High-selectivity butyrylcholine esterase inhibitor derived from moringa seed extract as well as screening method and application of high-selectivity butyrylcholine esterase inhibitor

Abstract

The invention belongs to the technical field of medicines, and particularly relates to a group of butyrylcholine esterase (BChE) inhibitors with high selectivity and a screening method thereof, in particular to two BChE specific inhibitors, namely methyl 4-hydroxybenzyl carbamate and moringa seed extract, which are screened from a moringa seed extract. The invention also relates to application of the BChE specific inhibitor in preparation of drugs for preventing or treating Alzheimer's disease. The invention provides a brand new strategy for efficiently screening the anti-Alzheimer disease medicine.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to a group of highly selective butyrylcholinesterase (BChE) inhibitors and their screening methods, particularly to two BChE-specific inhibitors, methyl 4-hydroxybenzylcarbamate and moringa seed extract, obtained from Moringa seed extract. This invention also relates to the use of the aforementioned BChE-specific inhibitors in the preparation of drugs for the prevention or treatment of Alzheimer's disease. Background Technology

[0002] Alzheimer's disease (AD) is a neurodegenerative disease characterized by memory decline, language impairment, and a range of other cognitive impairments. The pathological changes in AD patients primarily include decreased levels of acetylcholine in the brain. β Amyloid deposition, hyperphosphorylated tau protein forming neurofibrillary tangles, biometal ion dysregulation, and inflammatory responses are among the factors contributing to Alzheimer's disease (AD). Several hypotheses exist regarding the pathogenesis of AD, with the classic cholinergic hypothesis posits that acetylcholine, a key neurotransmitter, is crucial for neural functions related to learning and memory. Numerous studies have shown a close correlation between abnormally low acetylcholine levels in the hippocampus and cortex and cognitive decline in AD patients. Acetylcholine is readily hydrolyzed into choline, a form with no neurotransmitting activity, by cholinesterase within the synaptic cleft. Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) are two key enzymes catalyzing the hydrolysis of acetylcholine and are important targets for existing AD treatments. In the cholinergic system, BChE typically acts as a complementary enzyme to AChE. Studies have reported that in AD patients, AChE enzyme activity levels may decrease to 62%-90% of normal physiological levels, while BChE enzyme activity levels may increase to 165%, indicating that BChE plays a compensatory role in the reduction of AChE activity during acetylcholine hydrolysis and participates in the hydrolysis of acetylcholine under AD pathological conditions. Furthermore, both BChE knockout and BChE inhibition produce neuroprotective effects. More importantly, BChE is considered a relatively safe therapeutic target. Most currently approved cholinesterase inhibitors (such as donepezil and galantamine) have good inhibitory effects on both AChE and BChE, but these drugs are often accompanied by various peripheral cholinergic side effects and other adverse reactions. Therefore, developing selective BChE inhibitors holds promise for both reducing peripheral cholinergic side effects and more effectively treating advanced AD. In addition to cholinergic function, inhibiting BChE can also reduce… β -Amyloid deposition further demonstrates the important role of BChE as a therapeutic target for AD.

[0003] Covalent inhibitors are drugs whose reactive functional groups can form irreversible or reversible covalent bonds with nucleophilic amino acid residues (such as cysteine, lysine, and serine) in target proteins, thereby affecting the biological function of the target proteins and producing a long-lasting and potent pharmacological effect. In the early stages of drug development, researchers tend to avoid designing covalent drugs due to concerns about potential off-target effects and adverse reactions. However, compared to traditional reversible inhibitors, covalent inhibitors have advantages such as higher occupancy rates, longer duration of action, and the potential to overcome certain drug resistance. With the development of drug design concepts and technologies, many marketed covalently bound drugs, such as ibrutinib, afatinib, and zanubrutinib, have shown superior efficacy in disease treatment, thus covalent drugs have gradually become a hot topic in new drug development. Carbamate drugs can undergo a nucleophilic substitution reaction between the carbamate fragment in their structure and the serine (Ser) residue in the active site of cholinesterase, forming a carbamylated enzyme complex, thereby leading to cholinesterase inactivation. Rosavirin is a representative drug developed based on this mechanism; however, it inhibits both AChE and BChE and may cause side effects in the digestive, nervous, and cardiovascular systems, and has gradually faded from clinical use.

