A method for high-throughput screening and activity detection of compounds based on thin-layer chromatography

Abstract

The present application relates to a kind of based on thin layer chromatography compound high-throughput screening and activity detection method.The screening method of the present application includes the following operating steps: 1. according to the way of first by row combination, then by column combination, mix part of compound in compound library;2. recover the lead compound;3. trace back the position of "lead compound".The present application also provides a method for verifying the influence of lead compound on the enzyme activity reaction process of protease based on thin layer chromatography, and a method for verifying the influence of lead compound on the binding of protein and small molecule substrate or polypeptide based on thin layer chromatography.The method of the present application has the characteristics of high efficiency, low cost, low technical threshold of experimental operation.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a high-throughput screening and activity verification method for compounds based on thin-layer chromatography. Background Technology

[0002] From lead compounds to candidate drugs, and finally to clinical application, drug development is an extremely lengthy process. The design and discovery of lead compounds is the initial step in new drug development. Selecting and establishing effective high-throughput methods based on screening targets to capture potential lead compounds from a vast library is a crucial first step in the development process. "The most important discovery is the scientific method itself, because it has unlimited potential." Currently, numerous high-throughput screening methods have been developed, leading to a wide variety of flexible screening schemes to meet the demands for "more scientific," "more efficient," and "more precise" compound screening. In terms of screening targets, compound libraries can be divided into physical compound libraries and virtual compound databases. Correspondingly, high-throughput screening methods for small molecule compounds can be divided into physical screening and virtual screening. In terms of screening models, high-throughput screening methods can be divided into screening targeting proteins, nucleic acids, and other biomolecules, and screening based on cell phenotypes. There is also screening using animal models, but with lower throughput compared to the former two. For purified target proteins or nucleic acids, biochemical and biophysical screening methods are mainly used.

[0003] Biochemical screening methods typically target proteins or nucleic acids, with known substrate molecules, interacting proteins, or nucleic acids as targets. Based on the specific characteristics of the target, substrate-like probes are designed and synthesized. Common methods involve detecting the inhibition of protein-probe binding by candidate compounds, or detecting the inhibition of target protein activity by compounds. Screening machines offer high sensitivity for detecting light signals such as absorbance, fluorescence intensity, and luminescence intensity. The reaction system volume is between 10 and 40 μL, requiring low amounts of protein, compound, and probe. 384-well plates are often used as the reaction and detection containers, thus facilitating high-throughput screening.

[0004] In screening methods for detecting absorbance, changes in the absorbance of the system at a specific absorption wavelength can indicate the consumption of raw materials or the formation of products in an enzyme activity reaction, thus characterizing the extent of the enzyme activity reaction. Screening methods for detecting fluorescence intensity mainly include total fluorescence intensity analysis and fluorescence ratio methods. Of these two methods, total fluorescence intensity analysis is easily interfered with by the fluorescence produced by the compound being tested, and the shorter the excitation wavelength of the fluorescent probe, the stronger the interference from the compound. Fluorescence ratio methods, such as commonly used fluorescence anisotropy (FP) and fluorescence resonance energy transfer (FRET), are less affected by the fluorescence interference from the compound. Screening methods for detecting luminescence intensity differ from those for detecting fluorescence intensity in that the former does not require excitation light but directly detects the fluorescence emitted after luciferin is hydrolyzed by luciferase. This is an irreversible enzyme-catalyzed reaction, therefore, it is impossible to perform multiple detections of the reaction process as in experiments like FP and FRET.

[0005] Biophysical methods are a series of approaches for detecting interactions between biomacromolecules and small molecule compounds, or between biomacromolecules themselves. Unlike biochemical screening methods, for screening small molecule compounds targeting proteins, biophysical methods are typically used to characterize the binding between proteins and small molecules. Although, under certain circumstances, the influence of small molecules on target activity can be indirectly characterized through clever design, this indirect characterization is mostly achieved through direct detection of the binding. Furthermore, in practical applications, most biophysical methods do not require the design of specific probes as in biochemical methods; the reaction system often contains only two main components: the protein and the compound. Some methods utilize dye-labeled proteins to visualize the signal.

[0006] Commonly used biophysical methods include surface plasmon resonance (SPR), protein thermal shift (PTS), microscale thermophoresis (MST), isothermal titration calorimetry (ITC), nuclear magnetic resonance (NMR), and mass spectrometry (MS). Isothermal titration calorimetry is considered the "gold standard" for identifying binding compounds. Compared to biochemical methods, general biophysical methods are time-consuming to detect single samples and often struggle to achieve high-throughput detection. In recent years, based on classical biophysical detection methods, some high-throughput compound screening techniques have been developed, such as affinity-selection mass spectrometry (AS-MS), which relies on mass spectrometry detection.

[0007] In general, the screening and validation of lead compounds typically rely on the technical principles of classical experiments, combined with the structural and functional characteristics of specific targets, to rationally design corresponding screening schemes. Multiple methods are often employed in combination, integrating biochemical and biophysical approaches to comprehensively confirm the activity of lead compounds, clarify their mechanisms of action, and provide reliable data support for subsequent design modification and in-depth research.

[0008] Thin-layer chromatography (TLC) is a method that separates sample components by coating a stationary phase onto a plate and utilizing the different adsorption capacities of the components on the stationary phase. As the mobile phase flows over the stationary phase surface, the sample components undergo varying degrees of adsorption-dissociation-adsorption processes. The earliest recorded application of TLC dates back to 1938, and after nearly ninety years of development, it remains a widely used separation and identification method. Commonly used stationary phase materials include silica gel, alumina, polyamide, cellulose, and dextran gel. Different stationary phases are paired with corresponding mobile phases to identify different types of samples. Thin-layer adsorption chromatography using silica gel and alumina as adsorbents is currently the most commonly used TLC method.

[0009] In recent years, many derivative methods have been developed based on the classical thin-layer chromatography theory. High-performance thin-layer chromatography (HPTLC) uses smaller diameter gel particles (4–6 μm) as the stationary phase, increasing the number of interactions with analyte molecules and achieving better separation. Ultra-thin-layer chromatography (UTLC) uses an even thinner stationary phase (10 μm) to reduce lateral diffusion of analytes. Pressurized thin-layer chromatography (OPLC) uses a liquid chromatography (LC) pump to drive the mobile phase through the stationary phase. Microemulsion thin-layer chromatography (ME-TLC) uses a water-in-oil microemulsion as the mobile phase.

[0010] The mitochondrial shift (Rf) value is the most important parameter used in thin-layer chromatography to determine the properties of analytes. Rf = distance traveled by the solute / distance traveled by the developing solvent; it represents the relative distance the analyte travels on the stationary phase under the influence of the developing solvent. During development, substances are subjected to two main effects: adsorption by the stationary phase and dissolution by the mobile phase. Under these two effects, substances repeatedly undergo adsorption-dissociation-adsorption on the stationary phase surface. These two effects are specific to different substances and closely related to their physical / chemical properties, making the Rf value a characteristic value for the qualitative analysis of compounds.

