Affinity capillary electrochromatography-based effective mobility ratio screening method and applications

Abstract

The present application relates to the technical field of drug screening, in particular to an effective mobility ratio screening method based on affinity capillary electrochromatography and application. A target protein is fixed by a MOF material, and a capillary electrochromatography column is prepared by in-situ growth method; a to-be-tested substance is passed through the column, and screening is realized according to the affinity between the to-be-screened component and the protein; the present application adopts effective mobility ratio as a quantitative screening strategy, and the binding affinity between the to-be-screened component and the target protein is directly and quantitatively represented by calculating the effective mobility ratio of the drug in the column containing the fixed phase and the empty capillary column, so that efficient, stable and sensitive screening of multiple active ingredients in a complex system is realized, the stability and reusability of the affinity capillary electrochromatography platform are improved, the drug screening strategy is improved, and the present application is expected to become an effective and reliable strategy for screening of thrombin inhibitors in natural products.

Description

Technical Field

[0001] This invention relates to the field of drug screening technology, specifically to a screening method and application based on the effective mobility ratio of affinity capillary electrochromatography. Background Technology

[0002] Thrombin (Thr) is a serine protease that plays a crucial role in the coagulation cascade and has become a key target in the development of antithrombotic drugs. Inhibiting thrombin activity is considered a highly effective treatment strategy for the prevention and treatment of thrombotic diseases. Currently, commonly used thrombin inhibitors such as dabigatran etexilate, argatroban, and bivalirudin suffer from drawbacks including high production costs, gastrointestinal toxicity, and the need for frequent monitoring of coagulation parameters. Currently, some natural components derived from blood-activating and stasis-removing plants and animals exhibit significant thrombin inhibitory activity. Techniques such as ultrafiltration centrifugation mass spectrometry, magnetic bead immobilization of thrombin, fluorescence and resonance light scattering of peptide microarray chips, and drug molecule docking have been used for the rapid detection and screening of thrombin and its inhibitors. However, given the complexity and diversity of biological samples and plant and animal components, these methods are cumbersome, costly, and difficult to widely apply. Currently, traditional affinity capillary electrophoresis based on the determination of the dissociation constant between drugs and proteins is one of the commonly used drug screening methods, which typically uses free-state target proteins for analysis. However, using free target proteins has significant drawbacks: First, this method relies on the binding-dissociation equilibrium between the protein and the drug, and the detection range of the dissociation constant is typically narrow (usually 10⁻³~10⁻). 5 The concentrations of free proteins in the thrombin are typically between mol·L⁻¹. For drugs with extremely low and extremely high affinity, it is difficult to establish detectable differences in migration rates, thus limiting the screening scope. Secondly, due to the lack of spatial constraints on proteins and drugs, the binding modes of different drugs to free proteins are small, resulting in insignificant differences in migration rates during electrophoresis, low peak capacity, and difficulty in effectively separating and distinguishing multiple active ingredients in complex systems. Furthermore, free proteins are susceptible to conformational denaturation or loss of activity due to fluctuations in environmental factors (such as pH and temperature), leading to poor reproducibility and stability of experimental results. Therefore, developing an efficient and sensitive method for screening thrombin inhibitors from natural products is an urgent and important task in disease diagnosis and drug evaluation. Summary of the Invention

[0003] To address the aforementioned issues, this invention provides an effective mobility ratio screening method and application based on affinity capillary electrochromatography, which accurately distinguishes the interaction strength of multiple components in complex systems, achieving efficient, stable, and sensitive screening of various active ingredients in complex systems.

[0004] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0005] On one hand, the present invention provides a method for screening effective mobility ratios based on affinity capillary electrochromatography, comprising the following steps:

[0006] S1. Chromatographic columns for high performance liquid chromatography, gas chromatography, or capillary electrochromatography are prepared using biomacromolecules @MOFs as the stationary phase via in-situ growth or direct packing methods.

[0007] S2. Pass the component to be screened through the chromatographic column and empty capillary column prepared in S1. Based on the effective mobility ratio Rem of the drug in the two columns, the binding affinity of the drug to thrombin is quantitatively characterized, thereby achieving the screening of candidate components.

[0008] The effective mobility ratio Rem is calculated using equation I:

[0009] Formula I

[0010] In the formula, Rem represents the effective mobility ratio; This indicates the effective mobility of the analyte on the chromatographic column; This indicates the effective mobility of the analyte on an empty capillary column.

[0011] Furthermore, in S1, the biological macromolecule is selected from one or more of proteins, enzymes, polypeptides, and nucleic acids, or from one of lipase, alkaline protease, human serum albumin (HSA), α-acid glycoprotein (AGP), ovomucoid (OVM), cellulase, glucosidase, and thrombin.

[0012] Furthermore, in S1, the MOFs are selected from ZIF-type, UiO-type, MIL-type, and HKUST-type.

[0013] Class, PCN-class or other metal-organic framework materials with microporous / mesoporous structures.

