Atherosclerosis risk factor TMAO intervention key site and inhibitor screening

Abstract

The invention discloses an atherosclerosis risk factor TMAO intervention key site and inhibitor screening, and belongs to the field of biological medicine. The invention provides an inhibitor aiming at the generation of TMA and / or TMAO, the inhibitor is cannabidiol, and the Cys48 site in intestinal trimethylamine lyase CutC can be targeted. By using the inhibitor, the CutC enzyme activity can be effectively reduced, and the conversion of choline to TMA is inhibited, so that the TMAO level is reduced, the promotion effect of TMAO on atherosclerosis is reduced, and the atherosclerosis risk is reduced.

Description

Technical Field

[0001] The present invention relates to the key intervention sites of TMAO, a risk factor for atherosclerosis, and the screening of inhibitors, and belongs to the field of biomedicine. Background Art

[0002] Cardiovascular disease (CVD) is the leading cause of death in humans today, and atherosclerosis (AS) is the pathological basis of CVD. In recent years, increasing evidence has shown that intestinal bacteria are influencing factors in the development and deterioration of AS. Among them, trimethylamine (TMA) is a metabolite derived from intestinal bacteria. Dietary nutrients rich in TMA, such as choline, L-carnitine or betaine, are broken down to produce TMA. TMA is further oxidized by flavin monooxygenase (mainly FMO3 enzyme) in the liver to produce trimethylamine-N-oxide (TMAO). Since the discovery that TMAO is associated with the onset of cardiovascular disease, TMAO has attracted attention as an independent biomarker of cardiovascular disease risk. A large number of human and animal studies have also shown that the TMAO pathway is the first intestinal microbiome-centered CVD drug target with strong clinical prognostic value. TMAO can promote the development of diseases such as AS, thrombosis, coronary heart disease, heart failure, and type 2 diabetes. Unlike traditional risk factors, TMAO is an independent predictor and promoter of AS. TMAO promotes AS through various mechanisms, including enhancing foam cell formation, promoting endothelial dysfunction, aggravating platelet hyperreactivity and thrombosis risk, activating inflammatory response and inhibiting reverse cholesterol transport, etc. Therefore, inhibiting the generation of TMAO is considered a potential target for intervening in the occurrence and development of AS.

[0003] Dietary precursors of TMA structure are metabolized to TMA by intestinal trimethylamine lyase (cutC, cntA and yeaW), and then oxidized to TMAO by liver flavin monooxygenase (FMOs, mainly FMO3). The production of TMA by intestinal microorganisms is the first and rate-limiting step in the formation of TMAO. Inhibiting the activity of intestinal trimethylamine lyase can effectively reduce the production of TMA in the intestine. The development of inhibitors of intestinal trimethylamine lyase can be used as an effective intervention to inhibit the production of TMAO. Dietary polyphenols have pharmacological effects such as antioxidant, anti-inflammatory, anticoagulant, anti-atherosclerotic and anti-tumor effects. They are believed to have the effect of improving cardiovascular metabolic health and are widely present in various fruits, vegetables, tea, red wine and other foods. Studies have found that resveratrol, choline hydrochloride, berberine and the like can reduce the production of TMAO in choline diets. Therefore, it is necessary to explore the key sites of intestinal trimethylamine lyase and intervene in these key sites to achieve effective inhibition of atherosclerosis. Summary of the invention

[0004] First, the present invention provides an intervention method for intestinal flora, and finds key strains for TMAO production in patients with atherosclerosis. One of the key strains is C. sporogenes, and subsequent methods such as microbial transplantation, antibacterial agents, and probiotics can be used to change the content of C. sporogenes in the intestine and reduce the production of TMAO.

[0005] Secondly, the present invention provides a key site for TMAO intervention, which can effectively reduce the synthesis of TMAO in the body, thereby reducing the risk of atherosclerosis in patients. The key intervention site is cutC cleavage enzyme and its 48th cysteine ​​(Cys48).

[0006] Finally, the present invention also provides a method for developing an inhibitor based on a key site. By screening the compound library, a compound with the ability to inhibit TMAO synthesis was found. One of the inhibitors is cannabidiol (CBD), which can bind to the Cys48 site on CutC and affect the enzymatic reaction.

[0007] The first object of the present invention is to provide the use of cannabidiol CBD in preparing a CutC enzyme targeted inhibitory binder.