[0011] By definition, the piezoresistive value ranges from 0 to 1. In a developing system, the piezoresistive value is primarily determined by three factors: the adsorption strength of the fixed compound, the polarity of the compound, and the polarity of the developing solvent. In addition, environmental factors such as temperature and humidity also affect the development results. To accurately determine the analyte's affiliation, a good development result typically requires a piezoresistive value between 0.2 and 0.8. This is usually achieved by adjusting the polarity of the developing solvent.

[0012] Thin-layer chromatography (TLC) colorimetric methods include those dependent on the physical properties of compounds, using visible light, ultraviolet light, and fluorescence, and those dependent on chemical properties, using chromogenic reagents. Colorimetric development under 254 nm and 365 nm ultraviolet light is a broad-spectrum, sensitive, and non-destructive method, generally the preferred method. Iodine, potassium permanganate solution, and sulfuric acid solution, among other reducing agents or strong oxidizing agents, are commonly used broad-spectrum chromogenic reagents. In addition, some specific chromogenic reagents are used for compounds with corresponding characteristic structures. Under some colorimetric methods, different compounds show different colors, which can be combined with Rf values ​​to aid in compound identification.

[0013] Thin-layer chromatography (TLC) is commonly used for qualitative analysis, content determination, and separation and purification of compounds. TLC is simple to operate, highly reliable, and sensitive, often serving as a direct means of monitoring reaction progress during chemical synthesis. In qualitative analysis, the analyte and its standards are spotted in parallel, and color development occurs at appropriate positions. The relative shifts are compared to determine if a product has formed. The approximate concentration ratio of the reactants and products is compared to assess the reaction progress. For more precise quantification, a standard concentration gradient is set, a concentration-color intensity standard curve is fitted, and the analyte concentration is calculated colorimetrically. Preparative-grade TLC plates can withstand high loading rates (500 mg). The target compound and impurities are separated during development. After development, the stationary phase at the target compound band is scraped off, and the product is eluted with organic reagents.

[0014] Thin-layer chromatography (TLC) is a simple, rapid, and economical method, making it an excellent tool for screening lead compounds, especially for the preliminary screening of active ingredients in natural products. Bioautochromatography is an application of TLC in lead compound screening. Bioautochromatography is an effect-directed analysis (EDA) method that combines chemical analysis with biological detection. In this method, TLC separates mixtures of compounds; the TLC plate serves as a carrier of the compounds and also as the site of the biological reaction. By coating biological materials, such as enzymes or bacteria, onto the TLC plate carrying the compounds, the activity of the active compounds is inhibited, thus suppressing enzyme activity or bacterial growth. This prevents enzyme-catalyzed color-changing reactions from occurring or colonies from forming, resulting in a different color or blank area at that location compared to the background.

[0015] In reported screening methods involving one-to-many incubation of proteins and compounds, specialized detection methods are often required to identify lead compounds. For example, affinity selection mass spectrometry and autoradiography ultimately rely on mass spectrometry to detect compound mass and structure. In establishing our screening method, we solved the problem of lead compound identification by orderly mixing compounds from a compound library. Compounds in a library are typically stored in standardized compound plates, each with its own information record—an electronic file corresponding to the compound's location (plate number and well number). Therefore, determining the compound's location information identifies the compound. The orderly mixing of compounds allows us to trace the location information of lead compounds, thereby obtaining their electronic files. Summary of the Invention

[0016] The technical objective of this invention is to provide a high-throughput screening and activity verification method for compounds based on thin-layer chromatography. This method is convenient, rapid, cost-effective, and efficient.

[0017] On one hand, the present invention provides a high-throughput screening method for compounds based on thin-layer chromatography, the method comprising:

[0018] 1) Divide the compounds in the compound library into equal groups. Store the stock solutions of these compounds in a multi-well plate, with each well containing one compound at a concentration of 1 mM to 10 mM. Mix each group of compounds using the following two steps:

[0019] Step 1.1: Merge by row

[0020] A multi-channel gun is used to extract a certain volume from each hole in each row of the perforated plate and merge them into a single row; Step 1.2: Merging by column

[0021] Take a certain volume from each well in each column of the compounds merged in step 1.1 and merge them into one column.

[0022] After two merging processes, each resulting compound mixture contains m*n compounds, where m is the number of rows merged and n is the number of columns merged, with m ranging from 8 to 96 and n ranging from 5 to 10.

[0023] 2) Dilute and mix the target protein and the mixture of compounds obtained in step 1) with the reaction solution, incubate, and then perform desalting to separate the protein-compound complex.

[0024] 3) Denature the protein using a protein denaturation method to release the lead compounds in the complex, and then freeze-dry it;

[0025] 4) Detection of lead compounds using thin-layer chromatography: Dissolve the freeze-dried sample from step 3) and the m*n compounds obtained in step 1) in ethyl acetate and spot them on the same silica gel plate. After spotting, wait for the ethyl acetate to completely evaporate, and then develop the compounds sequentially with the developing solvent until the ratio shift value of the "lead compound" is between 0.2 and 0.8. This determines which column of the original compound master plate the "lead compound" belongs to. Then, dissolve the m compounds in that column in ethyl acetate and spot them together with the lead compound sequentially until the ratio shift value of the "lead compound" is between 0.2 and 0.8. This determines which row of the original compound master plate the "lead compound" belongs to.

[0026] 5) Determine the information of the lead compound based on the electronic file of the original compound master plate, wherein the electronic file of the original compound master plate is the information recorded when the compound library was established, which corresponds one-to-one with the position of each compound in the storage container.

[0027] In the specific implementation scheme, in step 1), m is 16 and n is 10. Specifically, 160 compounds are mixed using the following two-step method: The 160 compounds correspond to the total number of compounds in two 96-well plates in the compound library, with 80 compounds in each plate. First, they are merged by row, using a 10-channel pipette to take 1 μL of the compound from each well in each row of the two plates and merge them into one row. At this point, each well in the merged plate contains 16 compounds. Then, they are merged by column, taking 0.5 μL of the compound from each well in each column obtained in the previous step and merging them into one column. After these two merges, a mixture of 160 compounds is obtained.

[0028] In a specific implementation, in step 1), the plurality of compounds are dissolved in dimethyl sulfoxide before mixing. After mixing, the dimethyl sulfoxide is removed using a freeze dryer, and a small amount of dimethyl sulfoxide is added to redissolve the compounds. The volume of dimethyl sulfoxide added when redissolving the compound sample is between 1% and 12% of the total volume of the reaction solution in step 2, preferably 10%, depending on the tolerance of the target protein to dimethyl sulfoxide.

[0029] In a specific implementation scheme, in step 2), the target protein may be a protein molecule that is closely related to the occurrence and development of the disease and has drug targeting significance, such as a full-length or truncated somatic protein expressed by eukaryotic or prokaryotic cells.