[0014] Furthermore, the preparation method of the chromatographic column in S1 includes the following steps:

[0015] S11. Rinse the activated uncoated fused silica capillary tube sequentially with NaOH, deionized water, HCl, and deionized water, then dry it with nitrogen and set aside.

[0016] S12. Inject the sulfur-containing amino acid solution into the capillary prepared in S11, introduce surfactant groups containing thiol, amino or carboxyl groups into the inner wall of the capillary, and then rinse with deionized water and dry with nitrogen.

[0017] S13. Continuously add a metal-organic ligand solution into the capillary tube prepared in S12, and seal both ends of the capillary tube.

[0018] The reaction was carried out in an oven at 100-120 ℃ for 7-9 h to allow meso-MOF-1 crystals to grow in situ on the inner wall of the capillary, thus obtaining meso-MOF-1@capillary column;

[0019] S14. Continuously inject a precursor solution containing biomacromolecules into the meso-MOF-1@capillary column, seal both ends of the capillary, and let it stand at room temperature and in the dark for 3-5 h to obtain the chromatographic column.

[0020] Furthermore, the sulfur-containing amino acid solution is selected from one of methionine, cysteine, homocysteine, and glutamic acid;

[0021] Furthermore, the metal-organic ligand solution is prepared by the following method: copper nitrate trihydrate in a mass ratio of (2-4):1 is thoroughly mixed with H3TATAB and dissolved in N,N-dimethylformamide to obtain a precursor solution.

[0022] Furthermore, in S2, the effective mobility value ( Calculate using the formulas shown in Equations II-III:

[0023] II

[0024] In the formula, μ eof Indicates electroosmotic mobility; μ ap Indicates the apparent migration rate of the analyte;

[0025] III

[0026] In the formula, L eff The effective length of the capillary is represented by V; the total length of the capillary is represented by L; the applied voltage is represented by V; and the migration time of the electroosmotic marker is represented by t.

[0027] Secondly, the present invention provides a stationary phase for the above screening method, comprising MOFs and biomacromolecules, wherein the MOFs encapsulate the biomacromolecules in situ to form biomacromolecules@MOFs;

[0028] Biological macromolecules are selected from one or more of proteins, enzymes, polypeptides, and nucleic acids, or from one of lipases, alkaline proteases, human serum albumin (HSA), α-acid glycoprotein (AGP), ovomucoid (OVM), cellulase, glucosidase, and thrombin; and / or,

[0029] The MOFs are selected from ZIF-class, UiO-class, MIL-class, HKUST-class, PCN-class, or other types.

[0030] Metal-organic framework materials with microporous / mesoporous structures.

[0031] Thirdly, the present invention provides a chromatographic column for the above-described screening method, comprising the above-described stationary phase.

[0032] Fourthly, the present invention provides a method for preparing the above-mentioned chromatographic column, comprising the following steps:

[0033] S11. Rinse the activated uncoated fused silica capillary tube sequentially with NaOH, deionized water, HCl, and deionized water, then dry it with nitrogen and set aside.

[0034] S12. Inject the sulfur-containing amino acid solution into the capillary prepared in S11, introduce surfactant groups containing thiol, amino or carboxyl groups into the inner wall of the capillary, and then rinse with deionized water and dry with nitrogen.

[0035] S13. Continuously add a metal-organic ligand solution into the capillary tube prepared in S12, and seal both ends of the capillary tube.

[0036] The reaction was carried out in an oven at 100-120 ℃ for 7-9 h to allow meso-MOF-1 crystals to grow in situ on the inner wall of the capillary, thus obtaining meso-MOF-1@capillary column;

[0037] S14. Continuously inject a precursor solution containing biomacromolecules into the meso-MOF-1@capillary column, seal both ends of the capillary, and let it stand at room temperature and in the dark for 3-5 h to obtain the chromatographic column.

[0038] Furthermore, the sulfur-containing amino acid solution is selected from one of methionine, cysteine, homocysteine, and glutamic acid;

[0039] Further, the metal-organic ligand solution is prepared by the following method: copper nitrate trihydrate and H3TATAB are thoroughly mixed at a mass ratio of (2-4):1 and dissolved in N,N-dimethylformamide.

[0040] The precursor solution was obtained.

[0041] Fifthly, the application of the aforementioned stationary phase and chromatographic column in affinity-based drug screening, target protein binding studies, inhibitor screening, ligand library screening, or bioactivity assessment also falls within the scope of protection of this invention.

[0042] Compared with the prior art, the present invention has the following beneficial effects:

[0043] (1) In this invention, meso-MOF-1 is selected as the thrombin immobilization carrier. Its mesoporous structure is highly matched with the size of thrombin molecules, which can form a precise spatial confinement effect, effectively protecting the conformation and activity of biological macromolecules and making them less susceptible to denaturation due to environmental factors.

