Screening method of targeted drug for inhibiting accumulation of trimethylamine and / or trimethylamine-n-oxide

Abstract

The invention provides a screening method of a targeted drug for inhibiting accumulation of trimethylamine and / or trimethylamine-N-oxide. The screening method comprises the following steps: determining an action target on a metabolic pathway of trimethylamine and / or trimethylamine-N-oxide; if the target is protein, preliminarily screening candidate components based on the three-dimensional structure of the target; the candidate component is validated to confirm the targeted drug. According to the method, large-scale screening of targeted drugs aiming at targets can be achieved, the targeted drugs inhibit accumulation of trimethylamine and / or trimethylamine-N-oxide in the body by influencing the metabolic pathway of trimethylamine and / or trimethylamine-N-oxide, side effects are small, and the normal metabolic process of the human body is prevented from being influenced.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of medical microbiology technology, in particular, the present application relates to a screening method for a target drug for inhibiting trimethylamine and / or trimethylamine-N-oxide accumulation. BACKGROUND

[0002] Trimethylamine (TMA) comes from certain foods (such as fish, meat and eggs) or foods containing TMA precursors, such as trimethylamine-N-oxide (TMAO), choline and L-carnitine, TMA is a biological metabolite; Trimethylamine N-oxide (TMAO) is a derivative of TMA, which is oxidized by flavin-containing monooxygenase 3 (FMO3) in the liver, and is usually an intermediate product in the metabolism of trimethylamine.

[0003] The human gut microbiota can metabolize dietary precursors into trimethylamine (TMA) through specific enzyme complexes (such as CutC / D, CntA / B and YesW / X). Among them, there are mainly two TMA synthesis pathways in bacteria, one of which takes choline as the substrate and is catalyzed by CutC / CutD enzyme complex, and this enzyme is mainly distributed in the phyla of Proteobacteria, Firmicutes and Actinobacteria; The other takes L-carnitine as the substrate and is mediated by the two-component Rieske-type oxygenase / reductase CntA / B, and this enzyme mainly exists in the phylum of Proteobacteria.

[0004] Both TMA and TMAO are closely related to intestinal microorganisms and human metabolism, and their excess may have a negative impact on health. Abnormal accumulation and excretion of TMA in the body can cause trimethylamineuria (TMAU), also known as fishy syndrome or old fish syndrome, and its pathogenesis is usually flavin-containing monooxygenase 3 (FMO3) deficiency or dysfunction. As a metabolite of TMA, the abnormal accumulation of TMAO is associated with a variety of health problems, especially cardiovascular disease and kidney disease. Elevated circulating levels of TMAO are associated with increased risk of cardiovascular disease (CVD) and mortality, and elevated levels of TMAO in the blood promote the accumulation of lipids in the arteries, leading to atherosclerosis, which has been identified as an independent predictor and initiator of atherosclerosis. In addition, TMAO is a potential biomarker for chronic kidney disease.

[0005] Currently, broad-spectrum antibiotic intervention in the TMA production pathway can effectively reduce the level of TMAO and inhibit the development of cardiovascular diseases such as atherosclerosis in the short term. However, long-term use of antibiotics can lead to drug resistance, disrupt the balance of intestinal flora, and even cause side effects, and the level of TMAO may recover after drug withdrawal. Therefore, broad-spectrum antibiotics are feasible as a short-term intervention means, but there are obvious limitations as a long-term treatment plan, and should be used with caution. SUMMARY

[0006] The present application is directed to the shortcomings of the prior art, and proposes a screening method for targeted drugs for inhibiting trimethylamine and / or trimethylamine-N-oxide accumulation, to solve the technical problem of the lack of targeted drugs for inhibiting trimethylamine and / or trimethylamine-N-oxide accumulation in the related art.

[0007] The present application provides a screening method for targeted drugs for inhibiting trimethylamine and / or trimethylamine-N-oxide accumulation, which comprises the following steps: determining an action target on the metabolic pathway of trimethylamine and / or trimethylamine-N-oxide; if the target is a protein, screening candidate components based on the three-dimensional structure of the target; verifying the candidate component to confirm the targeted drug.

[0008] Alternatively, the three-dimensional structure of the target includes the protein crystal structure of the target and / or the protein crystal structure of the ligand of the target.

[0009] Further, the screening of candidate components based on the three-dimensional structure of the target includes detecting the interaction of the candidate components with the target by molecular docking and binding mode.