[0030] In a specific embodiment, in step 2), the working concentration of the target protein is between 1 and 50 μM, preferably 10 μM. The working concentration of the compound is between 10 and 100 μM, preferably 50 μM. The total volume of the reaction solution is between 20 and 120 μL, preferably 100 μL.

[0031] In a specific implementation, in step 2), the reaction solution contains one or more of the following: 4-hydroxyethylpiperazine ethanesulfonic acid buffer (HEPES), tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl), 2-morpholine ethanesulfonic acid buffer (MES), 3-morpholine propanesulfonic acid buffer (MOPS), disodium hydrogen phosphate / potassium dihydrogen phosphate buffer, sodium chloride, potassium chloride, magnesium chloride, calcium chloride, EDTA, DTT, Tween-20, NP-40, Triton X-100, and bovine serum albumin (BSA), preferably phosphate buffer (1x PBS buffer).

[0032] In a specific implementation, in step 2), the reaction temperature is between 15 and 45°C, preferably 37°C. The reaction time is between 20 and 60 minutes, preferably 30 minutes.

[0033] In a specific implementation, in step 2), the desalination method is to use Zeba Spin Desalting Columns (Thermo Fisher Scientific #89883) or Zeba Spin Desalting Plate (Thermo Fisher Scientific #89807) for desalination.

[0034] In a specific embodiment, in step 3), the denaturation method is selected from the heat denaturation of proteins, the organic reagent extraction of compounds, and the organic reagent denaturation of proteins, preferably the organic reagent denaturation of proteins. The organic reagent can be ethanol, acetonitrile, methanol, dimethyl sulfoxide, etc., more preferably acetonitrile, and the volume of acetonitrile is 2 to 5 times the volume of the reaction solution, preferably 3 times.

[0035] In a specific embodiment, in step 4), the plate used in the thin-layer chromatography is a silica gel plate.

[0036] In a specific implementation, in step 4), the colorimetric method used in the thin-layer chromatography is iodine colorimetry, potassium permanganate solution colorimetry, or ultraviolet light colorimetry, preferably ultraviolet light colorimetry.

[0037] In a specific embodiment, in step 4), the developing solvent used in the thin-layer chromatography is one or more of petroleum ether, ethyl acetate, dichloromethane, methanol, triethylamine, acetic acid, and water.

[0038] In a specific implementation, in step 4), the compound is developed sequentially using developing solvents of increasing polarity (from #1 to #5). Preferred developing solvent formulations (developing solvents #1 through #5 with increasing polarity) are as follows:

[0039]

[0040] In the specific implementation scheme, in step 4), the freeze-dried sample and the 10 groups (16 compounds per group) of 160 compounds from the first round of mixing in step 1 are dissolved in 5 μL of ethyl acetate and spotted onto the same silica gel plate. After spotting, the ethyl acetate is allowed to evaporate completely, and the compounds are developed sequentially with 5 polar gradient developing solvents (from #1 to #5) until the ripple shift value of the "leading compound" is between 0.2 and 0.8. This determines which group the "leading compound" belongs to (i.e., which column of the original compound master plate the compound belongs to). Then, the 16 compounds in that column are dissolved in ethyl acetate and spotted sequentially together with the leading compound until the ripple shift value of the "leading compound" is between 0.2 and 0.8. This determines which group the "leading compound" belongs to (i.e., which row of the original compound master plate the compound belongs to).

[0041] In specific implementation schemes, the information includes, but is not limited to, the CAS number, structure, molecular formula, and relative molecular mass of each compound.

[0042] On the other hand, the present invention provides a method for verifying the effect of a seed compound on the enzymatic activity process of a protease, the method comprising the following steps:

[0043] 2.1: Dilute the test protease, the precursor compound, and DMSO (corresponding volumes to the precursor compound stock solution) with the reaction solution to obtain protease solution, precursor compound solution, and DMSO solution, respectively. Mix the diluted protease solution and the precursor compound solution as the experimental group; mix the diluted protease solution and the DMSO solution (corresponding volumes to the experimental group) as the NCC (no compound control) group; mix the blank reaction solution and the diluted DMSO solution (corresponding volumes to the experimental group) as the NPC (no protein control) group.

[0044] 2.2: Add the corresponding substrate diluted with the reaction solution to the mixed solution of the experimental group, NCC group and NPC group in step 2.1 respectively to start the catalytic reaction;

[0045] 2.3: Heating the solution from step 2.2 terminates the catalytic reaction;

[0046] 2.4: Thin-layer chromatography was used to analyze the samples taken from step 2.3. The samples taken from step 2.3 were spotted on a silica gel plate, and after development with the developing solvent, the differences in substrate consumption / product generation between the experimental group, the NCC group, and the NPC group were observed to determine the inhibitory / stimulatory effect of the compound on protease activity.

[0047] In a specific implementation scheme, the lead compound can be obtained by screening using the method described above.

[0048] In a specific implementation scheme, in step 2.1, the working concentration of the protease is between 20 nM and 20 μM, the working concentration of the seed compound is between 2 μM and 2 mM, the incubation time is between 20 and 60 min, and the incubation temperature is between 15 and 45°C.

[0049] In the specific implementation scheme, in step 2.2, the working concentration of the substrate is between 2 and 200 μM, the total volume of the reaction system is between 10 and 100 μL, the incubation time is between 20 and 60 min, and the incubation temperature is between 15 and 45 °C.

[0050] In a specific implementation, the reaction solutions in steps 2.1 and 2.2 contain one or more of the following: 4-hydroxyethylpiperazine ethanesulfonic acid buffer (HEPES), tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl), 2-morpholine ethanesulfonic acid buffer (MES), 3-morpholine propanesulfonic acid buffer (MOPS), disodium hydrogen phosphate / potassium dihydrogen phosphate buffer, sodium chloride, potassium chloride, magnesium chloride, calcium chloride, EDTA, DTT, Tween-20, NP-40, Triton X-100, and bovine serum albumin (BSA), preferably phosphate buffer (1xPBS buffer) or Tris-HCl buffer.

[0051] In a specific implementation, in step 2.3, the heating temperature is 80-100℃, preferably 100℃, and the heating time is 3-5 minutes, preferably 3 minutes.

[0052] In the specific implementation plan, the developing agent used in step 2.4 is not particularly limited. For example, as described in step 4) above, developing agents of different polarities from the aforementioned 1# to 5# can be selected according to the properties of the substance to be developed.

[0053] In another aspect, the present invention provides a method for verifying the effect of a lead compound on the binding of a protein to a small molecule substrate or peptide, the method comprising the following steps:

[0054] 3.1: The protein diluted with the reaction solution and the precursor compound were mixed evenly and incubated for a certain period of time to form the experimental group. At the same time, a control group was set up. The control group was different from the experimental group only in that it did not contain the precursor compound.