[0004] Moringa seeds are derived from the Moringa plant, a member of the Moringa family. Moringa oleifera The dried seeds of *L.*, rich in carbamates and isothiocyanates, represent a potential resource of covalent inhibitors of beta-carbamate ethoxylates (BChE). Previous studies have reported that *Moringa oleifera* seed extract possesses various pharmacological activities, including inhibiting beta-carbamate ethoxylates (AChE), improving behavioral indicators in dementia mice, and reducing hippocampal neuronal damage, demonstrating potential therapeutic value for Alzheimer's disease (AD). However, the specific active components in *Moringa oleifera* seed extract that exhibit BChE inhibitory activity remain unclear. Summary of the Invention

[0005] To overcome the deficiencies and shortcomings of existing technologies, this invention aims to provide a rapid and accurate method for screening and characterizing BChE covalent inhibitors based on a chemical peptide mapping strategy. This invention also provides two BChE-specific inhibitors, methyl 4-hydroxybenzylcarbamate and moringa seed extract, obtained by the aforementioned method from Moringa seed extract, and their use in the preparation of drugs for the prevention or treatment of Alzheimer's disease.

[0006] Specifically, the present invention is achieved through the following technical solutions: According to a first aspect of the present invention, a method for screening BChE covalent inhibitors based on a chemical peptide mapping strategy is provided. The core idea is to directly react the target protein with a library of covalent compounds to be screened. Subsequently, the target protein is enzymatically digested into "compound-peptide" covalent complexes. Then, high-throughput liquid chromatography-high-resolution mass spectrometry (LC-HRMS) combined with protein data analysis software is used to identify all modified peptides and their corresponding bound compounds.

[0007] The method includes the following steps: Step 1: Preparation of Moringa seed extract: Moringa seed powder was extracted by heating and reflux with 70% methanol, and concentrated under reduced pressure until no alcohol odor was detected. Then, it was extracted three times each with petroleum ether, dichloromethane, ethyl acetate and n-butanol at a volume ratio of 1:1. The extracts were combined, concentrated and dried under reduced pressure to obtain Moringa seed extracts of different polarities. Step 2: Initial screening of enzyme inhibitory activity in different extracts of Moringa seeds: The modified Ellman method was used to determine the inhibition rate of BChE and AChE in different extracts of Moringa seeds, and the extracts with higher inhibition rates of BChE were screened for subsequent screening of active ingredients. Step 3: Co-incubation of BChE protein with Moringa seed extract: Incubate BChE protein with the Moringa seed extract to be screened in a suitable buffer system. After the reaction is completed, add an aqueous solution containing 0.1% formic acid to inactivate the protein and terminate the reaction. The DMSO control group is treated in the same way, that is, BChE protein is incubated with an equal amount of DMSO in a suitable buffer system. Step 4: Preparation of BChE polypeptide group: BChE protein in the DMSO control group or the Moringa seed extract group was fully enzymatically digested with trypsin to obtain the corresponding BChE polypeptide components of the DMSO control group and the Moringa seed extract group, respectively. Step 5: LC-HRMS detection and analysis: BChE peptide samples from the DMSO control group and the Moringa seed extract treatment group were analyzed using LC-HRMS to obtain primary and secondary mass spectrometry information of free peptides and compound-peptide covalent complexes. Step Six: Data Analysis and Identification of "Hit" Molecules: Exporting Mass Spectrometry Data (MS) 1 (Retention time, response value, etc.) Differential peptides were screened by volcano plot analysis. The Proteome Discoverer software and Sequest HT search engine were used to identify covalently modified peptide sequences and bound small molecule structures. The screened small molecules were used as candidate "hit" molecules. Step 7: Selectivity Verification: The "hit" molecules obtained from the initial screening are analyzed against BChE or AChE according to steps 2 and 3 through 6 above, respectively, to evaluate the selective inhibition ability of the compound against BChE; and Step 8; Inhibition type and enzyme kinetics study: Based on the experimental methods in Step 2, the inhibition type and enzyme kinetic parameters of the "hit" compound were determined by combining time-dependent curves, enzyme concentration-reaction rate curves, and the Lineweaver-Burk double reciprocal curve method.

[0008] Alternatively, the HRMS can be the Q Exactive MS or an instrument of the same series, or the LTQ-Orbitrap MS or an instrument of the same series, etc.

[0009] As an alternative, the "hit" molecules screened in the above steps can be isolated from Moringa seed extract, or obtained by purchasing reference standards, or by chemical synthesis, for subsequent verification.

[0010] According to a second aspect of the present invention, a group of highly selective BChE inhibitors obtained by screening using the method described in the first aspect is provided, which are methyl 4-hydroxybenzylcarbamate as shown in formula (I) or moringa seed extract as shown in formula (II): .

[0011] According to a third aspect of the invention, the use of the highly selective BChE inhibitor described in the second aspect above in the preparation of a medicament for the prevention or treatment of Alzheimer's disease is provided.