[0010] Further, the interaction of the candidate components with the individual protein crystal structure is detected by molecular docking and binding mode, respectively.

[0011] Alternatively, the target includes an enzyme complex of intestinal microorganisms metabolizing trimethylamine and / or trimethylamine-N-oxide.

[0012] Further alternatively, the enzyme complex includes at least one of CutC / D, CntA / B and YesW / X; and the candidate components include flavonoid glycosides, isoflavones and / or santalane components.

[0013] Alternatively, the flavonoid glycoside component targeting the CntA / B enzyme complex includes at least one of daidzein, genistin and glycitein; the isoflavone component targeting the CntA / B enzyme complex includes at least one of 7-hydroxyisoflavone, biochanin, apigenin, formononetin, genistein and daidzein; the santalane component targeting the CntA / B enzyme complex includes maackiain.

[0014] Further, the targeted drug is used for inhibiting the conversion of L-carnitine into trimethylamine, and / or inhibiting the conversion of trimethylamine into trimethylamine-N-oxide.

[0015] Still further, the verification of the candidate component includes detecting the ex vivo activity of the candidate component, and verifying the in vitro and / or in vivo physiological effect of the candidate component.

[0016] Alternatively, the targeted drug is used for intervening in cardiovascular diseases, kidney diseases and / or liver diseases.

[0017] The technical scheme provided by the embodiments of the present application has the following beneficial technical effects: (1) The method of the present application can realize large-scale screening of the targeted drug targeting the target, the targeted drug inhibits the accumulation of trimethylamine and / or trimethylamine-N-oxide in vivo by affecting the metabolic pathway of trimethylamine and / or trimethylamine-N-oxide, has less side effects, and avoids affecting the normal metabolic process of the human body; (2) The method of the present application can preliminarily verify a batch of natural compounds with potential effects, so as to directly use the candidate component or the chemically modified candidate component to prepare the targeted drug, and improve the efficiency of drug development.

[0018] Additional aspects and advantages of the present application will be in part apparent and in part pointed out hereinafter. BRIEF DESCRIPTION OF DRAWINGS