[0055] 3.2: Add the small molecule substrate or polypeptide corresponding to the protein diluted with the reaction solution to the mixed solution of the experimental group and the control group obtained in step 3.1, and desalt after incubation for a certain period of time;

[0056] 3.3: Denatured proteins, the desalted effluent was freeze-dried;

[0057] 3.4: Thin-layer chromatography was used to analyze the sample from step 3.3. The sample from step 3.3 was redissolved in solvent and spotted onto a silica gel plate. After development with a developing solvent, the changes in the substrate / peptide recovery in the experimental and control groups were observed to determine the effect of the compound on the binding of the protein to the small molecule substrate or peptide.

[0058] In a specific implementation scheme, the lead compound can be obtained by screening using the method described above.

[0059] In the specific implementation scheme, in step 3.1, the working concentration of the protein is between 20 nM and 20 μM, the working concentration of the spur compound is between 2 μM and 2 mM, the incubation time is between 20 and 60 min, and the incubation temperature is between 15 and 45°C.

[0060] In a specific implementation, the reaction solution in steps 3.1 and 3.2 comprises one or more of the following: 4-hydroxyethylpiperazine ethanesulfonic acid buffer (HEPES), tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl), 2-morpholine ethanesulfonic acid buffer (MES), 3-morpholine propanesulfonic acid buffer (MOPS), disodium hydrogen phosphate / potassium dihydrogen phosphate buffer, sodium chloride, potassium chloride, magnesium chloride, calcium chloride, EDTA, DTT, Tween-20, NP-40, Triton X-100, and bovine serum albumin (BSA), preferably phosphate buffer (1x PBS buffer).

[0061] In a specific implementation, in step 3.1, the total volume of the reaction solution is between 20 and 120 μL, preferably 100 μL.

[0062] In a specific implementation scheme, in step 3.2, the concentration of the small molecule substrate / peptide is between 10 and 200 μM, the incubation time is between 20 and 60 min, and the incubation temperature is between 15 and 45 °C.

[0063] In a specific implementation scheme, in step 3.3, the protein denaturation method is selected from the heat denaturation method, the organic reagent extraction method, and the organic reagent denaturation method, preferably the organic reagent denaturation method. The organic reagent can be ethanol, acetonitrile, methanol, dimethyl sulfoxide, etc., and more preferably acetonitrile.

[0064] In a specific implementation plan, the solvent used in step 3.4 includes, but is not limited to, water, ethyl acetate, dichloromethane, etc., with a volume of 5 to 10 μL.

[0065] In the specific implementation plan, the developing agent used in step 3.4 is not particularly limited. For example, as described in step 4) above, developing agents of different polarities from the aforementioned 1# to 5# can be selected according to the properties of the substance to be developed.

[0066] Beneficial effects

[0067] This invention provides a high-throughput method for screening lead compounds from mixed compounds by separating and visualizing them using thin-layer chromatography. The two-step screening method used in this invention is highly efficient, rapid, cost-effective, and requires minimal experimental expertise.

[0068] Furthermore, by using thin-layer chromatography to separate and visualize enzyme substrates / products, a method is provided to indicate the effect of compounds on enzyme activity / enzyme reaction progress; by using thin-layer chromatography to separate and visualize target protein small molecule substrates / peptides, a method is also provided to verify the effect of compounds on the binding of target proteins to substrates / peptides. These methods can further confirm the exact biological activity of the lead compounds screened through the above screening methods. The verification methods of this application can eliminate false-positive lead compounds that do not affect the biological activity of the target protein; they can effectively separate compounds and substrates, avoiding the problem of fluorescence interference that may occur when all components of the system are detected together in biochemical experiments. Attached Figure Description

[0069] Figure 1 : Schematic diagram of the principle of high-throughput screening of compounds based on thin-layer chromatography.

[0070] Figure 2 : A schematic diagram of the "two-step method" for mixing 160 compounds in this application.

[0071] Figure 3 : Schematic diagram of the process for determining the location information of lead compounds in a high-throughput screening method based on thin-layer chromatography.

[0072] Figure 4 High-throughput screening results for compounds targeting BPTF.

[0073] Figure 5 High-throughput screening results for compounds targeting USP7.

[0074] Figure 6 Experimental results of the inhibition of USP7 enzyme activity by the positive compound GNE-6776 based on thin-layer chromatography.

[0075] Figure 7 Experimental results of the validation compound's inhibition of OGT enzyme activity based on thin-layer chromatography.

[0076] Figure 8 Experimental results of the positive compound's inhibition of OGT homotope binding based on thin-layer chromatography.

[0077] Figure 9 Experimental results of the positive compound's inhibition of OGT binding to peptides based on thin-layer chromatography. Detailed Implementation

[0078] The following embodiments will further illustrate the present invention, but are not intended to limit the scope of the invention.

[0079] Example 1: High-throughput screening of compounds targeting BPTF

[0080] Seed compounds were recovered from a mixture of compounds using the BPTF (bromodomain PHD finger transcription factor) protein, and their original positions in the compound library were determined by thin-layer chromatography.

[0081] (1) Materials:

[0082] GST-BPTF (2914-3037) protein, 96-well plate (Thermo Fisher Scientific #249944), Zeba Spin Desalting Plate (Thermo Fisher Scientific #89807), PBS buffer (Meilun #MA0015), centrifuge (Eppendorf, 5417R), freeze dryer (Alpha 3-4LSC basic), ethyl acetate (Sinopharm #10009418), methanol (Sinopharm #10014159), petroleum ether (Sinopharm #10015218), dichloromethane (Sinopharm #80047318), thin-layer chromatography silica gel plate (Bailingwei #956419).

[0083] (2) Operation steps:

[0084] ① Mixed Compounds: The total number of compounds in 60 96-well plates (80 compounds per plate) from a compound library of 4800 compounds (all compounds in this application were purchased from Topscience and constitute a compound library of 4800. It should be noted that the compound library is not limiting in this application; anyone skilled in the art can obtain a compound library of any size as long as they have compound information) was mixed. The 160 compounds in each pair of plates were ultimately mixed into a group. First, the plates were merged by row, using a 10-channel pipette to take 1 μL of the compound from each well in each row of the two plates, merging them into one row and mixing thoroughly. At this point, each well contained 16 compounds. Then, the plates were merged by column, taking 0.5 μL of the compound from each well in each column of the compounds obtained in the previous step, merging them into one column. After two merges, 30 groups of 160 compounds each were obtained. The samples from both merges were freeze-dried to remove the DMSO (dimethyl sulfoxide) solvent.