[0044] (2) By combining MOF cage confinement technology with open-tube capillary electrochromatography, a chromatographic column containing biomacromolecules@MOFs stationary phase is prepared, which realizes high loading capacity, high activity retention and uniform fixation of biomacromolecules. Its separation performance is significantly better than that of traditional capillary columns. It can produce a size-based sieving effect on the components to be sieved. Combined with the differential affinity of biomacromolecules for different components, the retention behavior of different components in the column is significantly different, thereby greatly improving the separation ability and peak capacity, and can effectively separate multiple active components in complex systems.

[0045] (3) This invention uses the effective mobility ratio (Rem) as a quantitative screening strategy. By calculating the effective mobility ratio of the component to be screened in a chromatographic column containing a stationary phase and an empty capillary column, the binding affinity of the drug to the biomolecule is directly and quantitatively characterized. This ratio is not affected by peak overlap or migration time fluctuations in a single electrophoresis pattern, and can accurately distinguish the differences in binding strength of different components, solving the problem that traditional ACE technology cannot accurately distinguish the intensity of multi-component interactions in complex systems;

[0046] (4) This invention has high screening efficiency, wide applicability, excellent stability and repeatability, broadens the detection range of drug-protein interactions, and realizes accurate differentiation and sorting of the affinity of multiple active ingredients to proteins in complex systems; it improves the stability and reusability of the affinity capillary electrochromatography platform, reduces drug screening costs, and is expected to become an effective and reliable strategy for screening thrombin inhibitors in natural products. Attached Figure Description

[0047] Figure 1 The images show the electrophoretic patterns of the three drugs in Example 1 on an empty capillary column, a Thr@ capillary column, a meso-MOF-1@ capillary column, and a Thr / meso-MOF-1@ capillary column, respectively; where 1 is benzalkonium hydrochloride; 2 is argatroban; and 3 is rutin.

[0048] Figure 2 The images show the electrophoretic patterns of the three drugs in Example 2 on an empty capillary column and a Thr / meso-MOF-1@capillary column, respectively; where 1 is benzalkonium hydrochloride; 2 is argatroban; and 3 is rutin.

[0049] Figure 3 The curves show the changes in the effective mobility ratio (Rem) of different active ingredients with concentration; where A is the curve of benzalkonium hydrochloride, B is the curve of argatroban, and C is the curve of rutin.

[0050] Figure 4The images show the electrophoretic patterns of the six drugs in Example 3 on an empty capillary column and a Thr / meso-MOF-1@capillary column, respectively; wherein, 1 is benzalkonium hydrochloride; 2 is argatroban; 3 is rutin; 4 is kaempferol; 5 is quercetin; and 6 is luteolin.

[0051] Figure 5 This is a schematic diagram of the binding model of thrombin with six components; where a is benzalkonium hydrochloride; b is argatroban; c is rutin; d is kaempferol; e is quercetin; and f is luteolin.

[0052] Figure 6 The effects of six drugs on plasma coagulation parameters are illustrated in Example 6. Detailed Implementation

[0053] To enable those skilled in the art to better understand the technical solutions of the present invention, the present invention will be described in detail below with reference to specific embodiments. Experimental methods in the following embodiments that do not specify specific conditions are generally performed under conventional conditions or as recommended by the manufacturer. Unless otherwise specified, the test materials used in the following embodiments were purchased from conventional biochemical reagent stores. Unless otherwise stated, percentages and parts are by weight. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those familiar with the art. Furthermore, any methods and materials similar to or equivalent to those described herein can be applied to the present invention. The preferred embodiments and materials described herein are for illustrative purposes only.

[0054] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0055] The inventors have optimized existing CEC drug screening methods, providing an effective mobility ratio (Rem) screening method based on affinity capillary electrochromatography. The core of this method lies in using biomacromolecules @MOFs as the stationary phase to prepare the chromatographic column and introducing the effective mobility ratio (Rem) as a quantitative screening index. This overcomes the problems of narrow detection range, insufficient separation capability for complex samples, and poor repeatability associated with ACE, thus broadening its application in the efficient screening of active ingredients from complex systems such as natural products.

[0056] In some specific embodiments, an effective mobility ratio screening method based on affinity capillary electrochromatography is provided, including the following steps:

[0057] S1. A chromatographic column for capillary chromatography is prepared using biomacromolecules @MOFs as the stationary phase via in-situ growth or direct packing.

[0058] S2. The drug to be tested is passed through the chromatographic column and empty capillary column prepared in S1. The binding affinity of the drug to thrombin is quantitatively characterized based on the effective mobility ratio Rem of the drug in the two columns, thereby achieving the screening of candidate drugs.