[0019] The above and / or additional aspects and advantages of the present application will become apparent and be readily appreciated from the following description, including the appended drawings. Figure 1 The three-dimensional binding schematic diagram generated by using the method of the present application for molecular docking and binding mode prediction shows the binding schematic diagram of CntA enzyme and daidzin, daidzein, genistin and Indian yellow sand glycoside, respectively; Figure 2 The three-dimensional binding schematic diagram generated by using the method of the present application for molecular docking and binding mode prediction shows the binding schematic diagram of CntA enzyme and 3'-O-methyl aubepyrone, apigenin and calycosin glycoside, respectively; Figure 3 The three-dimensional binding diagrams generated by the method of molecular docking and binding mode prediction of the present application show the binding diagrams of CntA enzyme and genistin, puerarin, puerarin and 2'-hydroxy genistein, respectively; Figure 4 The three-dimensional binding diagrams generated by the method of molecular docking and binding mode prediction of the present application show the binding diagrams of CntA enzyme and 7-hydroxyisoflavone, biochanin and daidzein, respectively; Figure 5 The three-dimensional binding diagrams generated by the method of molecular docking and binding mode prediction of the present application show the binding diagrams of CntA enzyme and formononetin, glycitein, irilone and trifolin, respectively; Figure 6 The three-dimensional binding diagrams generated by the method of molecular docking and binding mode prediction of the present application show the binding diagrams of CntA enzyme and irilone, puerarin and sophorin, respectively; Figure 7 The three-dimensional binding diagrams generated by the method of molecular docking and binding mode prediction of the present application show the binding diagrams of CntA enzyme and genistein, irilone, olopol and prunetin, respectively; Figure 8 The three-dimensional binding diagrams generated by the method of molecular docking and binding mode prediction of the present application show the binding diagrams of CntA enzyme and 6a-hydroxy medyntanosine, isomedyntanosine and medyntanosine, respectively; Figure 9 The three-dimensional binding diagrams generated by the method of molecular docking and binding mode prediction of the present application show the binding diagrams of CntB enzyme and daidzin, glycitein, ononin and rottlerin, respectively; Figure 10 The three-dimensional binding diagrams generated by the method of molecular docking and binding mode prediction of the present application show the binding diagrams of CntB enzyme and 3'-O-methylsophoranone, apigenin and calycosin glycoside, respectively; Figure 11 The three-dimensional binding diagrams generated by the method of molecular docking and binding mode prediction of the present application show the binding diagrams of CntB enzyme and genistin, puerarin, puerarin and 2'-hydroxy genistein, respectively; Figure 12 The three-dimensional binding diagrams generated by the method of molecular docking and binding mode prediction of the present application show the binding diagrams of CntB enzyme and 7-hydroxyisoflavone, biochanin and daidzein, respectively; Figure 13The three-dimensional binding diagrams generated by the method of the present application for molecular docking and binding mode prediction show the binding diagrams of CntB enzyme and formononetin, daidzein, irilone and trifolin respectively; Figure 14 The three-dimensional binding diagrams generated by the method of the present application for molecular docking and binding mode prediction show the binding diagrams of CntB enzyme and formononetin, daidzein, irilone and trifolin respectively; Figure 15 The three-dimensional binding diagrams generated by the method of the present application for molecular docking and binding mode prediction show the binding diagrams of CntB enzyme and formononetin, daidzein, irilone and trifolin respectively; Figure 16 The three-dimensional binding diagrams generated by the method of the present application for molecular docking and binding mode prediction show the binding diagrams of CntB enzyme and formononetin, daidzein, irilone and trifolin respectively; Figure 17 The three-dimensional binding diagrams generated by the method of the present application for molecular docking and binding mode prediction show the binding diagrams of CntB enzyme and formononetin, daidzein, irilone and trifolin respectively; Figure 18 The three-dimensional binding diagrams generated by the method of the present application for molecular docking and binding mode prediction show the binding diagrams of CntB enzyme and formononetin, daidzein, irilone and trifolin respectively; Figure 19 The three-dimensional binding diagrams generated by the method of the present application for molecular docking and binding mode prediction show the binding diagrams of CntB enzyme and formononetin, daidzein, irilone and trifolin respectively; Figure 20 The three-dimensional binding diagrams generated by the method of the present application for molecular docking and binding mode prediction show the binding diagrams of CntB enzyme and formononetin, daidzein, irilone and trifolin respectively; Figure 21 The three-dimensional binding diagrams generated by the method of the present application for molecular docking and binding mode prediction show the binding diagrams of CntB enzyme and formononetin, daidzein, irilone and trifolin respectively; Figure 22 The three-dimensional binding diagrams generated by the method of the present application for molecular docking and binding mode prediction show the binding diagrams of CntB enzyme and formononetin, daidzein, irilone and trifolin respectively; Figure 23 The three-dimensional binding diagrams generated by the method of the present application for molecular docking and binding mode prediction show the binding diagrams of CntB enzyme and formononetin, daidzein, irilone and trifolin respectively; Figure 24 The three-dimensional binding diagrams generated by the method of the present application for molecular docking and binding mode prediction show the binding diagrams of CntB enzyme and formononetin, daidzein, irilone and trifolin respectively; Figure 25 The three-dimensional binding diagrams generated by the method of the present application for molecular docking and binding mode prediction show the binding diagrams of CntB enzyme and formononetin, daidzein, irilone and trifolin respectively; Figure 26 The three-dimensional binding diagrams generated by the method of the present application for molecular docking and binding mode prediction show the binding diagrams of CntB enzyme and formononetin, daidzein, irilone and trifolin respectively. DETAILED DESCRIPTION

[0020] The embodiments of the present application will be described below in conjunction with the accompanying drawings. It should be understood that the embodiments described below in conjunction with the accompanying drawings are exemplary descriptions of the technical solutions of the embodiments of the present application, and do not constitute a limitation on the technical solutions of the embodiments of the present application.

[0021] Those skilled in the art can understand that "said" and "the" used herein can also include plural forms unless specifically stated. It should be further understood that the use of the word "comprise" in the specification of the present application means that the features, integers, steps, operations, elements and / or components exist, but does not exclude other features, information, data, steps, operations, elements, components and / or combinations thereof supported by the present technology. The term "and / or" used herein means at least one of the items defined by the term, for example, "A and / or B" can be implemented as "A", or as "B", or as "A and B".

[0022] In order to make the purpose, technical solutions and advantages of the present application clearer, the embodiments of the present application will be described in further detail below in conjunction with the accompanying drawings.