[0012] Compared with the prior art, the present invention has the following advantages: This invention provides a rapid and accurate method for screening and characterizing BChE covalent inhibitors based on a chemical peptide mapping strategy, offering a novel strategy for efficiently screening anti-Alzheimer's disease drugs. Attached Figure Description

[0013] Figure 1 Inhibition rates of different extracts from Moringa seeds and positive control drugs donepezil and rivastigmine on AChE (A) and BChE (B); Figure 2 BChE amino acid sequence and mass spectrometry analysis to identify coverage; Figure 3 Differential volcano plot of BChE trypsin-hydrolyzed peptides after reaction of dichloromethane (A) and ethyl acetate (B) extracts from Moringa seeds; Figure 4 Differential peptides m / zMS values ​​of 733.7307 (A), 837.4246 (B), 783.4138 (C), and 832.0990 (D) 2 Spectrum; Figure 5 Synthetic route of methyl 4-hydroxybenzylcarbamate; Figure 6 Differential volcano plots of BChE trypsin-hydrolyzed peptides after the reaction of methyl 4-hydroxybenzylcarbamate (A) and moringa seed extract (B); Differential volcano plots of AChE trypsin-hydrolyzed peptides after the reaction of methyl 4-hydroxybenzylcarbamate (C) and moringa seed extract (D). Figure 7 Inhibition rates of methyl 4-hydroxybenzylcarbamate (A) and moringa seed extract (B) on BChE; Inhibition rates of methyl 4-hydroxybenzylcarbamate (C) and moringa seed extract (D) on AChE; Figure 8 Dose-inhibition curves of rivastigmine (A), donepezil (B), methyl 4-hydroxybenzylcarbamate (A) and moringa seed extract (B) against BChE after pre-incubation for 3, 63 and 93 min; Figure 9 Methyl 4-hydroxybenzylcarbamate (A) and Moringa seed extract (B) at different concentrations: BChE enzyme concentration-reaction rate curves; Figure 10 Enzyme kinetics of methyl 4-hydroxybenzylcarbamate (A) and moringa seed extract (B) on BChE using Lineweaver-Burk double reciprocal curves. Detailed Implementation

[0014] Example 1: Preparation of Moringa Seed Extract Moringa seeds were pulverized using a grinder and sieved. 5 g of moringa seed powder was extracted three times with 6 times the volume of 70% methanol under reflux for 2 hours each time. The extracts were combined and concentrated under reduced pressure until no alcohol odor remained. Subsequently, the extracts were extracted three times each with petroleum ether, dichloromethane, ethyl acetate, and n-butanol. The extracts were combined, concentrated under reduced pressure, and dried to obtain 4.51 mg of petroleum ether fraction, 48.34 mg of dichloromethane fraction, 64.00 mg of ethyl acetate fraction, 94.83 mg of n-butanol fraction, and 538.21 mg of water fraction from moringa seeds.

[0015] Example 2: Determination of enzyme inhibitory activity from different extracts of Moringa seeds The inhibition rates of different extracts from Moringa seeds against BChE and AChE were determined using a modified Ellman method. Different extracts from Moringa seeds were dissolved in dimethyl sulfoxide (DMSO) to prepare 200 mg / mL stock solutions. The covalently bound positive control drug rivastigmine (purchased from Chengdu Lemeitian Pharmaceutical Technology Co., Ltd., purity >98%) and the non-covalently bound positive control drug donepezil hydrochloride (purchased from Shanghai Yuanye Biotechnology Co., Ltd., purity >98%) were prepared in DMSO and water to prepare 20 mmol / L stock solutions, respectively. BChE protein (derived from horse serum, 553 U / mg, purchased from Shanghai Yuanye Biotechnology Co., Ltd.) and AChE protein (derived from electric eel, 1872 U / mg, purchased from Sigma-Aldrich, USA) were prepared in phosphate-buffered saline (PBS, 0.1 mol / L, pH 8.0) to prepare 1000 U / mL stock solutions, respectively. All stock solutions were stored at –20 °C.