[0059] The effective mobility ratio Rem is calculated using equation I:

[0060] Formula I In the formula, R em Indicates the effective mobility ratio; This indicates the effective mobility of the analyte on the chromatographic column; This indicates the effective mobility of the analyte on an empty capillary column. The mesoporous structure of metal-organic frameworks (MOFs) allows for the in-situ encapsulation and confinement of biomolecules within their pores. This confinement effect maximizes the preservation of the native conformation and activity of the biomolecules, providing a stable and uniformly distributed interaction interface. When the component to be screened flows through this chromatographic column, it undergoes affinity interactions with the immobilized biomolecules. The stronger the interaction, the longer the retention time of the component within the column, and the higher its effective mobility value (…). Compared to the value in an empty capillary column ( The greater the decrease in the ratio of the component to be screened, the greater the Rem value becomes. Therefore, the Rem value can directly and quantitatively reflect the binding affinity between the component to be screened and the target biomacromolecule, and this ratio effectively avoids the errors caused by peak overlap or migration time fluctuations in a single electrophoresis pattern.

[0061] In some embodiments of this implementation, the biomacromolecule is selected from one or more of proteins, enzymes, peptides, and nucleic acids, or from one of lipases, alkaline proteases, human serum albumin (HSA), α-acid glycoprotein (AGP), ovomucoid (OVM), cellulase, glucosidase, and thrombin. When thrombin is selected as the biomacromolecule, it is a key target for the development of antithrombotic drugs as a serine protease. Immobilization of thrombin allows for rapid screening of thrombin inhibitors from complex natural product systems. The immobilization process benefits from the confinement protection of MOFs, resulting in high thrombin activity retention, thus ensuring the accuracy and reliability of the screening results. Besides thrombin, other types of biomacromolecules (such as HSA and AGP) can be selected to study the plasma protein binding rate of drugs or to screen inhibitors for different targets, broadening the applicability of this method.

[0062] In some embodiments of this implementation, the MOFs are selected from ZIF-type, UiO-type, MIL-type, HKUST-type, PCN-type, or other metal-organic framework materials with microporous / mesoporous structures; in some preferred embodiments, the MOFs are meso-MOF-1 with a regular mesoporous structure. meso-MOF-1 is composed of copper ions and the organic ligand H3TATAB, and its pore size matches the hydrodynamic diameter of most biomolecules (such as thrombin), enabling efficient loading and confinement of biomolecules. Its ultra-high specific surface area not only provides a huge immobilization capacity but also increases the theoretical plate number during capillary electrochromatographic separation and exerts a size-based sieving effect on drug molecules through its pores. Synergistically, its affinity for biomolecules enhances the separation performance of the chromatographic column.

[0063] This application also provides the stationary phase and chromatographic column used in the above screening method. The stationary phase includes MOFs and a biomacromolecule resolving agent; the MOFs encapsulate biomolecules in situ, forming biomacromolecules@MOFs. The chromatographic column containing this stationary phase is prepared by the following steps:

[0064] S11. Rinse the activated uncoated fused silica capillary tube sequentially with NaOH, deionized water, HCl, and deionized water, then dry it with nitrogen and set aside.

[0065] In some embodiments of this implementation, each solution is rinsed for a certain period of time, for example, NaOH is rinsed for 2 h, water is rinsed for 30 min, HCl is rinsed for 1 h, water is rinsed again for 30 min, and finally the capillary is purged with high-purity nitrogen for 10 min to dry it thoroughly, so as to obtain an activated bare capillary column. The purpose is to clean and activate the silanol groups on the inner wall of the capillary, so as to provide active sites for subsequent modification.

[0066] S12. Inject the sulfur-containing amino acid solution into the capillary prepared in S11, introduce surfactant groups containing thiol, amino or carboxyl groups into the inner wall of the capillary, and then rinse with deionized water and dry with nitrogen.

[0067] In some embodiments of this implementation, the sulfur-containing amino acid solution is selected from methionine, cysteine, homocysteine, and glutamic acid; specifically, for example, a 0.5 M cysteine ​​solution can be injected and reacted at room temperature for a certain period of time, so that cysteine ​​binds to the activated capillary inner wall through its thiol group, thereby introducing active sites on the surface that can be used for MOF crystal growth.

[0068] S13. Continuously add a metal-organic ligand solution into the capillary tube prepared in S12, and seal both ends of the capillary tube.

[0069] The reaction was carried out in an oven at 100-120 ℃ for 7-9 h to allow meso-MOF-1 crystals to grow in situ on the inner wall of the capillary, thus obtaining meso-MOF-1@capillary column;

[0070] In some embodiments of this implementation, the baking temperature can be selected as 100 ℃, 110 ℃, or 120 ℃; other specific values ​​within this range can also be selected, and will not be described in detail here; the reaction time can be selected as 7 h, 8 h, or 9 h; other specific values ​​within this range can also be selected, and will not be described in detail here.

[0071] In some embodiments of this implementation, the metal-organic ligand solution is prepared by the following method: copper nitrate trihydrate and H3TATAB are thoroughly mixed in a mass ratio of (2-4):1 and dissolved in N,N-dimethylformamide to obtain a precursor solution.

[0072] S14. Continuously inject a precursor solution containing biomacromolecules into the meso-MOF-1@capillary column, and seal the capillary.