[0023] Due to the limitations of broad-spectrum antibiotic intervention in TMA, clinical treatment urgently needs to develop natural source inhibitors with high efficiency, low toxicity and specific targeting CntA / B enzyme. Some studies suggest using analogues to inhibit TMAO synthesis. Hui et al. proposed the inhibition of phospholipase-self-classification factor pathway, which produces choline and lysophosphatidic acid from lysophosphatidylcholine. Deoxycholic acid (DMB) as a choline analogue can inhibit choline TMA lyase, reduce the conversion of choline, carnitine and crotonbetaine to TMA, but cannot completely prevent the synthesis of TMAO.

[0024] Natural dietary compounds have become an ideal way of health intervention due to their high safety, low toxicity, multi-target effect and promotion of intestinal microbiota health. Especially isoflavones (such as genistein), santalanes (such as pterostilbene) and flavonoid glycosides (such as rutin), which not only can effectively regulate various physiological processes of the body, such as antioxidant, anti-inflammatory, immune regulation, etc., but also can assist in the prevention and treatment of chronic diseases, and have good long-term use sustainability. Therefore, natural dietary compounds have significant advantages in promoting health and preventing diseases. Natural dietary compounds can effectively inhibit TMA generation by regulating intestinal microbial ecology and interfering with the activity of key TMA synthesis enzymes (such as CutC / D, CntA / B). However, the interaction mechanism of isoflavones, santalanes and flavonoid glycosides with the active center of CntA / B enzyme needs to be further analyzed.

[0025] I. Molecular docking and binding simulation prediction (1) Data acquisition: 1. This example takes CntA / B enzyme complex as the target, CntA / B is a two-component Rieske-type oxygenase / reductase expressed by intestinal flora, which is composed of two subunits CntA and CntB. The crystal structure of the target protein was obtained from the Protein DataBank (PDB), and the PDB numbers of the crystal structure of CntA and its ligand were 6Y8J, 6Y8S, 6Y9C, 6Y9D, and 6ZGP.

[0026] 2. This example takes a variety of natural dietary compounds as candidate components, and obtains small molecule compound structures from the PubChem database for subsequent molecular docking and binding mode prediction. The selected compound information is shown in Table 1.

[0027] Table 1 CID numbers of flavonoid glycosides, isoflavones and santalane drugs

[0028] (2) Preparation of docking files: 1. Use AutoDock Tools 1.5.6 to preprocess the protein receptor and small molecule ligand, including removing water molecules, adding charges and defining flexible bonds; 2. Generate PDBQT format input files suitable for AutoDock Vina; (3) Molecular docking and energy evaluation: 1. Perform molecular docking by AutoDock Vina 1.0.2, set the search space parameters as follows:

[0029] 2. Screen the optimal binding conformation based on the binding energy (ΔG, unit: kcal / mol), and the formula for calculating the binding energy is ΔG = Cinter1 / (1 + 0.0585 × Nrot).

[0030] (4) Key residue identification and visualization: 1. Use PyMOL software to analyze the hydrogen bonds, hydrophobic interactions and π-π stacking interactions of the protein-ligand complex; 2. Determine the key binding residues by residue contact frequency analysis: arginine at position 320 (ARG-320), aspartic acid at position 323 (ASP-323), tyrosine at position 203 (TYR-203), etc. (5) Results output: A comprehensive report containing binding conformation, energy score and key residue information is generated, and the interaction mode is displayed in a three-dimensional visual model. Figures 1-16 ).

[0031] The various candidate components of flavonoid glycosides, isoflavones and santalans are subjected to molecular docking with CntA protein and CntB protein, respectively, so as to identify the key binding residues of each candidate component and protein crystal and to calculate the affinity, and the specific results are shown in Figures 17-19 and Table 2. The results show that there are relatively fixed key binding residues in the drug types of each candidate component. Other information not shown in the tables and figures of the present application can also provide valuable data for the screening of candidate components, and the present application does not limit the data used for decision-making.

[0032] Table 2. List of binding affinity of flavonoid glycosides, isoflavones and santalans drugs to CntA and CntB proteins

[0033] II. CntA / B protein purification In order to further verify the results of molecular docking and binding mode prediction, in vitro verification of the candidate components and the target is also needed to further screen the targeted drugs and investigate the characteristics of the targeted drugs. The specific steps are as follows: (1) Optimization of recombinant expression system: 1. Host strain: Recombinant E. coli BL21(DE3) is used, and LB agar plates containing ampicillin (50 μg / ml) are incubated at 37°C overnight to screen recombinant E. coli BL21(DE3) with resistance genes.