[0016] BChE and AChE stock solutions were diluted to 1 U / mL and 0.1 U / mL, respectively, with different extracts from Moringa seeds diluted to 1 mg / mL. S-iodinated butyrylthiocholine (BTCI, BChE substrate, purchased from Shanghai Yuanye Biotechnology Co., Ltd., purity >98%) or iodothioacetylcholine (ATCI, AChE substrate, purchased from Shanghai Yuanye Biotechnology Co., Ltd., purity >98%) were prepared into 0.6 mmol / L stock solutions with water. 5,5-dimercapto-2,2-dinitrobenzoic acid (DTNB, purchased from Shanghai Yuanye Biotechnology Co., Ltd., purity >98%) was dissolved in PBS buffer to prepare a 0.6 mmol / L stock solution. The BTCI or ATCI stock solutions were then mixed thoroughly with the DTNB stock solution at a 1:1 ratio, stored in the dark, and used immediately. Activity was measured using 96-well plates, with three replicates for each extract, plus drug control wells, blank reaction wells, and blank control wells. The specific conditions for adding the solution are as follows: Drug reaction well: 50 µL drug solution + 50 µL enzyme solution Drug control wells: 50 µL drug solution + 50 µL PBS solution Blank reaction wells: 50 µL PBS solution + 50 µL enzyme solution Blank control wells: 50 µL PBS solution + 50 µL PBS solution After adding the drug solution and enzyme solution to the 96-well plate, mix thoroughly and incubate at 37 °C for 90 min. Then add 100 µL of a mixed solution of BTCI and DTNB or ATCI and DTNB, mix thoroughly, and incubate at 37 °C for 15 min. Measure the absorbance (OD) at 405 nm using a microplate reader.

[0017] The results are as follows Figure 1 As shown, different extracts from Moringa seeds exhibited varying degrees of inhibitory activity against BChE and AChE. Different positive control agents showed inhibition rates exceeding 50% against both AChE and BChE. The total extract and aqueous fraction (W) of Moringa seeds showed inhibitory activities of approximately 14% and 27% against AChE, respectively, while the petroleum ether (P), dichloromethane (C), ethyl acetate (E), and n-butanol fractions (B) showed little or no inhibitory activity. Figure 1 A). The inhibitory activity of different extracts of Moringa seeds against BChE showed drastically different results, with dichloromethane (C), ethyl acetate (E), n-butanol (B), and water (W) all showing varying degrees of BChE inhibitory activity. Figure 1 The inhibition rates of different extracts of Moringa seeds, especially the dichloromethane and ethyl acetate fractions, were relatively high, reaching 30% and 74%, respectively. These results indicate that different extracts of Moringa seeds, particularly the dichloromethane and ethyl acetate fractions, have the potential to screen for selective inhibitors of BChE.

[0018] Example 3: Screening of BChE covalent inhibitors in the dichloromethane and ethyl acetate extracts of Moringa seeds based on chemical peptide mapping The dichloromethane and ethyl acetate extracts of Moringa seeds, which exhibited strong BChE inhibitory activity, were selected for screening as covalent inhibitors of BChE. The dichloromethane and ethyl acetate extracts of Moringa seeds were dissolved in DMSO to prepare 200 mg / mL stock solutions. 50 µg of commercially available BChE protein was incubated with 2 µL of the Moringa seed dichloromethane extract stock solution in 200 µL of PBS buffer (pH 7.4) for 12 h. The control group was incubated simultaneously with 2 µL of DMSO solution. Each sample was tested in triplicate. After incubation, 0.1% formic acid was added to inactivate the protein and terminate the reaction. BChE protein in the DMSO control group or the Moringa seed extract group was fully digested with trypsin to obtain BChE peptide solutions for the DMSO control group or the Moringa seed extract group, respectively. The digested peptide solutions were concentrated and dried using a vacuum concentrator, and then reconstituted with an aqueous solution containing 0.1% formic acid to a final volume of 30 µL. Subsequently, LC-Q Exactive MS was used to analyze the BChE-digested peptides in the DMSO control group or the Moringa seed extract group. Chromatographic column: AcquityUPLC HSS T3 column (2.1 mm × 100 mm, 1.8 μm, Waters Corporation, USA); mobile phase: 0.1% (v / v) formic acid water (A) and 0.1% (v / v) formic acid acetonitrile (B); elution program: 0–30 min, 5–28% B; 30–35 min, 28–45% B; 35–38 min, 45–95% B; 38–40 min, 95% B; 40–40.1 min, 95–5% B; 40.1–45 min, 5% B; flow rate: 0.25 mL / min; column temperature: 40℃; injection volume: 10 μL. The mass spectrometry acquisition mode was set to Full-MS / DDA mode. 1 Scan range m / z 200–2000, resolution 70,000, MS 2 The resolution was 17,500 nm, and the normalized collision energy (NCE) was 20%, 30%, and 40%. Primary mass spectrometry (MS) spectra of free peptides and compound-peptide covalent complexes were finally obtained. 1 ) and secondary mass spectrometry (MS) 2 Information. MS data from the DMSO control group or the Moringa seed extract group will be collected. 1Information, retention time, and response values ​​were derived, and t-tests were used to calculate the fold change (FC) and p-value of peptides between the control and reaction groups. Volcano plot analysis was performed, with |log2FC| ≥ 2.323 and p-value ≤ 0.01, to screen upregulated and downregulated peptides. Further analysis using Proteome Discoverer software and the Sequest HT search engine was conducted to retrieve the raw mass spectrometry data, identifying upregulated and downregulated peptides. The MS values ​​of the upregulated and downregulated peptides were compared. 1 Mass-to-charge ratio difference and MS 2 Fragment ion differences are used to identify covalently bound small molecule fragments and binding sites, and the screened small molecules are used as candidate "hit" molecules.