[0073] The two ends of the tube are left to stand at room temperature and in the dark for 3-5 hours to obtain the chromatographic column.

[0074] In some embodiments of this implementation, the precursor solution for the biomacromolecule is a 30-50 μM thrombin phosphate buffer solution; the concentration of the thrombin phosphate buffer solution can be selected as 30 μM, 40 μM, or 50 μM; other specific values ​​within this range can also be selected, and will not be elaborated here; the thrombin is allowed to diffuse into the mesoporous cages of MOFs and be confined and fixed, and finally a chromatographic column packed with biomacromolecules@MOFs stationary phase is obtained, namely Thr / meso-MOF-1@capillary column.

[0075] In one specific embodiment, the effective mobility value involved in S2 ( Calculated using Equation II:

[0076] Formula II

[0077] In the formula, μ eof Indicates electroosmotic mobility; μ ap Indicates the apparent migration rate of the analyte;

[0078] Formula III

[0079] In the formula, L eff V represents the effective length of the capillary; L represents the total length of the capillary; V represents the applied voltage; t represents the migration time of the electroosmotic marker.

[0080] Finally, the drug was calculated on the Thr / meso-MOF-1@capillary column. Recorded as Calculated on an empty capillary column Recorded as Substituting into Equation I yields the effective flow rate for quantitative characterization of affinity.

[0081] Degree ratio Rem.

[0082] The technical solution of the present invention will be further described in detail below with reference to several preferred embodiments and accompanying drawings.

[0083] However, the described embodiments are merely some, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention. It should be understood that the following embodiments are merely illustrative of the present invention and are not intended to limit the present invention.

[0084] Example 1: Preparation of Thr / meso-MOF-1@capillary column

[0085] A fused silica capillary with a total length of 50 cm (effective length 41.5 cm) and an inner diameter of 75 μm was used. The empty capillary was then sequentially rinsed with 0.1 M sodium hydroxide solution for 2 h, ultrapure water for 30 min, and 0.1 M hydrochloric acid solution for 1 h, followed by rinsing with ultrapure water for 30 min. Finally, it was purged with nitrogen for 10 min and dried to obtain a bare capillary column for later use. The activated capillary was modified with cysteine ​​(Cys). Then, 25.85 mg of copper nitrate trihydrate (Cu(NO3)2·3H2O) and 10.0 mg of H3TATAB were thoroughly mixed and dissolved in 3 mL of N,N-dimethylformamide (DMF) to prepare a precursor solution. The precursor solution was injected into the cysteine-modified capillary, and both ends of the capillary were sealed with heat-resistant rubber stoppers. The capillary was then placed in a 120 ℃ oven for 8 hours. h, allowing meso-MOF-1 crystals to grow in situ on the inner wall of the capillary; after the reaction, the capillary was rinsed with methanol for 30 min and dried with nitrogen to obtain a meso-MOF-1@capillary column. 40 μM thrombin solution was injected into the meso-MOF-1@capillary column using a syringe, and both ends of the capillary were sealed with sealing film. The column was then allowed to stand at room temperature in the dark for 4 h to finally obtain the Thr / meso-MOF-1@capillary column.

[0086] Example 2: Investigating the separation performance of Thr / meso-MOF-1@capillary column

[0087] Using benzalkonium hydrochloride, argatroban, and rutin as test subjects, electrophoretic analysis was performed on Thr / meso-MOF-1@capillary column (from Example 1), empty capillary column (Bare@capillary), capillary column with only thrombin immobilization (Thr@capillary), and capillary column with only meso-MOF-1 growth (meso-MOF-1@capillary). The results are as follows: Figure 1 As shown in Table 1.

[0088] Electrophoresis conditions: 100 mbar, 5 s, 20 kV

[0089] Table 1. Resolution of the three drugs on different capillary columns (Ri) s )data

[0090] Bare@capillary 10.14 12 Thr@capillary 12.43 13.69 meso-MOF-1@capillary 29.33 13.76 Thr / meso-MOF-1@capillary 31.11 14.57

[0091] The difference in drug migration time reflects the separation ability of each capillary column. Figure 1 Electrophoretic patterns showed that the drug migration time difference was smallest in the empty capillary column; compared with the empty capillary column, the drug migration time difference was increased in both the meso-MOF-1@capillary column and the Thr@capillary column, while the largest drug migration time difference was observed in the Thr / meso-MOF-1@capillary column.

[0092] Table 1 shows the separation efficiency (R) of benzalkonium hydrochloride, argatroban, and rutin on four different capillary columns. s The data shows that the empty capillary column has the worst separation performance, with R0... s1 =10.14、R s2 =12.00; Thr@ capillary column separation performance improved, R s1 =12.43、R s2 =13.69, improved resolution and better peak shape; the separation performance of the meso-MOF-1@capillary column is significantly better than that of the empty capillary column, R s1 =29.33、R s2 =13.76. The Thr / meso-MOF-1@capillary column exhibits the best separation performance, with R0 = 13.76. s1 =31.11、R s2=14.57. This improved separation performance is attributed to the abundant mesoporous structure and large specific surface area of ​​meso-MOF-1. Immobilizing it on a capillary column increases the theoretical plate number during the separation process. The mesoporous structure also facilitates size-based sieving of drug molecules. Furthermore, thrombin exhibits varying affinities for different drugs, leading to interactions of varying strengths between different drugs and the capillary column. This indicates that Thr / meso-MOF-1@capillary column achieves optimal separation performance.