[0034] 2. Induction conditions: In LB liquid medium, 0.5 mM IPTG is used to induce expression at 37°C for 12 hours to ensure high-efficiency soluble expression of the protein.

[0035] (2) Lysis buffer and cell disruption: 1. Buffer formula: 50 mM NaH2PO4, 30 mM NaCl, 1 mM PMSF (pH 7.4), to inhibit protease activity and maintain protein stability.

[0036] 2. Disruption method: ultrasonic treatment (power 200 W, working 7 seconds / intermittent 8 seconds, total time 40 minutes) to ensure complete cell lysis and protein denaturation.

[0037] (3) Affinity purification process: Chromatography resin: Reducing chelating His-tag purification resin, suitable for imidazole-containing reducing environment.

[0038] Binding and washing: Balanced and washed with 20 mM Tris base + 3 mM imidazole + 0.5 M NaCl (pH 8.0), to remove non-specific impurities.

[0039] Elution conditions: 20 mM Tris base + 500 mM imidazole + 0.5 M NaCl (pH 8.0), one-step elution of target protein, and verified by SDS-PAGE.

[0040] As shown in Figure 20 SDS-PAGE, CntA (~ 37 kDa) and CntB (~ 42 kDa) bands are single, indicating that the extracted CntA and CntB proteins have high quality, which is beneficial for subsequent screening steps.

[0041] III. Enzyme activity detection experiment (1) Optimized enzyme activity detection system reaction 1. System composition: 10 mM HEPES buffer (pH 7.4), 200 μg purified CntA / B enzyme, 0.2 mM L-carnitine, 0.1 mM NADH, total volume 200 μL.

[0042] 2. Detection conditions: Incubate the reaction mixture at 37°C for 1 hour, measure OD340 value by ultraviolet spectrophotometer, calculate NADH oxidation rate (ΔOD / h).

[0043] (2) Screening method: 1. Add the test compounds (such as BCA, GEN, GLY) or controls (DMSO) to the 96-well plate respectively; 2. Prepare the reaction solution according to the above system, record the initial OD value; 3. Measure the final OD value after incubation, calculate the NADH oxidation inhibition rate (formula: inhibition rate = 1 - (treatment group consumption ratio / baseline value).

[0044] The results of the enzyme activity detection experiment are shown in Figure 21 and Table 3. The candidate components with high binding energy determined by molecular docking and binding mode prediction were all proved to have high inhibition rate on CntA / B enzyme in vitro, which improved the screening efficiency of targeted drugs.

[0045] Table 3 Enzyme activity inhibition rate of flavonoid glycosides, isoflavones and santalane drugs on CntA / B enzyme

[0046] Further, the above drug screening method can be extended to the screening of inhibitory compounds for other enzymes or enzyme complexes (e.g., CutC / D and YesW / X).

[0047] The daidzein with the highest inhibition rate (97.20%) in Table 3 was selected for further verification: IV. In vitro incubation of bacteria with daidzein (1) Bacterial culture 1. In this example, Escherichia coli A1 (A1) and Acinetobacter baumannii (AB) were used as CntA / B enzyme high-expression bacteria. Escherichia coli A1 and Acinetobacter baumannii are gram-negative bacilli. The base medium was used to prepare an Escherichia coli A1 and Acinetobacter baumannii bacterial solution with an OD 600 = 0.5. Escherichia coli Acinetobacter baumannii

[0048] 2. d3-L-carnitine was diluted with the bacterial solution of Escherichia coli A1 and Acinetobacter baumannii prepared with the base medium at a ratio of 1:600, and d3-L-carnitine was added to the bacterial solution.

[0049] 3. In this example, dimethyl sulfoxide (DMSO), phosphate buffered saline (PBS) were used as control groups, and isoflavone drug glycitein (GLY) was used as the experimental group. DMSO, PBS and GLY were diluted with the above-prepared bacterial solution of Escherichia coli A1 and Acinetobacter baumannii at a ratio of 1:1000, and the corresponding drugs were added to the bacterial solution.

[0050] 4. To reduce experimental errors and ensure data reliability, three replicate wells were set up for each sample.