[0085] ② Recovery of lead compounds: The 160 lyophilized compound mixture samples were redissolved in 10 μL DMSO, and 40 μL 1x PBS was added to dilute the compounds to a concentration of 100 μM. The BPTF protein was diluted to 20 μM with PBS. Equal volumes of compound and protein solutions (50 μL + 50 μL) were mixed, and an NCC group (10 μL DMSO + 40 μL PBS + 50 μL protein solution) was set up and incubated at room temperature for 30 min. After incubation, the mixture was centrifuged at 13000 rpm for 10 min at 4 °C. The supernatant was desalted using a ZebaSpin Desalting Plate. Three volumes of acetonitrile-denatured protein were added to the effluent, and the mixture was centrifuged at 13000 rpm for 10 min at 25 °C to remove the protein precipitate. The supernatant was then lyophilized.

[0086] ③ Tracing the position of the "leading compound": Dissolve the freeze-dried sample and the 160 compounds from the first round of mixing in the "mixed compounds" step in 5 μL of ethyl acetate and spot them on the same silica gel plate. After spotting, wait for the ethyl acetate to completely evaporate, and then develop the compounds sequentially with the five polar gradient eluents (from #1 to #5) as described above until the ripple shift value of the "leading compound" is between 0.2 and 0.8. This determines which group the "leading compound" belongs to (i.e., which column of the original compound master plate it belongs to). Dissolve the freeze-dried sample and the 16 compound standards in that column in 5 μL of ethyl acetate and spot them on the same silica gel plate. After spotting, wait for the ethyl acetate to completely evaporate, and then develop the compounds sequentially with the five polar gradient eluents as well until the ripple shift value of the "leading compound" is between 0.2 and 0.8. This determines which group the "leading compound" belongs to (i.e., which row of the original compound master plate it belongs to).

[0087] (3) Analysis of experimental results

[0088] This screening method determines the structural information of lead compounds by their location. The two-step approach effectively ensures convenient and orderly backtracking of compound positions. The 160 basic screening units correspond precisely to the total number of compounds in the two 96-well plates in the compound library. Based on the correspondence between the instrument and consumables (multichannel pipettes, 96-well plates, and 96 pipette tips / boxes), this method employs a hybrid strategy of "merging rows first, then columns." Conversely, when determining the location of lead compounds, an analytical method of "determining the number of columns first, then the number of rows" is used. Figure 4As shown, taking compound 02-H06 (CAS No.: 2758411-46-8) as an example, the above method first determines that the lead compound comes from column 6, and secondly determines that the compound belongs to row H of compound plate 2. The position of the lead compound 02-H06 is accurately determined among 160 compounds, and then the corresponding lead compound structure information is retrieved from the compound library information table based on its position.

[0089] Example 2: High-throughput screening of compounds targeting USP7

[0090] The USP7 protein was used to recover the lead compound from the mixed compounds, and the original location of the lead compound in the compound library was determined by thin-layer chromatography.

[0091] (1) Materials:

[0092] His-USP7 (208-560) protein, 96-well plate (Thermo Fisher Scientific #249944), Zeba Spin Desalting Plate (Thermo Fisher Scientific #89807), PBS buffer (Meilun #MA0015), centrifuge (Eppendorf, 5417R), freeze dryer (Alpha3-4LSC basic), ethyl acetate (Sinopharm #10009418), methanol (Sinopharm #10014159), petroleum ether (Sinopharm #10015218), dichloromethane (Sinopharm #80047318), thin-layer chromatography silica gel plate (Bailingwei #956419).

[0093] (2) Operation steps

[0094] ① Compound Mixing: 4800 compounds (the corresponding compounds and compound library are the same as in Example 1) were ultimately mixed into groups of 160 compounds each. First, they were merged by row: using a 10-channel pipette, 1 μL of the compound was taken from each well in each row of both plates and merged into one row, then thoroughly mixed. At this point, each well contained 16 compounds. Then, they were merged by column: 0.5 μL of the compound was taken from each well in each column of the compounds obtained in the previous step and merged into one column. After two merges, 30 groups of 160 compounds each were obtained. The merged samples were freeze-dried separately to remove the DMSO solvent.

[0095] ② Recovery of lead compounds: The 160 lyophilized compound mixture samples were redissolved in 10 μL DMSO, and 40 μL 1x PBS was added to dilute the compounds to a concentration of 100 μM. USP7 protein was diluted to 20 μM with PBS. Equal volumes of compound and protein solutions (50 μL + 50 μL) were mixed, and an NCC group (10 μL DMSO + 40 μL PBS + 50 μL protein solution) was set up and incubated at room temperature for 30 min. After incubation, the mixture was centrifuged at 13000 rpm for 10 min at 4 °C. The supernatant was desalted using a ZebaSpin Desalting Plate. Three volumes of acetonitrile-denatured protein were added to the effluent, and the mixture was centrifuged at 13000 rpm for 10 min at 25 °C to remove the protein precipitate. The supernatant was then lyophilized.

[0096] ③ Tracing the position of the "leading compound": Dissolve the freeze-dried sample and the 160 compounds from the first round of mixing in the "mixed compounds" step in 5 μL of ethyl acetate and spot them on the same silica gel plate. After spotting, wait for the ethyl acetate to completely evaporate, and then develop the compounds sequentially with the five polar gradient eluents (from #1 to #5) until the ripple shift value of the "leading compound" is between 0.2 and 0.8. This determines which group the "leading compound" belongs to (i.e., which column of the original compound master plate it belongs to). Dissolve the freeze-dried sample and the 16 compound standards in that column in 5 μL of ethyl acetate and spot them on the same silica gel plate. After spotting, wait for the ethyl acetate to completely evaporate, and then develop the compounds sequentially with the five polar gradient eluents until the ripple shift value of the "leading compound" is between 0.2 and 0.8. This determines which group the "leading compound" belongs to (i.e., which row of the original compound master plate it belongs to).

[0097] (3) Analysis of experimental results

[0098] like Figure 5 As shown, taking compound 158-B10 (5-(5-pyrimidinyl)indoline) as an example, the first step determined that the lead compound belonged to column 10 of compounds 158 and 159. The second step further determined that the compound belonged to row B of plate 158. Thus, the original position of the lead compound is 158-B10.

[0099] Example 3: Testing the inhibitory effect of positive compounds on the enzyme activity of USP7 (ubiquitin-specific protease 7).

[0100] Thin-layer chromatography was used to separate USP7 catalytic products to indicate the enzyme activity reaction process. By comparing the difference in the amount of hydrolysis products within a fixed time period, the inhibition of USP7 enzyme activity by positive compounds was verified.

[0101] (1) Material preparation:

[0102] His-USP7 (208-560) protein, ubiquitin-rhodamine 110 (Keshengjingtai), GNE-6776 (MCE#HY-107986), reaction solution (50mM Tis-HCl, pH 7.4, 100mM NaCl, 0.05% CHAPS, 1mM EDTA, pH 8.0), Zeba Spin Desalting Plate (Thermo Fisher Scientific #89807), centrifuge (Eppendorf, 5417R), freeze dryer (Alpha 3-4LSC basic), ethyl acetate (Sinopharm #10009418), methanol (Sinopharm #10014159), petroleum ether (Sinopharm #10015218), dichloromethane (Sinopharm #80047318), thin-layer chromatography silica gel plate (Bailingwei #956419).