[0019] Using the Proteome Discoverer software, a total of 25 peptides belonging to BChE were identified. The amino acid sequences of BChE are as follows: Figure 2 As shown, the identification coverage reached 50.87%. The peptide fragments obtained by reacting the dichloromethane and ethyl acetate extracts of Moringa seeds with BChE were analyzed by MS. 1 Changing volcano map as follows Figure 3 As shown, the upregulated and downregulated peptides selected from the two groups are quite similar. Based on the matching results of the Proteome Discoverer software, the downregulated peptides were further analyzed. m / z 733.7307 ([M+3H]) 3+ ) belongs to the sequence 191 SVTLFGESAGAASVSLHLLSPR 213 [M+3H] 3+ The quasi-molecular ion peak indicates that this peptide is a characteristic peptide containing the active site Ser198 of BChE. The key to BChE's physiological function lies in the hydroxyl group of Ser198 acting as an electron donor, leading to a nucleophilic substitution reaction with butyrylcholine or acetylcholine. The acyl fragment forms a covalent bond with the hydroxyl group of Ser198, resulting in the hydrolysis of butyrylcholine or acetylcholine. The upregulated peptides mainly include... m / z 837.4246 ([M+3H]) 3+ ), 783.4138 ([M+3H]) 3+ ) and 832.0990 ([M+3H] 3+ The molecular mechanism of BChE covalent inhibitors involves their covalent binding to the Ser198 active site of the BChE enzyme, thereby occupying the active site and preventing it from exerting its physiological function. Based on this, it is speculated that... m / z 837.4246 ([M+3H]) 3+ ), 783.4138 ([M+3H])3+ ) and 832.0990 ([M+3H] 3+ All three upregulated peptides are polypeptides. 191 SVTLFGESAGAASVSLHLLSPR 213 Covalent complexes formed with small molecules. Further analysis. m / z 733.7307 and MS of three upregulated peptides 2 Spectra were used to analyze the covalently bound small molecule structures and possible binding sites. m / z The process of identifying small molecule structures and binding sites is illustrated using 837.4246 as an example. m / z 733.7307 and m / z MS 837.4246 2 Spectrum as Figure 4 A and Figure 4 As shown in B, overall m / z 837.4246 fragment ions and m / z 733.7307 contains some shared fragment ions such as y1 + ~y 14 + With b2 + ~ b5 + . m / z 837.4246 generated m / z Elemental analysis of the 1182.6010 fragment ion suggests that the small molecule-peptide covalent complex lost a rhamnose residue (C6H). 10 The doubly charged fragment ions formed by O4 (146.05 Da) suggest that this small molecule fragment structure contains a rhamnose group. The remaining key fragment ions, after assignment, are y + or b + The ion loses a rhamnose residue to form, with m / z 733.7307 corresponds to y + or b + Compared to each other, the molecular weights of the ions differ by 165.03 Da. (Based on y...) 14 + ( m / z 1378.7791) and [y 15 –C6H 10 O4] 2+ ( m / zTwo key fragment ions (815.9272) can... m / z 837.4246 was identified as a small molecular fragment containing a rhamnose group in a certain molecular structure that binds to the Ser198 residue. 191 SVTLFGESAGAASVSLHLLSPR 213 Peptide-small molecule covalent complex. Further calculations. m / z 837.42 and m / z The molecular weight difference between 733 and 73 is 311.0817 Da. Through elemental analysis, it is inferred that this polypeptide sequence binds to a peptide with the molecular formula C. 14 H 17 A fragment of NO5S. Using the same method, we can obtain... m / z 783.4138 ( Figure 4 C) and 832.0990 ( Figure 4 D) are C8H7NO2 and C respectively. 14 H 17 NO6 and 191 SVTLFGESAGAASVSLHLLSPR 213 The quasi-molecular ion peaks of the formed polypeptide-small molecule covalent complexes all had Ser198 residues as their binding sites. Since small molecules may undergo nucleophilic substitution or nucleophilic addition reactions with Ser198 residues—meaning small molecules can bind to Ser198 residues through their entire structure or a partial structural fragment—a series of compounds derived from Moringa plants with the aforementioned molecular formula fragments were screened in literature and databases such as PubMed. Ultimately, Moringa seed extract and methyl 4-hydroxybenzylcarbamate were preliminarily classified as "hit" molecules.