[0008] In one embodiment, the binding target of the CBD is covalently bound to a cysteine ​​residue in the CutC enzyme.

[0009] In one embodiment, the CutC enzyme has Gene ID: 69425759 on NCBI.

[0010] In one embodiment, the binding target of the CBD is located at the Cys48 site in the CutC enzyme.

[0011] In one embodiment, the CutC enzyme targeted inhibition binding agent comprises a pharmaceutically acceptable carrier.

[0012] The second object of the present invention is to provide a method for inhibiting CutC enzyme activity, wherein the method comprises inhibiting CutC using CBD.

[0013] In one embodiment, CBD is contacted with CutC to inhibit CutC enzymatic activity.

[0014] In one embodiment, the method is not used for the purpose of disease treatment, but is applied in scientific research such as molecular experiments or cell experiments.

[0015] The third object of the present invention is to provide the use of cannabidiol CBD in the preparation of TMA production inhibitors.

[0016] In one embodiment, the CBD inhibits the conversion of choline to TMA.

[0017] The third object of the present invention is to provide the use of cannabidiol CBD in the preparation of TMAO production inhibitors.

[0018] In one embodiment, the CBD inhibits the decomposition of choline into TMA by inhibiting the activity of CutC enzyme, and further affects the oxidation of TMA to generate TMAO.

[0019] Beneficial Effects

[0020] 1. Inhibition of TMAO production: The invention provides an inhibitor targeting the key site of the intestinal trimethylamine lyase CutC that produces TMAO. By using this inhibitor, the activity of CutC enzyme can be effectively reduced, the conversion of choline to TMA can be inhibited, thereby reducing the level of TMAO and reducing the promoting effect of TMAO on atherosclerosis, thereby reducing the risk of atherosclerosis.

[0021] 2. Reduce the risk of cardiovascular and cerebrovascular diseases: The inhibitor of this invention has the effect of reducing the formation of TMAO, a risk factor for cardiovascular and cerebrovascular diseases, thereby alleviating the occurrence of atherosclerosis, and can reduce the incidence and mortality of cardiovascular and cerebrovascular diseases.

[0022] 3. Provide new drug research and development direction: This invention provides new directions and ideas for the development of TMAO intervention key sites and inhibitors. The research and development of related drugs can not only play an important role in the prevention and treatment of atherosclerosis, but also be applied to other related diseases, such as hypertension, diabetes, etc., and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 is the plasma level of TMAO-related metabolites in patients with atherosclerosis.

[0024] Figure 2 is the relative expression level of trimethylamine lyase gene in the feces of patients with atherosclerosis.

[0025] Figure 3 Relative expression level of CutC gene of TMA-producing Clostridium in patients with atherosclerosis.

[0026] Figure 4 The results of correlation analysis among TMAO, CutC and C.sporogenes.

[0027] Figure 5 The results of polyphenols inhibiting the production of TMA by microorganisms using choline in vitro. Figure a shows the in vitro culture of C. sporogenes, and b shows the in vitro culture of human fecal microorganisms.

[0028] Figure 6Are the plasma TMA / TMAO levels in mice, **: p<0.01, ***: p<0.001.

[0029] Figure 7 The results show that CBD inhibits CutC enzyme activity in vitro.

[0030] Figure 8 The results of the construction of the CutC enzyme activity assay system. In the figure, a is the reaction conditions of CutC / D enzyme, b is the effect of different components on the reaction, c is the NADH absorbance change curve corresponding to different YADH concentrations, d is the change in NADH absorbance corresponding to different YADH concentrations, and e is the OD340 change of NADH under different YADH concentrations within 25s 20s.

[0031] Fig. 9 The results of the binding of CBD to CutC. In the figure, a is the binding energy of choline and CutC analyzed by microthermophoresis, b is the binding energy of choline and CutC analyzed by molecular dynamics simulation, c is the binding of CBD and CutC analyzed by microthermophoresis, and d is the binding of CBD and CutC analyzed by molecular dynamics simulation.

[0032] Fig.10 This is the molecular docking result of CutC protein and CBD.

[0033] Fig.11 The results of protein molecular weight determination before and after CutC binds to CBD. In the figure, a is the deconvolution diagram, and b is the quality and purity of the peak.

[0034] Fig.12 The results of the identification of the binding site between CBD and CutC protein. In the figure, a is the coverage of the enzymatic peptide segment, b is the peptide segment of the binding site, and c is the identification result of the binding site.