[0103] (2) Operation steps

[0104] ① Dilute USP7 protein to 400 nM with a reaction solution containing 4% DMSO, and dilute compound GNE-6776 to 800 μM. Then, serially dilute to seven concentrations at two-fold volume ratios. Mix equal volumes of His-USP7 protein and compound (2.5 μL + 2.5 μL) and incubate at room temperature for 30 min as the experimental group. An NCC (no compound control) group (2.5 μL reaction solution containing 4% DMSO + 2.5 μL His-USP7 protein solution) and an NPC (no protein control) group (2.5 μL reaction solution containing 4% DMSO + 2.5 μL reaction solution) were also set up. (Note that to offset the potential influence of DMSO, the solvent used in the compound stock solution, on the experiment, in the NPC and NCC groups, "DMSO solution obtained by diluting the corresponding volume of DMSO solvent with the reaction solution" was used instead of "compound stock solution diluted with the reaction solution" in the experimental group.)

[0105] ② Add 5 μL of 2 μM ubiquitin-rhodamine 110 to each group of solutions. At this point, the protein concentration is 100 nM, and the highest concentration of the compound is 200 μM. The enzyme activity reaction is carried out at 37℃ for 1 h.

[0106] ③ Heat each group of solutions to 100℃ and maintain for 3 minutes to terminate the reaction and precipitate the protein. Centrifuge at 25℃ and 13000 rpm for 10 minutes to remove the precipitate. Spot the supernatant of each group onto a silica gel plate and evaporate the solvent with a hot air gun. Develop the samples using solvent #4 (ethyl acetate:methanol = 3:2). After the solvent reaches the defined front, remove the silica gel plate, evaporate the solvent, and observe the color development of Rhodamine 110 in each group under UV light.

[0107] (3) Analysis of experimental results

[0108] In this experiment, the amount of Rhodamine 110 detected by thin-layer chromatography indicated the extent of enzyme activity, such as... Figure 6 As shown, compared with the NCC (no compound control) group, the amount of Rhodamine 110 detected in the corresponding experimental group gradually decreased with increasing compound concentration, indicating that the compound inhibited the hydrolytic activity of USP7 deubiquitination. Simultaneously, the thin-layer chromatography process separated the compound and Rhodamine 110, which differs from the classic method of detecting all components together in an enzyme activity reaction system, effectively avoiding interference from the compound's own fluorescence on the detection results.

[0109] Example 4: Testing the inhibitory effects of positive and lead compounds on the enzymatic activity of OGT (O-GlcNAc transferase).

[0110] Thin-layer chromatography was used to separate the OGT substrate O-GlcNAc to indicate the enzyme activity reaction process. By comparing the difference in the amount of substrate consumed within a fixed time, the inhibitory effect of the compound on OGT enzyme activity was verified.

[0111] (1) Materials

[0112] His-OGT (313-1031) protein, reaction solution (50mM Tris-HCl, pH 7.8, 20mM CaCl2, 0.05% Tween-20, 0.05% NP-40, 0.1% BSA, 500μM TCEP), UDP-GlcNAc (Sigma, U4375), OMSI-4 (MCE#HY-114361), centrifuge (Eppendorf, 5417R), freeze dryer (Alpha 3-4LSC basic), ethyl acetate (Sinopharm #10009418), methanol (Sinopharm #10014159), petroleum ether (Sinopharm #10015218), dichloromethane (Sinopharm #80047318), thin-layer chromatography silica gel plate (Bailinwei #956419).

[0113] (2) Operation steps

[0114] ① Dilute OGT protein to 20 μM with 12.5% ​​DMSO reaction solution, dilute compound B (CAS No.:2645-32-1) and OMSI-4 to 4 mM, dilute peptide (SEQ ID No.:1: STPVSRANMK, with biotin modification) to 200 μM, and dilute UDP-GlcNAc to 200 μM.

[0115] Equal volumes of protein and compound (2.5 μL + 2.5 μL) were mixed and incubated at room temperature for 30 min. Two groups were set up: the NCC (no compound control) group (2.5 μL reaction solution containing 12.5% ​​DMSO + 2.5 μL OGT protein solution) and the NPC (no protein control) group (2.5 μL reaction solution containing 12.5% ​​DMSO + 2.5 μL reaction solution).

[0116] ② Add 2.5 μL of diluted peptide and 2.5 μL of UDP-GlcNAc to each group of solutions. The enzyme activity reaction was carried out at 25℃ for 1 h.

[0117] ③ Heat each group of solutions to 100℃ and maintain for 3 minutes to terminate the reaction and precipitate the protein. Centrifuge at 25℃ and 13000 rpm for 10 minutes to remove the precipitate. Spot the supernatant of each group onto a silica gel plate and evaporate the solvent using a hot air gun. Develop the samples in solvent #5 (dichloromethane:methanol:water = 3:2:0.5). After the solvent reaches the defined front, remove the silica gel plate, evaporate the solvent, and observe the color development of each group of UDP-GlcNAc under UV light.

[0118] (3) Analysis of experimental results

[0119] First, by comparing the NCC (no compound control) group and the NPC (no protein control) group... Figure 7 The results showed that the spot corresponding to UDP-GlcNAc (shown in the white dashed box) in the NCC group was extremely weak, indicating that the enzyme activity reaction proceeded normally and consumed most of the substrate UDP-GlcNAc. In addition to the consumption of UDP-GlcNAc, a significantly newly added band was observed in the NCC group. Based on the OGT reaction principle, it is speculated that the compound corresponding to this band should be UDP (shown in the white dashed box), one of the products of the OGT enzyme activity reaction. In experimental group 1 treated with OMSI-4 and experimental group 2 treated with compound B, the amount of UDP-GlcNAc was comparable to that in the NPC group, indicating that compounds OMSI-4 and compound B effectively inhibited the OGT-catalyzed glycosylation reaction. Meanwhile, as... Figure 7As shown, the unfolding process effectively separates the compound, substrate, and product, thus avoiding the fluorescence interference that may occur when all components of the system are detected together, as is the case in classic biochemical experiments.

[0120] Example 5: Testing the effect of positive and lead compounds on the binding of OGT homosubstrate O-GlcNAc.

[0121] Thin-layer chromatography was used to separate the substrate O-GlcNAc, and the difference in O-GlcNAc recovery was compared to indicate the effect of the compound on the binding of OGT to the substrate O-GlcNAc.

[0122] (1) Material preparation

[0123] His-OGT (313-1031) protein, PBS buffer (Meilun), UDP-GlcNAc (Sigma, U4375), OMSI-4 (MCE#HY-114361), Zeba Spin Desalting Columns (Thermo Fisher Scientific #89883), centrifuge (Eppendorf, 5417R), freeze dryer (Alpha 3-4LSC basic), ethyl acetate (Sinopharm #10009418), methanol (Sinopharm #10014159), petroleum ether (Sinopharm #10015218), dichloromethane (Sinopharm #80047318), thin-layer chromatography silica gel plates (Bailingwei #956419).