[0093] Example 3: Screening of active ingredients based on effective mobility ratio (Rem)

[0094] Three active ingredients, benzalkonium hydrochloride, argatroban, and rutin, were prepared into a mixed sample and analyzed by electrophoresis on an empty capillary column and a Thr / meso-MOF-1@capillary column, respectively. The effective mobility values ​​of each component in the two columns were calculated. and the effective mobility ratio Rem. The results are as follows: Figure 2 As shown in Table 2.

[0095] Electrophoresis conditions: 100 mbar, 5 s, 20 kV

[0096] Table 2 Effective mobility values and effective mobility ratio Rem

[0097]

[0098] Note: This indicates the effective mobility of the analyte on the Thr / meso-MOF-1@capillary column; Rem represents the effective mobility of the analyte on an empty capillary column; Rem represents the ratio of the effective mobility of the analyte on a Thr / meso-MOF-1@capillary column to its effective mobility on an empty capillary column.

[0099] like Figure 2 As shown in Table 2, compared with the empty capillary column, the effective mobility of the three active substances in the Thr / meso-MOF-1@capillary column decreased to varying degrees: the effective mobility of benzalkonium hydrochloride decreased from 1.596 to 1.427, argatroban from -0.159 to -0.144, and rutin from -1.125 to -0.785. This is because the thrombin immobilized on the inner wall of the Thr / meso-MOF-1@capillary column exhibited a specific affinity interaction with the active substances, which led to changes in their effective mobility, and the degree of interaction varied among different components.

[0100] The effective mobility ratio (Rem) for benzalkonium hydrochloride was 0.894, for argatroban it was 0.907, and for rutin it was 0.697. These results indicate that argatroban has the strongest interaction with thrombin, followed by benzalkonium hydrochloride, while rutin has the weakest interaction with thrombin. This ranking is consistent with known pharmacological activities. It is evident that the stronger the binding interaction, the higher the proportion of the active ingredient forming a complex with thrombin within the column, and the longer the retention time, thus increasing the effective mobility ratio (Rem), indicating a stronger binding interaction between the active ingredient and thrombin. This demonstrates that the introduction of the effective mobility ratio (Rem) in this invention can enable the evaluation and screening of the interaction strength between different types of active ingredients and thrombin. Furthermore, the effective mobility ratio (Rem) parameter avoids errors caused by peak overlap and migration time fluctuations in single electrophoresis patterns, accurately characterizing the binding strength between the drug and the target.

[0101] Figure 3 The effect of active ingredient concentration on the effective mobility ratio (Rem) was further investigated. The results showed that within the concentration range of 0–1000 μM, the Rem values ​​of the three components fluctuated significantly with concentration, but their relative order remained unchanged. Within the concentration range of 1000–5000 μM, the effective mobility ratio (Rem) did not fluctuate significantly with concentration: benzalkonium hydrochloride remained stable between 0.88 and 0.90, argatroban remained stable between 0.90 and 0.92, and rutin remained stable between 0.69 and 0.70. These results confirm that even in mixed systems with fluctuating active ingredient concentrations, the binding strength between the drug and thrombin can be accurately distinguished.

[0102] Example 4 Screening of complex multi-component systems

[0103] To further verify the applicability of the method in complex systems, a mixed sample containing six drugs—benzamidin hydrochloride, argatroban, rutin, kaempferol, quercetin, and luteolin—was prepared and analyzed by electrophoresis on an empty capillary column and a Thr / meso-MOF-1@capillary column. The effective mobility of each component in the two columns was calculated. and the effective mobility ratio Rem. The results are as follows: Figure 4 As shown in Table 3.

[0104] Table 3 Effective mobility ratios (Rem) of six drugs

[0105]

[0106] like Figure 4As shown in Table 3, the Rem value of argatroban was 0.738, and that of benzalkonium hydrochloride was 0.704, confirming that argatroban has a stronger binding affinity to thrombin than benzalkonium hydrochloride. The Rem values ​​of rutin (0.67), kaempferol (0.646), quercetin (0.658), and luteolin (0.685) indicate that flavonoids have relatively weaker binding affinity to thrombin. Among the four flavonoids, luteolin showed the highest affinity for thrombin, followed by rutin, then quercetin, with kaempferol showing the lowest affinity. The results show that the natural flavonoid products luteolin, rutin, quercetin, and kaempferol all have moderate binding affinity to thrombin.