[0035] Fig.13 Figure 1 shows the effect of CutC and Cys48 mutations on the production of TMA and TMAO. Figure 2 shows the effect of CutC and Cys48 mutations on the production of TMA by C. sporogenes using choline in vitro, and Figure 3 shows the effect of colonization of C. sporogenes with CutC and Cys48 mutations on the production of TMA and TMAO in mice. DETAILED DESCRIPTION

[0036] Mega medium preparation is shown in Table 1

[0037] Table 1

[0038]

[0039]

[0040] Example 1. Quantitative analysis of plasma TMAO and related metabolites in patients with atherosclerosis

[0041] Sample processing: Take 20 μL of plasma sample, add 4 volumes (80 μL) of ice methanol (containing 5 μg / mL Fmoc-glycine-15N as internal standard), vortex mix for 30 seconds to precipitate plasma proteins, centrifuge at 14000 rpm, 4°C for 10 minutes, centrifuge the supernatant again under the same conditions, and collect the supernatant for subsequent analysis.

[0042] UPLC-MS / MS detection conditions: The samples were separated on a BEH Amide (100 mm × 2.1 mm, 1.7 μm, Waters) column. Mobile phase A was 0.1% formic acid in water, and mobile phase B was acetonitrile. The elution program was as follows: 0-0.5 min, 95% B; 0.5-6 min, 95% B-40% B; 6-8 min, 40% B; 8-8.1 min, 40% B-95% B; 8.1-11 min, 95% B. Column temperature: 40°C; flow rate: 0.3 mL / min; injection volume: 2 μL.

[0043] Mass spectrometry conditions: electrospray ion source (ESI), positive ion mode, multiple reaction (MRM) monitoring, capillary voltage 4.0 kV, pyrolyzer voltage 110 V, drying gas temperature 200 ° C, flow rate 0.3 mL / min, nebulizer pressure 25 Psi. The detection parameters of TMAO-related metabolite standards are shown in Table 2.

[0044] The absolute quantitative method was used to detect the content of TMAO and its precursor (TMA) in patients with atherosclerosis. It was found that the TMAO content in patients with atherosclerosis was significantly increased, and the betaine content was significantly decreased ( Figure 1 ). Studies have reported that the ratio of betaine to choline is inversely proportional to the occurrence of CVD, that is, the higher the betaine level, the lower the risk of cardiovascular disease. TMAO has also been reported to be an independent biomarker for cardiovascular disease risk.

[0045] Table 2 Detection parameters of TMAO-related metabolite standards in plasma of patients with atherosclerosis

[0046]

[0047] Example 2: Determination of TMA lyase gene expression in patients with atherosclerosis

[0048] Feces from healthy people and patients with atherosclerosis were collected, and fecal genomic DNA was extracted using a fecal genomic DNA extraction kit. The purity and concentration of fecal genomic DNA were determined and used as template DNA for quantitative amplification reactions. Primers were designed using DNA star and primer 5.0 software and synthesized by Shanghai Bioengineering. The primer design is shown in Table 3. Using the 16S gene as an internal reference, qPCR was used to analyze the relative abundance of three cleavage enzyme genes CutC, CntA, and YeaW in feces. It was found that the CutC gene was significantly increased in patients with atherosclerosis ( Figure 2 ), while there was no significant difference in the expression of CntA and YeaW. CutC is a key enzyme in the production of TMA and a potential target for reducing TMAO. Figure 3 As shown in Figure 2, by comparing the relative abundance of CutC genes in TMA-producing Clostridia, it was found that C. sporogenes and C. asparagiforme increased significantly in patients with atherosclerosis, with the change in C. sporogenes being more significant ( Figure 3 ).

[0049] Table 3 qPCR analysis primers

[0050]

[0051] Example 3. Correlation analysis between CutC, C.sporogenes and TMAO

[0052] The present invention found that CutC and C.sporogenes are more abundant in patients with atherosclerosis, and at the same time, the level of TMAO in patients with atherosclerosis is also higher. The present invention conducted a correlation analysis on the three indicators of CutC, C.sporogenes and TMAO and found that there was a strong positive correlation between the three ( Figure 4 ), indicating that the elevated TMAO levels in patients with atherosclerosis may be due to the increased relative expression of CutC and C.sporogenes.