[0124] (2) Operation steps

[0125] ① Add 17.67 μL of 283 μM OGT protein to a 100 μL reaction mixture. Then add 1.25 μL of 20 mM compound B (CAS No.: 2645-32-1) (Experimental Group 1, i.e., EG1) and OMSI-4 (Experimental Group 2, i.e., EG2), respectively, to achieve a working protein concentration of 50 μM and a working compound concentration of 250 μM. After thorough mixing, incubate at room temperature for 30 min. Set up the NCC group (17.67 μL OGT protein solution + 1.25 μL DMSO + 81.08 μL PBS buffer).

[0126] ② After incubation, add 0.5 μL of 10 mM UDP-GlcNAc solution (working concentration 50 μM) to the system, mix well, and incubate at room temperature for 30 min. Take 10 μL of the reaction solution from each group as an "input" control. Centrifuge the remaining reaction solution, desalt, denature, and freeze-dry the supernatant.

[0127] ③ Redissolve the dried sample in 10 μL of ethyl acetate and spot the samples onto the same silica gel plate. The standard for compound B (cpd1), experimental group 1 (EG1, components: compound B, OGT, UDP-GlcNAc), OMSI-4 standard (cpd2), experimental group 2 (EG2, components: OMSI-4, OGT, UDP-GlcNAc), UDP-GlcNAc standard, and the NCC group are all spotted. First, develop the sample using the developing solvent corresponding to the substrate UDP-GlcNAc. After development, observe the color development of the experimental group UDP-GlcNAc under UV light and record the results by taking photos.

[0128] ④ Develop the samples using compound B and the corresponding developing solvent for OMSI-4. After development, observe the color development of the experimental group compounds under UV light. To ensure that the initial amount of OGT protein in each group is consistent, add an equal volume of loading buffer to the 10 μL "input" control samples reserved from each group, and develop the protein samples using polyacrylamide gel electrophoresis. After electrophoresis, stain with Coomassie Brilliant Blue.

[0129] (3) Analysis of experimental results

[0130] Literature reports that the OGT inhibitor OMSI-4 binds to the same region of OGT as its substrate UDP-GlcNAc, thus competing with UDP-GlcNAc for binding. As predicted, the addition of the positive compound OMSI-4 reduced the binding amount of the substrate UDP-GlcNAc, while OMSI-4 itself was detected as a binding compound to the OGT protein. Figure 8 Similarly, this experiment demonstrated that the lead compound B (CAS No.: 2645-32-1) also inhibited the binding of OGT to the substrate UDP-GlcNAc. This is consistent with the principle of classic biochemical experiments—the binding of an active compound to a protein leads to a reduction in the binding of the corresponding substrate analog fluorescent probe, thereby causing a change in the detected fluorescence signal value.

[0131] In this method, there is no need to design synthetic substrate analogs; thin-layer chromatography can directly detect compounds and substrates, and concisely determine the binding inhibition / competition of compounds to natural substrates. Simultaneously, as... Figure 8 As shown, by performing two developments, the compound and the substrate are effectively separated, avoiding the problem of fluorescence interference that may occur when all components of the system are detected together in biochemical experiments.

[0132] Example 6: Detection of OGT binding to peptides using thin-layer chromatography

[0133] Thin-layer chromatography was used to separate and visualize peptides, and the difference in the amount of recovered peptides was compared to reflect the binding of OGT to peptides.

[0134] (1) Materials

[0135] His-OGT (313-1031) protein and peptide (Genscript), PBS buffer (Merlen), centrifuge (Eppendorf, 5417R), Zeba Spin Desalting Columns (Thermo Fisher Scientific #89883), freeze dryer (Alpha 3-4 LSC basic), ethyl acetate (Sinopharm #10009418), methanol (Sinopharm #10014159), petroleum ether (Sinopharm #10015218), dichloromethane (Sinopharm #80047318), iodine (Maclean's #C13454535), thin-layer chromatography silica gel plates (Bailingwei #956419).

[0136] (2) Operation steps

[0137] ① Dilute His-OGT protein to 50 μM with 1x PBS, and dilute the peptide (SEQ ID No.:1: STPVSRANMK, with biotin modification) to 200 μM. Mix the protein and peptide in equal volumes (20 μL + 20 μL) as the experimental group. Set up a protein control group, i.e., mix PBS and protein in equal volumes (20 μL + 20 μL). Set up a peptide control group, i.e., mix PBS and peptide in equal volumes (20 μL + 20 μL). Take two 5 μL samples from each of the above groups. One sample is used as the "pre-desalting" sample group, and the other is used as the "OGT protein input group" (to verify that the amount of protein added in the experimental group, protein control group, and peptide control group is accurate). Incubate the remaining 30 μL solution at room temperature for 20 min.

[0138] ② Equilibrate Zeba Spin Desalting Columns with 1x PBS solution. Add 300 μL of 1x PBS solution to the desalting column, centrifuge at 4°C, 1000g for 1 min, discard the effluent, and repeat four times. Add 30 μL of the incubated sample solution from each group to the desalting column equilibrated with 1x PBS, centrifuge at 4°C, 1000g for 1 min, and discard the desalting column. Heat the effluent from each group to 100°C in a metal bath and maintain for 3 min to fully denature and precipitate the protein. Centrifuge the denatured protein solution at 4°C, 13000 rpm for 10 min, and collect the supernatant.

[0139] ③ Apply the obtained supernatant to a silica gel plate using a pipette, with a sample volume of 0.5 μL each time. After each application, dry the solvent (water) with a hot air gun. Repeat the application process, with a total sample volume of 2.5 μL. Develop the sample using solvent #5 (dichloromethane:methanol:water = 3:2:0.5). After development, dry the solvent, immerse the silica gel plate in iodine-silica gel powder for 10 seconds, and then observe the color development. For the "pre-desalting" sample, follow the same steps as above, denature the protein, collect the supernatant, and complete the thin-layer chromatography experiment. For the "OGT protein input group" sample, add an equal volume of loading buffer, complete the SDS-PAGE experiment, and stain with Coomassie Brilliant Blue staining solution.

[0140] (3) Analysis of experimental results

[0141] like Figure 9 As shown, both the "experimental group" and the "peptide control group" exhibited distinct bands corresponding to the peptides before desalting, with comparable color development, indicating that the amount of peptide added in both groups was similar. Due to the presence of the protein-peptide complex, the amount of peptide retained in the "experimental group" was significantly greater than that in the "peptide control group" after the desalting step, providing a sufficient detection window to verify the effect of the compound on protein-peptide binding. Thin-layer chromatography (TLC) detection eliminates the need to design and synthesize peptides with tags or fluorescent probes; peptides can generally be developed using iodine or potassium permanganate solutions, saving costs and reducing experimental limitations.

