectrometry analysis conditions are the same as embodiment 1

Test results: myostatin, Trapoxin (TPX), FK228, and trichostatin A all bind to HDAC6, and the binding rates are 2%, 0.5%, 1.2%, and 1%, respectively. Myostatin, Trapoxin (TPX), FK228, and trichostatin A are inhibitors of HDAC6, and they were used as positive drugs in this experiment to verify the screening method. It is preliminarily confirmed that the present invention is suitable for drug screening of tumor disease-related targets.

Claims (7)

1. active component high-throughput screening method based on the affine selection of target protein, comprise: the screening reactive compound, the reactive compound that screens is imported high performance liquid chromatogram-mass spectrometer system to be analyzed, carry out the compound structure conclusive evidence, and it is carried out quantitative test, calculate the combination rate of these active components, estimate its activity, it is characterized in that screening reactive compound and comprise the steps: for target protein

A, target protein and testing compound are hatched in damping fluid altogether, hatch solution and be splined on molecular-exclusion chromatography post or online solid-phase extraction column, use the buffer solution elution pillar, eluent stream detects and collects the eluent that at first goes out the peak through UV-detector under absorbing wavelength 280nm;

B, eluent mixed with dissociation solution dissociate, the solution that dissociates is splined on online solid-phase extraction column, and target protein and buffering salt flowage go out pillar, and active component is retained on the online solid-phase extraction column;

The online solid-phase extraction column of the retention activity composition that c, wash-out b step obtain, eluent enters the analysis of high performance liquid chromatogram-mass spectrometer system;

Wherein damping fluid is selected from the phosphate buffer of pH6.0～8.0, TRIS buffer, pH6.0～8.0 ammonium acetate buffers or pH6.0～8.0 ammonium bicarbonate buffers of pH6.0～8.0;

Also contain solubilizer in the incubation buffer, described solubilizer is selected from one or more in tween, dimethyl sulfoxide (DMSO), polyglycol, Qu Latong, methyl alcohol, the ethanol.

2. the high-throughput screening method of claim 1 also comprises with mass spectrometry method the active component that screens is carried out activity rating.

3. the high-throughput screening method of claim 1, wherein testing compound be monomeric compound, the compound group formed by monomeric compound or contain the not Chinese medical extract of principal component.

4. the high-throughput screening method of claim 1, wherein the molecular-exclusion chromatography column packing is selected from Sephadex G-25, SephadexG-50 or Sephadex LH-20, elution flow rate 0.3-0.5ml/min.

5. the high-throughput screening method of claim 1, wherein online solid-phase extraction column is selected from Oasis MCX on-line chromatograph post, OasisWCX on-line chromatograph post, Oasis MAX on-line chromatograph post, Oasis WAX on-line chromatograph post or Oasis HLB on-line chromatograph post.

6. the high-throughput screening method of claim 1, wherein online solid-phase extraction column elution flow rate is 2-4ml/min.

7. the high-throughput screening method of claim 1, wherein dissociation solution is the mixed solution of aqueous acid or aqueous acid and organic reagent, and described aqueous acid is selected from trifluoroacetic acid, formic acid, acetate or hydrochloric acid, and pH is 1-3; Described organic reagent is selected from methyl alcohol or acetonitrile.

Images