[0053] Example 4: Molecular docking screening for inhibitors of CutC enzyme

[0054] The SDF format structural data file of polyphenols was obtained from PubChem database, the lowest energy conformation was optimized, and the mol2 format file was converted at the MM2 level using Chem 3D software for preparing docking ligands. The crystal structure of CutC (PBD ID: 5FAU) was obtained from PDB protein database, and the receptor was dehydrated and unnecessary heteroatoms were removed using visualization software pymol. Then the AutoDock tool was used to add solvation parameters, hydrogenation, and charge. Finally, the binding force between polyphenols and CutC was calculated in the active site pocket, with the box center of X: -37.750, Y: -11.750, Z: 57.778, and the sizes of X, Y, and Z were 112, 42, and 126, respectively. CutC was used as the protein ligand and docked with polyphenols, and small molecule inhibitors DMB and FMC, which have been reported to have the activity of inhibiting TMA generation, were set as positive controls. As shown in Table 4, the binding energy between choline and CutC is -3.2 Kcal / mol, the binding energy between DMB and CutC is -4.0 Kcal / mol, and the binding energy between FMC and CutC is -3.4 Kcal / mol. Polyphenols show better affinity for CutC than DMB and choline, indicating that these polyphenols may serve as inhibitors of CutC enzyme.

[0055] Table 4 Molecular docking results

[0056]

[0057]

[0058] Example 5: Effect of inhibitors on TMA production ability

[0059] (1) In vitro experiments:

[0060] First, the polyphenols screened by molecular docking in Example 4 were used for in vitro experiments to verify:

[0061] According to the results of molecular docking in Table 4 , the top ten polyphenols with the highest binding energy (sesamolin, sesamin, quercetin, chlorogenic acid, oleuropein, resveratrol, cannabidiol (CBD), ferulic acid, caffeic acid or gentisic acid) were selected to culture C. sporogenes in vitro to verify the inhibitory effect of polyphenols on choline metabolism of intestinal flora.

[0062] C. sporogenes ATCC15579 was cultured in Mega medium containing 15 mmol / L choline and 100 μmol / L polyphenols. After anaerobic culture at 37°C for 12 hours, 0.2 mol / L formic acid was added to terminate the reaction. Protein was then precipitated with 80% cold methanol and centrifuged at 14000 rpm at 4°C for 20 minutes to obtain the supernatant. The TMA content was measured using the same UPLC-MS / MS method as in Example 1. The results are shown in Figure 1. Figure 5 As shown in a, compared with the control group without adding polyphenols, sesamelin, sesamin, quercetin, chlorogenic acid, oleuropein, resveratrol, CBD, ferulic acid, and fluoromethylcholine can inhibit choline metabolism, while the reported CutC inhibitor DMB has no inhibitory effect, which may be due to the fact that DMB cannot interact with the active site of CutC. Among them, CBD, oleuropein, and resveratrol have the strongest inhibitory effect, with inhibition rates of 92.63%, 81.75%, and 80.38%, respectively.

[0063] The inhibition rate was calculated as follows:

[0064] Polyphenol choline metabolism inhibition rate (%) = (TMA content of blank control - TMA content of positive drug) / TMA content of blank control × 100%

[0065] Similarly, an appropriate amount of fresh human fecal sample was placed in a sterile Mega (1:20 (w / v)) medium, vortexed for 1 min, and centrifuged at room temperature (100g, 30s). 50 μL of the supernatant was suspended in a 96-deep-well plate of Mega (15 mmol / L choline, 100 μmol / L polyphenols, 1 mL), and the plate was then incubated at 37°C under anaerobic conditions for 12 h. The reaction was terminated by adding 0.2 mol / L formic acid, followed by protein precipitation with 80% cold methanol, centrifuged at 14000 rpm, 4°C for 20 min, and the supernatant was obtained. The TMA content was measured by UPLC-MS / MS method. The inhibitory effect of polyphenols on choline metabolism in a complex microbial system was verified, and it was found that chlorogenic acid, oleuropein, resveratrol, CBD, ferulic acid, and caffeic acid showed significant inhibitory effects, among which CBD, chlorogenic acid, and ferulic acid had the strongest inhibitory effects ( Figure 5 b), the inhibition rates reached 83.34%, 60.88% and 57.25%.

[0066] Based on the results of single colony and feces in vitro culture, it can be seen that CBD shows a strong inhibitory effect, so CBD can be used as a candidate inhibitor for inhibiting CutC enzyme.