[0020] Example 4: Selectivity Validation of Candidate Covalent Inhibitors Moringa seed extract was purchased from Chengdu Lemeitian Pharmaceutical Technology Co., Ltd., and its purity was >98% as determined by HPLC. Methyl 4-hydroxybenzylcarbamate was synthesized in the laboratory, and its purity was verified to be >95% by HPLC. The synthetic route of methyl 4-hydroxybenzylcarbamate is as follows: Figure 5As shown. 1.0033 g of triphosgene (purchased from Shanghai Yuanye Biotechnology Co., Ltd., purity >99%) was dissolved in 25 mL of dry dichloromethane in a round-bottom flask and kept in an ice bath. 0.2991 g of p-hydroxybenzylamine (purchased from Shanghai Yuanye Biotechnology Co., Ltd., purity >98%) dissolved in 30 mL of dry dichloromethane and 70 mL of tetrahydrofuran was added dropwise to the round-bottom flask, and the reaction was allowed to proceed for approximately 2 h. Subsequently, the mixture was heated under reflux in an oil bath at 50 °C for 1 h. After the reaction was completed, the mixture was concentrated and dried under reduced pressure to obtain a light brown oily substance, which was 4-hydroxybenzyl isocyanate. 15 mL of dry dichloromethane and 15 mL of methanol were added to this product, and the mixture was stirred at room temperature for 4 h. After concentration and drying under reduced pressure, 0.4893 g of a yellow oily product was obtained. Purification was performed using normal-phase silica gel, with the eluent transitioning from dichloromethane:methanol (100:1) to dichloromethane:methanol (1:1), yielding 276.90 mg of crude methyl 4-hydroxybenzylcarbamate. Further purification using a preparative buffer yielded 220.00 mg of a pale yellow oily final product.

[0021] 4-hydroxybenzylcarbamate and moringa seed extract were each prepared into 1000 mmol / L stock solutions using DMSO and stored at –20°C protected from light. Following the steps in Example 3 above, the peptides of 4-hydroxybenzylcarbamate and moringa seed extract after co-incubation with BChE or AChE were analyzed. Figure 6 A and Figure 6 As shown in Figure B, the upregulated and downregulated peptides after co-incubation of methyl 4-hydroxybenzylcarbamate with moringa seed extract and BChE were basically consistent with the results from the ethyl acetate extract of moringa seeds. Furthermore, the peptide group after incubation of moringa seed extract and BChE also showed the presence of... m / z 837.4246 ([M+3H]) 3+ ) and 832.0990 ([M+3H] 3+ This indicates that moringa seed extract may have been oxidized during the incubation process, generating a product with C... 14 H 17 The active structure of the NO6 fragment allows it to covalently bind to the Ser198 residue of BChE. Methyl 4-hydroxybenzylcarbamate ( Figure 6 C) and Moringa seed extract ( Figure 6 D) After co-incubation with AChE, no related upregulated or downregulated peptides were screened, indicating that methyl 4-hydroxybenzylcarbamate and moringa seed extract may not have covalently bound to AChE. These results suggest that methyl 4-hydroxybenzylcarbamate and moringa seed extract, as candidate inhibitors, can selectively covalently bind to BChE but not to AChE.

[0022] Furthermore, based on the Ellman experiment in Example 2, the selective inhibitory effects of methyl 4-hydroxybenzylcarbamate and moringa seed extract on BChE and AChE enzymes were verified. methyl 4-hydroxybenzylcarbamate and moringa seed extract were prepared into 1000 mmol / L stock solutions using DMSO, and then serially diluted with PBS buffer to 2000, 1000, 500, 250, and 125 µmol / L. Donepezil hydrochloride and rivastigmine stock solutions were diluted with PBS buffer to 1 µmol / L solutions, respectively, and used as positive control agents for AChE and BChE enzyme activity assays. The inhibition rates of methyl 4-hydroxybenzylcarbamate and moringa seed extract on AChE and BChE at different concentrations were determined according to the method in Example 2. Figure 7 A and Figure 7 As shown in Figure B, the inhibition rates of moringa seed extract and methyl 4-hydroxybenzylcarbamate on the enzyme activity of BChE gradually increased with increasing concentration, reaching over 50% inhibition at a concentration of 2000 µmol / L. However, at the same concentration, moringa seed extract (… Figure 7 C) showed an inhibition rate of less than 4% against AChE, while methyl 4-hydroxybenzylcarbamate ( Figure 7 D) The inhibition rate of AChE is less than 20%. This indicates that methyl 4-hydroxybenzylcarbamate and moringa seed extract have a stronger selective inhibitory effect on BChE, and have the potential to become highly selective BChE inhibitors and play a role in the treatment of AD.