Claims

1. A high-throughput screening method for compounds based on thin-layer chromatography, the method comprising: 1) Multiple compounds from the compound library are evenly grouped, and the stock solutions of these compounds are stored in multi-well plates, with each well containing one compound at a concentration of 1 mM to 10 mM. Each group of compounds is then mixed using the following two steps: Step 1.1: Merge by row A multi-channel gun is used to extract a certain volume from each hole in each row of a perforated plate and combine them into a single row. Step 1.2: Merge by column Take a certain volume from each well in each column of the compounds merged in step 1.1 and merge them into one column. After two merging processes, each resulting compound mixture contains m*n compounds, where m is the number of rows merged and n is the number of columns merged, with m ranging from 8 to 96 and n ranging from 5 to 10. 2). Dilute and mix the target protein and the mixture of compounds obtained in step 1) with the reaction solution, incubate, and then perform desalting to separate the protein-compound complex; 3) Denature the protein using a protein denaturation method to release the lead compounds in the complex, and then freeze-dry it; 4) Detection of lead compounds using thin-layer chromatography: The freeze-dried sample from step 3) and the m*n compounds obtained in step 1) were dissolved in ethyl acetate and spotted onto the same silica gel plate. After spotting, the ethyl acetate was allowed to evaporate completely. The compounds were then developed sequentially with the developing solvent until the ratio shift of the lead compound was between 0.2 and 0.

8. This determined which column of the original compound master plate the lead compound belonged to. Then, the m compounds in that column were dissolved in ethyl acetate and spotted sequentially together with the lead compound until the ratio shift of the lead compound was between 0.2 and 0.

8. This determined which row of the original compound master plate the lead compound belonged to. 5) Determine the information of the lead compound based on the electronic file of the original compound master plate, wherein the electronic file of the original compound master plate is the information recorded when the compound library was established, which corresponds one-to-one with the position of each compound in the storage container.

2. The method according to claim 1, wherein, In step 1), m is 16 and n is 10, and 160 compounds are mixed using the following two-step method: the 160 compounds correspond to the total number of compounds in two 96-well plates in the compound library, with 80 compounds in each plate. First, they are merged by row. 1 μL of compound is taken from each well in each row of the two plates using a 10-channel pipette and merged into one row. At this point, each well in the merged plate contains 16 compounds. Then, the compounds were merged by column. 0.5 μL of each well in each column of the compound obtained in the previous step was taken and merged into one column. After two merges, a mixture of 160 compounds was obtained.

3. The method according to claim 1, wherein, In step 1), the plurality of compounds are dissolved in dimethyl sulfoxide (DMSO) before mixing. After mixing, DMSO is removed using a freeze dryer, and a small amount of DMSO is added to redissolve the compounds. The volume of DMSO added when redissolving the compound sample is between 1% and 12% of the total volume of the reaction solution described in step 2).

4. The method according to claim 3, wherein, Taking into account the target protein's tolerance to dimethyl sulfoxide, the volume of dimethyl sulfoxide added when redissolving the compound sample is 10% of the total volume of the reaction solution described in step 2).

5. The method according to claim 1, wherein, In step 2), the target protein is a protein molecule that is closely related to the occurrence and development of the disease and has drug targeting significance.

6. The method according to claim 5, wherein, The protein molecule is a full-length or truncated somatic protein expressed by eukaryotic or prokaryotic cells.

7. The method according to claim 2, wherein, In step 4), the compound is developed sequentially using the following developing solvents, from low polarity to high polarity, numbered 1 to 5:

8. The method according to claim 7, wherein, In step 4), the freeze-dried sample and the 160 compounds from the first round of mixing in step 1) were dissolved in 5 μL of ethyl acetate and spotted onto the same silica gel plate. After spotting, the ethyl acetate was allowed to evaporate completely. The compounds were then developed sequentially with five polar gradient developing solvents from #1 to #5 until the ripple shift value of the "leading compound" was between 0.2 and 0.

8. This determined which group the "leading compound" belonged to, i.e., which column of the original compound master plate it belonged to. Then, the 16 compounds in that column were dissolved in ethyl acetate and spotted sequentially together with the leading compound until the ripple shift value of the "leading compound" was between 0.2 and 0.

8. This determined which group the "leading compound" belonged to, i.e., which row of the original compound master plate it belonged to.

9. The method according to claim 1, wherein, In step 5), the information includes the CAS number, structure, molecular formula, and relative molecular mass of each compound.

10. A method for verifying the effect of a seed compound screened according to claim 1 on the enzymatic activity reaction process of a protease, the method comprising the following steps: 2.1: Dilute the test protease, the seed compound, and DMSO (corresponding volume to the stock solution of the seed compound) with the reaction solution to obtain the protease solution, the seed compound solution, and the DMSO solution, respectively. Mix the diluted protease solution and the seed compound solution as the experimental group. Corresponding to the volume of the experimental group, the diluted protease solution and the DMSO solution were mixed to form the NCC (no compound control) group; corresponding to the volume of the experimental group, the blank reaction solution was mixed with the diluted DMSO solution to form the NPC (no protein control) group. 2.2: Add the corresponding substrate diluted with the reaction solution to the mixed solution of the experimental group, NCC group and NPC group in step 2.1 to start the catalytic reaction; 2.3: Heating the solution from step 2.2 terminates the catalytic reaction; 2.4: Thin-layer chromatography was used to analyze the samples taken from step 2.

3. The samples taken from step 2.3 were spotted on a silica gel plate, and after development with the developing solvent, the differences in substrate consumption / product generation between the experimental group, the NCC group, and the NPC group were observed to determine the inhibitory / stimulatory effect of the compound on protease activity.

11. A method for verifying the effect of a seed compound screened according to claim 1 on the binding of a protein to a small molecule substrate or peptide, the method comprising the following steps: 3.1: The protein diluted with the reaction solution and the precursor compound were mixed evenly and incubated for a certain period of time to form the experimental group. At the same time, a control group was set up. The control group was different from the experimental group only in that it did not contain the precursor compound. 3.2: Add the small molecule substrate or polypeptide corresponding to the protein diluted with the reaction solution to the mixed solution of the experimental group and the control group obtained in step 3.1, and desalt after incubation for a certain period of time; 3.3: Denatured proteins, the desalted effluent was freeze-dried; 3.4: Thin-layer chromatography was used to analyze the sample from step 3.

3. The sample from step 3.3 was redissolved in solvent and spotted onto a silica gel plate. After development with a developing solvent, the changes in the substrate / peptide recovery in the experimental and control groups were observed to determine the effect of the compound on the binding of the protein to the small molecule substrate or peptide.

12. The method according to claim 10 or 11, wherein in step 2.4 or step 3.4, the following developing agents of different polarities, from #1 to #5, are used sequentially:

Images