[0067] (2) In vivo experiments

[0068] Subsequently, the CBD screened in step (1) was used for animal experiments:

[0069] 8-week-old specific pathogen-free male C57BL / 6J mice were purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd., and standard mouse feed (LAD3001M) and 1.0% choline feed were purchased from Nantong Trophy Feed Technology Co., Ltd.

[0070] After one week of acclimatization, the animals were divided into three groups. The choline group was fed a 1% choline diet, and the remaining two groups were gavaged with broad-spectrum antibiotics (200 mg / kg vancomycin, 400 mg / kg ampicillin, 400 mg / kg metronidazole, and 400 mg / kg neomycin sulfate) to eliminate intestinal flora and fed a 1% choline diet. After two weeks of antibiotic intervention, C. sporogenes colonization (1×10 9 CFU, 200 μL, oral administration once every two days). Finally, a group of mice were randomly selected from the C. sporogenes colonization group and gavaged with CBD (50 mg / kg). After 4 weeks of CBD intervention, all mice were dissected and plasma was collected to analyze the effect of CBD intervention on TMA and TMAO levels in mice. As shown in the figure, C. sporogenes colonization led to significant accumulation of TMA and TMAO in mouse plasma. CBD treatment for 4 weeks significantly reversed the increase in TMA and TMAO levels caused by C. sporogenes colonization ( Figure 6 ).

[0071] Table 5 Animal grouping

[0072] Experimental Grouping Treatment Choline group 1.0% choline feed. Colonization group 1.0% choline feed, antibiotic intervention for 2 weeks, and C. sporogenes colonization for 2 weeks. CBD Group 1.0% choline feed, antibiotic intervention for 2 weeks, C. sporogenes colonization for 2 weeks, 50 mg / kg CBD intervention for 4 weeks.

[0073] Example 6: Determination of the binding site between cannabidiol and CutC enzyme

[0074] Choline generates TMA and acetaldehyde under the action of CutC / D, and acetaldehyde and NADH generate ethanol under the action of coenzyme YADH. At the same time, NADH loses H + Therefore, the absorption peak of NADH at 340nm can be used to measure the amount of NADH, which can indirectly reflect the amount of TMA.

[0075] (1) Establishment of CutC enzyme activity detection system

[0076] First, the feasibility of the CutC enzyme activity reaction system was verified. It was found that the reaction would only occur when acetaldehyde, NADH and YADH were present at the same time, and the reaction could be stabilized at 3 minutes ( Figure 8 b). At the same time, the amount of YADH enzyme required in the reaction was optimized, and it was found that too high or too low YADH enzyme would affect the rate of the catalytic reaction, and the final reaction degree was the same ( Figure 8c). Because the consumption of acetaldehyde in a short period of time was to be measured, the change in OD340 per unit time (ΔOD340) was analyzed. Figure 8 As shown in Figure d, medium concentration of YADH is more conducive to the occurrence of the reaction, and the reaction rate is extremely high in the initial short time, which will not affect the occurrence of subsequent reactions. Further, the present invention compares the effect of different YADH enzyme amounts on the change in OD340 per unit time within the initial 25s of the reaction, and finds that when there are 4.8 units of YADH in the system, the reaction rate is the fastest ( Figure 8 e).

[0077] Therefore, in the subsequent reaction, 4.8 units of YADH were added to the system to measure the enzyme activity. CutC was diluted and added to a mixture containing 0.04 μM NADH and 4.8 units of YADH, so that the final concentration of cutC monomer was 5 nM. The mixture was distributed in a 96-well plate, choline was added (final concentration was 200 μM), and the absorbance of NADH at 340 nm was immediately recorded.

[0078] (2) Effect of CBD on cutC enzyme activity

[0079] CutC was diluted and added to a mixture containing 0.04 μM NADH and 4.8 units of YADH, so that the final concentration of cutC monomer was 5 nM. The mixture was distributed into a 96-well plate, CBD (0.1 μmol / L to 100 μmol / L) was added to the 96-well plate, and choline (final concentration was 200 μM) was added. The absorbance of NADH at 340 nm was immediately recorded, and the EC was calculated using nonlinear regression fitting data. 50 It was found that CBD can effectively inhibit CutC enzyme activity in vitro, EC 50 =20.4μM( Figure 8 ).