[0023] Example 5: Determination of the type of inhibition of BChE by methyl 4-hydroxybenzylcarbamate and Moringa seed extract and enzyme kinetic study The time-dependent curves of the inhibitory activities of rivastigmine, donepezil, methyl 4-hydroxybenzylcarbamate, and moringa seed extract were determined using the method described in Example 2. Moringa seed extract was diluted with PBS to 5000, 2500, 1250, 625, 312.5, 156.25, 78.13, and 39.06 µM; methyl 4-hydroxybenzylcarbamate was diluted with PBS to 4000, 2000, 1000, 500, 250, 125, 62.5, and 31.25 µM; rivastigmine was diluted with PBS to 20, 10, 5, 2.5, 1.25, 0.63, 0.31, and 0.16 µM; and donepezil hydrochloride was diluted with PBS to 500, 250, 125, 62.5, 31.25, 15.63, 7.81, and 3.91 µM. The enzyme inhibition rate was measured at 3, 63 and 93 min after drug administration, and enzyme inhibition rate-concentration curves were constructed.

[0024] The inhibition rate curve of rivastigmine, a representative covalent inhibitor of BChE, shows a significant shift with increasing reaction time. Figure 8 A), IC 50The value decreased significantly. Conversely, the results for the non-covalent inhibitor donepezil showed the opposite; that is, with increasing reaction time, the IC50 value of donepezil for BChE decreased. 50 The values ​​did not change much, and even showed an increasing trend. Figure 8 B). Because the enzymatic reaction of covalent inhibitors requires two steps—an initial reversible binding and the formation of an irreversible covalent bond—the second step, forming a stable covalent bond, is a chemical reaction that requires overcoming an energy barrier and has a slower reaction rate. Therefore, the enzyme inhibition curve of covalent inhibitors exhibits a time-dependent characteristic. Non-covalent inhibitors, on the other hand, only require the formation of an enzyme-inhibitor complex through non-covalent interactions. This step occurs almost instantaneously, thus achieving maximum inhibition rate in a short time. Furthermore, because enzymes may become inactive under heating conditions with prolonged reaction time, their IC50 values ​​also decrease. 50 The value may gradually increase with increasing reaction time. The time dependence curves for methyl 4-hydroxybenzylcarbamate and moringa seed extract are shown below. Figure 8 C and Figure 8 D. Both are similar to rivastigmine, exhibiting a clear time-dependent characteristic, indicating that methyl 4-hydroxybenzylcarbamate and moringa seed extract are both BChE covalent binding inhibitors.

[0025] The inhibition types of methyl 4-hydroxybenzylcarbamate and moringa seed extract were determined using enzyme concentration-reaction rate curves. BChE was diluted with PBS to prepare solutions of 2, 1, 0.5, 0.25, and 0.1 U / mL. Moringa seed extract was diluted with PBS to 2000, 1000, and 500 µM, and methyl 4-hydroxybenzylcarbamate was diluted with PBS to 1000, 500, and 250 µM. The assay method was the same as in Example 2. The test compounds and enzymes were incubated at 37 °C for 90 min, followed by the addition of a mixed solution of BTCI and DTNB. The reaction was incubated at 37 °C for 20 min, and the absorbance OD1 was measured. The reaction was then incubated at 37 °C for another 10 min, and the absorbance OD2 was measured again. The reaction rate V was calculated as (OD2 - OD1) / time. Enzyme concentration-reaction rate curves for different concentrations of the test compounds were plotted. The results showed that with the increase of methyl 4-hydroxybenzylcarbamate (… Figure 9 A) and Moringa seed extract ( Figure 9 B) As the concentration increases, the slope of the enzyme concentration-reaction rate curve gradually decreases, indicating that methyl 4-hydroxybenzylcarbamate and moringa seed extract are both reversible inhibitors of BChE. This also indicates that methyl 4-hydroxybenzylcarbamate and moringa seed extract will not produce a continuous irreversible inhibitory effect, thus preventing toxicity.