[0051] 5. The above-prepared bacterial solution of Escherichia coli A1 and Acinetobacter baumannii was placed in a 37°C air environment for 24 h.

[0052] (2) Liquid chromatography-mass spectrometry (LC-MS) determination (sample processing) 1. The above-cultured bacterial solution of Escherichia coli A1 and Acinetobacter baumannii was centrifuged at 13500 rpm, 4°C for 15 min, and the supernatant was collected.

[0053] 2. In this example, mass spectrometry grade pure methanol was used. Mass spectrometry grade pure methanol usually needs to be separated and purified several times to remove impurities and control the isotopic ratio. The bacterial supernatant was mixed with mass spectrometry grade pure methanol at a ratio of 1:2.

[0054] ​​3. The mixture of the above bacterial supernatant and mass spectrometry grade pure methanol was centrifuged at 13500 rpm, 4°C, 15 min, the supernatant was collected and transferred to the sample bottle for mass spectrometry detection.

[0055] (3) LC-MS determination (standard curve processing) 1. The standard curve solution was prepared according to the following table:

[0056] 2. The prepared standard curve solution was centrifuged at 13500 rpm, 4°C, 15 min, and the supernatant was collected.

[0057] 3. The above supernatant was mixed with mass spectrometry grade pure methanol at a ratio of 1:2.

[0058] 4. The mixture of the above supernatant and mass spectrometry grade pure methanol was centrifuged at 13500 rpm, 4°C, 15 min, the supernatant was collected and transferred to the sample bottle for mass spectrometry detection.

[0059] (4) LC-MS determination of d3-TMA Results of the in vitro incubation of bacteria with daidzein: Reference Figure 22 and 23 DMSO, PBS, GLY were incubated with E. coli A1 and Acinetobacter baumannii in vitro, respectively, to confirm the content of d3-TMA produced by E. coli A1 and Acinetobacter baumannii after incubation with DMSO, PBS, GLY in vitro in an in vitro environment with d3-L-carnitine as the only carbon source. The results show that GLY significantly inhibits the production of d3-TMA by E. coli A1 and Acinetobacter baumannii in vitro.

[0060] Five, animal experiment of daidzein inhibiting the production of TMA and TMAO in vivo This example uses C57BL / 6 mice to construct an animal model with high carnitine, and the intervention group is fed with daidzein, and the positive control of the intervention group is fed with 1.3% L-carnitine drinking water.

[0061] Reference Figure 24The embodiments of the present application use 1.3% L-carnitine water to construct a high-carnitine mouse model, and observe the effect of daidzein on the plasma TMA and TMAO of the high-carnitine mouse model. All mice are randomly divided into four groups, 7-9 in each group. Experimental grouping: normal control group - ordinary drinking water, (Control), normal intervention group - daidzein 50 mg / kg gavage on the basis of ordinary drinking water (GLY), model group - 1.3% L-carnitine drinking water (1.3% L-carnitine), model intervention group - daidzein 50 mg / kg gavage on the basis of 1.3% L-carnitine drinking water (1.3% L-carnitine+GLY).

[0062] After the above-mentioned embodiment intervention for 2 weeks, serum is extracted from the blood of the mice, 50 μL of each sample is taken, and the contents of TMA and TMAO are detected.

[0063] Take 50ul serum, add 150ul d9-TMA and d9-TMAO internal standard (50% methanol), mix well, centrifuge for 10 minutes (13.3x1000 rpm, 4°C); take 50ul supernatant, add 100ul mobile phase A, mix well, then transfer the mixed solution to the sample bottle for determination; mobile phase preparation: A phase: 0.1% ammonium fluoride + 0.1% formic acid + water, B phase: 0.1% ammonium fluoride + 0.1% formic acid + methanol. Bubble removal before mobile phase on machine: after the mobile phase A and B are prepared, they are placed in an ultrasonic machine for ultrasonic treatment for 5 minutes to help remove bubbles. Mass spectrometry on machine detection. Chromatographic column: Phenomenex Luna Omega 3um Polar C18, 100 x 3.0 mm. Mobile phase: A phase: 2 mM ammonium fluoride + 0.1% formic acid aqueous solution; B phase: 2 mM ammonium fluoride + 0.1% formic acid methanol solution. Mass spectrometry parameters: APCI source, spray voltage: 5500 V; temperature: 400oC; atomization gas: 60 psi; auxiliary heating gas: 55 psi; gas curtain gas: 25 psi; CAD: 10.