[0080] (3) Investigation of the binding between CutC and CBD

[0081] The in vitro interaction between small molecules and proteins was analyzed by microcalorimetry. The results showed that the binding energy between CutC and choline was 11.8 nmol / L. After incubation with CBD, the binding energy between CutC and choline was significantly reduced (Kd = 869.2 nmol / L) ( Fig. 9 a); and CutC can bind to CBD with a Kd of 522.9nmol / L ( Fig. 9 c). Similarly, molecular dynamics simulation was used to study the interaction between choline and CutC. The results showed that when CBD was present, the binding energy between CutC and choline was significantly reduced ( Fig. 9b), and CBD can bind to CutC, with the main interaction force being van der Waals force ( Fig. 9 d).

[0082] Based on the previous results, it is speculated that CBD may affect the activity of CutC by binding to CutC. In order to explore the possible binding mode and binding site, molecular docking of CBD and CutC was performed. The results showed that CBD has a high inhibitory activity on CutC enzyme, which may be due to the covalent interaction between CBD and CutC. The double bond of CBD can covalently bind to the sulfhydryl group of free cysteine ​​residues and form hydrogen bonds with Tyr361, Glu365 and Glu60 on CutC ( Fig.10 ). Therefore, the present invention uses protein spectrometry to determine the molecular weight of the protein conjugate. CBD and CutC proteins are co-incubated and the molecular weight of the CutC-CBD complex is detected by UPLC / Q-TOFMS. It is found that the difference between the two molecules is 311Da, which is 3Da lower than the molecular weight of CBD, indicating that CBD and CutC are covalently bound ( Fig.11 ).

[0083] In order to determine the binding site, CutC and CutC-CBD complex were hydrolyzed with pronase, and after enzymatic treatment, the peptide coverage of CutC after enzymatic treatment was 51.7%, and the peptide coverage of CutC-CBD complex after enzymatic treatment was 53% ( Fig.12 b). Matching the peptides revealed that CBD can bind to the Cys48 site on CutC ( Fig.12 c).

[0084] Example 7: Effect of CutC and Cys48 on the Generation of TMA and TMAO

[0085] The CutC gene and the Cys48 site of the C. sporogenes strain were edited by CRISPR / Cas9 to obtain C. sporogenes with CutC and Cys48 sites deleted. The mutant strains were cultured in Mega medium containing 15mmol / L choline, and after anaerobic culture at 37℃ for 12h, 0.2mol / L formic acid was added to terminate the reaction. The protein was then precipitated with 80% cold methanol, centrifuged at 14000rpm, 4℃ for 20min, and the supernatant was obtained. The TMA content was measured by UPLC-MS / MS method. Fig.13 As shown in a, the ability of the mutant strain to utilize choline to produce TMA in vitro was significantly reduced compared with the non-mutated strain.

[0086] Using antibiotic-induced microbial consumption mice as the research object, the effects of wild-type, CutC and Cys48 mutant strain colonization on TMAO production in mice were investigated. It was found that compared with the wild-type, the TMAO content in the mice colonized with CutC and Cys48 mutant strains was significantly reduced ( Fig.13 b), the above results indicate that CutC and Cys48 are the key sites for TMAO generation.

[0087] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. Use of cannabidiol CBD in the preparation of CutC enzyme targeted inhibitor binding agent, characterized in that: The CutC enzyme has Gene ID: 69425759 on NCBI.

2. The use according to claim 1, characterized in that The binding target of the CBD is located at a cysteine ​​residue in the CutC enzyme.

3. The use according to claim 1, characterized in that The binding target of the CBD is located at the Cys48 site in the CutC enzyme.

4. The use according to claim 1, characterized in that The CutC enzyme targeted inhibition binding agent includes a pharmaceutically acceptable carrier.

5. A method for inhibiting CutC enzyme activity not for the purpose of treating a disease, characterized in that: The method is to use CBD to inhibit CutC.

6. The method according to claim 5, characterized in that CBD is brought into contact with CutC to inhibit the enzymatic activity of CutC.

7. Application of cannabidiol (CBD) in the preparation of TMA production inhibitors.

8. The use according to claim 7, characterized in that: The CBD inhibits the conversion of choline to TMA.

9. Application of cannabidiol (CBD) in the preparation of TMAO production inhibitors.

10. The use according to claim 9, characterized in that: The CBD inhibits the decomposition of choline into TMA by inhibiting the activity of CutC enzyme, and further affects the oxidation of TMA to generate TMAO.

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