[0107] Example 5 Molecular Dynamics Simulation

[0108] Spatial conformation simulation and prediction were performed on the binding modes between six candidate components and the thrombin active site. The desired protein structures were selected from the RCSB PDB database (PDB ID: 1AWH). Water molecules and irrelevant heteroatoms were removed using PyMOL software, retaining only the protein structure. The atomic charges of the proteins were calculated using AutoDock software, and hydrogen atoms were added to the proteins. The three-dimensional structures of small molecules were downloaded from the open-source PubChem database. Their conformations were optimized using Gaussian 16 software under a GAFF force field, and low-energy conformations were output and saved as PDB files. Molecular docking experiments were performed using AutoDock 4.2 software: the optimal binding site of the protein was predicted using PyMOL software, and conformation sampling and scoring were performed using a genetic algorithm. The protein was set as a semi-flexible entity, and the small molecule as a flexible entity, for semi-flexible docking. The conformations were ranked according to the docking scores, and the optimal conformation was selected. The results are shown below. Figure 5 As shown in Table 4.

[0109] Table 4 Combining free energy (ΔG) and dissociation constant (K) d )

[0110] ΔG, kcal / mol -8.38 -10.07 -7.71 -5.41 -7.54 -7.72 <![CDATA[K d ,μM]]> 0.72 0.042 2.25 18.18 2.96 2.2

[0111] Figure 5 Hydrogen bonds are represented by red dashed lines, amino acid residues by colored circles, van der Waals forces by light green, hydrogen bonds by green, and electrostatic interactions by orange. It can be seen that all six drugs can form stable complexes within the active pocket of thrombin. The formation of these complexes depends on non-covalent interactions such as hydrogen bonds, hydrophobic interactions, and electrostatic forces.

[0112] As shown in Table 4, the clinical drug argatroban had the lowest ΔG value (-10.07 kcal / mol). dThe value was 0.042 μM, indicating that it had the strongest affinity for thrombin; the ΔG value of benzalkonium hydrochloride was -8.38 kcal / mol, K d The value was 0.72 μM. In the flavonoid system, the ΔG value of luteolin was -7.72 kcal / mol, K... d The value was 2.2 μM, confirming its strongest binding activity with thrombin; the ΔG value of rutin was -7.71 kcal / mol, K d The value was 2.25 μM; the ΔG value of quercetin was -7.54 kcal / mol, K d The value was 2.96 μM; the ΔG value of kaempferol was -5.41 kcal / mol, K d The value was 18.18 μM, confirming that its binding to thrombin was the weakest. It is evident that the order of affinity of the six components to thrombin is completely consistent with the order of Rem values ​​obtained in Example 4, providing important theoretical support for the capillary electrochromatographic screening results and verifying the screening mechanism of the Thr / meso-MOF-1@capillary column system at the atomic level.

[0113] Example 6: In vitro anticoagulation validation of thrombin inhibitor screening

[0114] Blood was collected from the marginal ear artery of healthy male New Zealand white rabbits and transferred to centrifuge tubes containing 3.8% sodium citrate (anticoagulant to blood volume ratio 1:9). After thorough mixing, the blood was centrifuged at 3500 rpm for 15 min to obtain plasma. 495 μL of plasma samples were then mixed with 5 μL each of benzalkonium hydrochloride, argatroban, rutin, quercetin, kaempferol, and luteolin, and incubated at 37 ℃ for 3 min. Activated thromboplastin time (APTT), prothrombin time (PT), and thrombin time (TT) were measured using a STA Compact-Max coagulation analyzer to assess the anticoagulant activity of the drugs.

[0115] like Figure 6 As shown, the positive control drug argatroban (0.05 μg / mL) exhibited significantly prolonged APTT (51.3 s), TT (31.7 s), and PT (28.9 s) values ​​compared to the control group. Benadimidine hydrochloride (0.5 μg / mL) also showed significantly prolonged APTT values ​​compared to the control group, indicating its anticoagulant activity is similar to argatroban. The flavonoid active ingredients (rutin, quercetin, kaempferol, and luteolin) at 50 μg / mL all had APTT values ​​higher than 40 s, and PT and TT values ​​higher than 20 s. These results indicate that all four flavonoid compounds possess anticoagulant activity and could serve as candidates for drug development in the prevention of thrombosis.

[0116] Finally, it should be noted that the above description is only a preferred embodiment of the present invention. Those skilled in the art, under the guidance of the present invention, can make various similar representations without departing from the spirit and claims of the present invention, and such modifications all fall within the protection scope of the present invention.

Claims

1. A screening method for effective mobility ratio based on affinity capillary electrochromatography, characterized in that, Includes the following steps: S1. A chromatographic column for affinity capillary electrochromatography was prepared by in-situ growth method using biomacromolecules @MOFs as the stationary phase; S2. The component to be screened is passed through the chromatographic column and empty capillary column prepared in S1. The binding affinity of the component to be screened to thrombin is quantitatively characterized based on the effective mobility ratio Rem of the component in the two columns, thereby achieving the screening of candidate drugs. The effective mobility ratio Rem is calculated using equation I: Equation I In the formula, Rem represents the effective mobility ratio; This indicates the effective mobility of the analyte on the chromatographic column; This indicates the effective mobility of the analyte on an empty capillary column.