[0026] Enzyme kinetics were further studied using the Lineweaver-Burk double reciprocal curve method. BChE was prepared at 1 U / mL, and BTCI was prepared at concentrations of 100, 200, 400, 600, and 1000 µmol / L, respectively, and mixed with 0.6 mmol / L DTNB in ​​equal proportions. After incubating different concentrations of the test drug with BChE at 37 °C for 90 min, different concentrations of BTCI and DTNB mixed solutions were added, and absorbance values ​​were measured immediately, with measurements taken every 2 min for 30 min. Data from the first 15 min were used to plot a 1 / [S]-1 / [V] double reciprocal curve, where 1 / [S] is the reciprocal of the substrate BTCI concentration, and 1 / [V] is the reciprocal of the reaction rate. Figure 10 As shown in Figure A, with the increase of the concentration of methyl 4-hydroxybenzylcarbamate, the intercept of the ordinate gradually decreases, i.e., the maximum reaction rate V. max The Km value gradually decreases, while the intercept on the x-axis remains constant, indicating that methyl 4-hydroxybenzylcarbamate is a non-competitive inhibitor of BChE. Figure 10 As shown in Figure B, with the increase of moringa seed extract concentration, the intercepts of both the x-axis and y-axis gradually decrease, i.e., K... m and V max The decrease in size suggests that moringa seed extract is an anti-competitive inhibitor of BChE.

[0027] Obviously, the above embodiments are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation of the present invention. Those skilled in the art can make various modifications and variations to the present invention without departing from the spirit and scope of the invention. Therefore, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention also intends to include these modifications and variations.

Claims

1. A method for screening covalent inhibitors of BChE based on the chemical peptide mapping strategy, characterized by: The method comprises the following steps: Step one: Preparation of Moringa oleifera seed extract: Moringa oleifera seed powder is extracted by heating reflux with 70% methanol, concentrated under reduced pressure until no alcohol taste is left, then sequentially extracted with petroleum ether, dichloromethane, ethyl acetate, n-butanol at a volume ratio of 1:1, three times each, the extracts are combined and dried under reduced pressure to obtain Moringa oleifera seed extract fractions of different polarities; Step two: Preliminary screening of enzyme inhibitory activity of Moringa oleifera seed extract fractions: the inhibitory rates of Moringa oleifera seed extract fractions on BChE and AChE are determined by the modified Ellman method, and the Moringa extract fraction with a higher BChE inhibitory rate is selected for subsequent screening of active ingredients; Step three: Co-incubation of BChE protein and Moringa oleifera seed extract fraction: BChE protein and the Moringa oleifera seed extract fraction to be screened are incubated in a suitable buffer system, after the reaction is completed, 0.1% formic acid aqueous solution is added to inactivate the protein and thus terminate the reaction; Step four: Preparation of BChE polypeptide group: the BChE protein in the DMSO control group or the Moringa oleifera seed extract fraction group is fully enzymolyzed by trypsin to obtain polypeptide components of the corresponding control group and covalently modified group samples, respectively; Step five: LC-HRMS detection and analysis: the BChE polypeptide samples of the DMSO control group and the Moringa oleifera seed extract fraction group are analyzed by LC-HRMS to obtain the mass spectrum information of free polypeptides and compound-polypeptide covalent complexes; Step six: Data analysis and "hit" molecule identification: the mass spectrum data is derived, the differential peptides are screened by volcano plot analysis, the covalently modified peptide sequence and the combined small molecule structure are identified by Proteome Discoverer software and Sequest HT search engine, and the screened small molecules are used as candidate "hit" molecules; Step seven: Selectivity verification: the "hit" molecules obtained by preliminary screening are analyzed according to steps two and steps three to steps six with BChE or AChE, respectively, to evaluate the selective inhibitory ability of the compounds on BChE; and Step eight: Inhibition type and enzyme kinetics research: the inhibition type and enzyme kinetics parameters of the "hit" compound are determined according to the experimental method of step two combined with time-dependent curve, enzyme concentration-reaction rate curve and Lineweaver-Burk double-reciprocal curve method.

2. The method of claim 1, wherein: In step five, HRMS is Q Exactive MS or its same series instrument, or LTQ-Orbitrap MS or its same series instrument.

3. The method of claim 1, wherein: In step six, the mass spectrometry data includes MS 1 , retention time, response value.

4. The method of claim 1, wherein: In step seven, the screened "hit" molecules are separated from Moringa oleifera seed extract, or control samples are purchased, or obtained by chemical synthesis, so as to perform subsequent verification.

5. The highly selective BChE inhibitor screened by the method of any one of claims 1 to 4, characterized by: which are 4-hydroxybenzyl carbamate as shown in the following formula (I) or moringa seed extract as shown in the following formula (II): 。 6. Use of the BChE inhibitor with high selectivity according to claim 5 in the preparation of a medicament for preventing or treating Alzheimer's disease.

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