[0064] Reference Figure 25 and 26 The results show that the TMA and TMAO contents of the mice in the model group (1.3% L-carnitine) are the highest, and after using daidzein (1.3% L-carnitine+GLY), there is a significant effect on the contents of TMA and TMAO, and the contents decrease significantly, so the effect of daidzein on the clearance of TMA and TMAO is significant.

[0065] In summary, the application provides a method for screening targeted drugs for inhibiting trimethylamine and / or trimethylamine-N-oxide accumulation, comprising the following steps: determining the target in the metabolic pathway of trimethylamine and / or trimethylamine-N-oxide; if the target is a protein, screening candidate components based on the three-dimensional structure of the target; verifying the candidate component to confirm the targeted drug. The method of the application can realize large-scale screening of targeted drugs for the target, and the targeted drugs inhibit the accumulation of trimethylamine and / or trimethylamine-N-oxide in vivo by affecting the metabolic pathway of trimethylamine and / or trimethylamine-N-oxide, have less side effects, and avoid affecting the normal metabolic process of the human body.

[0066] Those skilled in the art can understand that the steps, measures, and schemes in various operations, methods, and processes discussed in the application can be alternated, changed, combined, or deleted. Further, other steps, measures, and schemes in various operations, methods, and processes discussed in the application can also be alternated, changed, rearranged, decomposed, combined, or deleted. Further, steps, measures, and schemes in various operations, methods, and processes in the related art can also be alternated, changed, rearranged, decomposed, combined, or deleted.

[0067] The terms "first", "second" are only for descriptive purposes, and cannot be understood as indicating or implying relative importance or implicitly indicating the number of indicated technical features. Therefore, the features defined with "first", "second" can explicitly or implicitly include one or more of the features. In the description of the application, unless otherwise stated, the meaning of "a plurality of" is two or more.

[0068] In the description of the specification, specific features, structures, materials or characteristics can be combined in any one or more embodiments or examples in a suitable manner.

[0069] The above only describes some embodiments of the application, and it should be pointed out that, for those skilled in the art, without departing from the technical concept of the application, other similar implementation means based on the technical idea of the application also belong to the protection scope of the embodiments of the application.

Claims

1. A method for screening of target drugs for inhibiting trimethylamine and / or trimethylamine-N-oxide accumulation, characterized in that, It comprises the following steps: determining a target on the metabolic pathway of trimethylamine and / or trimethylamine-N-oxide; if the target is a protein, screening candidate components based on the three-dimensional structure of the target; verifying the candidate components to confirm targeted drugs.

2. The method of claim 1, wherein, The three-dimensional structure of the target includes the protein crystal structure of the target and / or the protein crystal structure of the ligand of the target.

3. The method of claim 1, wherein, The screening of candidate components based on the three-dimensional structure of the target includes detecting the interaction of the candidate components with the target by molecular docking and binding mode.

4. The method of claim 3, wherein, The interaction of the candidate components with the single protein crystal structure is detected by molecular docking and binding mode, respectively.

5. The method of claim 1, wherein, The target includes the enzyme complex of intestinal microorganisms metabolizing trimethylamine and / or trimethylamine-N-oxide.

6. The method of claim 4, wherein, The enzyme complex includes at least one of CutC / D, CntA / B and YesW / X; the candidate components include flavonoid glycosides, isoflavones and / or santal components.

7. The method of claim 5, wherein, The flavonoid glycoside components targeting the CntA / B enzyme complex include at least one of daidzein, genistin and glycitein; the isoflavone components targeting the CntA / B enzyme complex include at least one of 7-hydroxyisoflavone, biochanin, apigenin, formononetin, genistein and daidzein; the santal components targeting the CntA / B enzyme complex include sophoranone.

8. The method of claim 1, wherein, The targeted drugs are used to inhibit the conversion of L-carnitine to trimethylamine, and / or inhibit the conversion of trimethylamine to trimethylamine-N-oxide.

9. The method of claim 1, wherein, The verification of the candidate components includes detecting the ex vivo activity of the candidate components, and verifying the in vitro and / or in vivo physiological effects of the candidate components.

10. The method of claim 1, wherein, The targeted drugs are used to intervene in cardiovascular diseases, kidney diseases and / or liver diseases.

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