2. The effective mobility ratio screening method according to claim 1, characterized in that... In S1, the biological macromolecule is selected from one or more of proteins, enzymes, polypeptides, and nucleic acids, or from one of lipases, alkaline proteases, human serum albumin, α-acid glycoproteins, ovomucoid, cellulase, glucosidase, and thrombin; and / or, The MOFs are selected from ZIF-type, UiO-type, MIL-type, HKUST-type, PCN-type, or other metal-organic framework materials with microporous / mesoporous structures.

3. The effective mobility ratio screening method according to claim 1, characterized in that... The preparation method of the chromatographic column in S1 includes the following steps: S11. Rinse the activated uncoated fused silica capillary tube sequentially with NaOH, deionized water, HCl, and deionized water, then dry it with nitrogen and set aside. S12. Inject the sulfur-containing amino acid solution into the capillary prepared in S11, introduce surfactant groups containing thiol, amino or carboxyl groups into the inner wall of the capillary, and then rinse with deionized water and dry with nitrogen. S13. Continuously add a metal-organic ligand solution into the capillary tube prepared in S12, and seal both ends of the capillary tube. The reaction was carried out in an oven at 100-120 ℃ for 7-9 h to allow meso-MOF-1 crystals to grow in situ on the inner wall of the capillary, thus obtaining meso-MOF-1@capillary column; S14. Continuously inject a precursor solution containing biomacromolecules into the meso-MOF-1@capillary column, seal both ends of the capillary, and let it stand at room temperature and in the dark for 3-5 h to obtain the chromatographic column.

4. The effective mobility ratio screening method according to claim 3, characterized in that, The sulfur-containing amino acid solution is selected from one of methionine, cysteine, homocysteine, and glutamic acid; and / or, The metal-organic ligand solution was prepared by the following method: copper nitrate trihydrate with a mass ratio of (2-4):1 was thoroughly mixed with H3TATAB and dissolved in N,N-dimethylformamide to obtain a precursor solution.

5. The effective mobility ratio screening method according to claim 3, characterized in that... In S2, Effective mobility value Calculate using the formula shown in Equation II-III: II In the formula, μ eof Indicates electroosmotic mobility; μ ap Indicates the apparent migration rate of the analyte; III In the formula, L eff The effective length of the capillary is represented by V; the total length of the capillary is represented by L; the applied voltage is represented by V; and the migration time of the electroosmotic marker is represented by t.

6. A stationary phase for use in the method of claim 1, characterized in that, Including MOFs and biomacromolecules, MOFs encapsulate biomacromolecules in situ to form biomacromolecule@MOFs; Biological macromolecules are selected from one or more of proteins, enzymes, polypeptides, and nucleic acids, or from one of lipases, alkaline proteases, human serum albumin, α-acid glycoproteins, ovomucoid, cellulase, glucosidase, and thrombin; and / or, The MOFs are selected from ZIF-class, UiO-class, MIL-class, HKUST-class, PCN-class, or other types. Metal-organic framework materials with microporous / mesoporous structures.

7. A chromatographic column, characterized in that: It includes the stationary phase as described in claim 6.

8. The method for preparing the chromatographic column according to claim 7, characterized in that, Includes the following steps: S11. Rinse the activated uncoated fused silica capillary tube sequentially with NaOH, deionized water, HCl, and deionized water, then dry it with nitrogen and set aside. S12. Inject the sulfur-containing amino acid solution into the capillary prepared in S11, introduce surfactant groups containing thiol, amino or carboxyl groups into the inner wall of the capillary, and then rinse with deionized water and dry with nitrogen. S13. Continuously add a metal-organic ligand solution into the capillary tube prepared in S12, and seal both ends of the capillary tube. The reaction was carried out in an oven at 100-120 ℃ for 7-9 h to allow meso-MOF-1 crystals to grow in situ on the inner wall of the capillary, thus obtaining meso-MOF-1@capillary column; S14. Continuously inject a precursor solution containing biomacromolecules into the meso-MOF-1@capillary column, seal both ends of the capillary, and let it stand at room temperature and in the dark for 3-5 h to obtain the chromatographic column.

9. The preparation method according to claim 8, characterized in that, The sulfur-containing amino acid solution is selected from one of methionine, cysteine, homocysteine, and glutamic acid; and / or, The metal-organic ligand solution was prepared by the following method: copper nitrate trihydrate with a mass ratio of (2-4):1 was thoroughly mixed with H3TATAB and dissolved in N,N-dimethylformamide to obtain a precursor solution.

10. The application of the stationary phase of claim 7 and the chromatographic column of claim 8 in affinity-based drug screening, target protein binding studies, inhibitor screening, ligand library screening, or bioactivity assessment.

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