Online screening of acetylcholinesterase inhibitors in natural products using monolith-based immobilized capillary enzyme reactors combined with liquid chromatography-mass spectrometry
Introduction
Traditional Chinese medicines (TCMs) are attracting increasing attention all over the world, due to their long historical clinical practice and appealing therapeutic efficacy. Moreover, TCMs possess high chemical scaffold diversity and can be considered as a huge and invaluable source of bioactive compounds for discovering promising new drugs [1,2]. However, because of the chemical complexity of TCMs, it is neither easy to identify bioactive constituents nor to elucidate their pharmacological mechanism. The conventional bioassay-guided fractionation approach has been a mainstream method for discovering bioactive compounds from natural products. Unfortunately, the isolation procedures central to this approach are usually labor-intensive, time-consuming and costly, and in many cases lead to the loss of bioactive compounds due to dilution and decomposition as well as sticking to vials, tubes, etc. [3]. Therefore, it is desirable to establish reliable and rapid methods for screening and identifying bioactive constituents from TCMs directly.
Taking advantage of good selectivity and high throughput, affinity-based approaches coupled to advanced chemical detectors have been frequently used to screen bioactive compounds in TCMs [[4], [5], [6], [7]]. Ligand fishing is a well-developed affinity-based technique in which selective binding of ligands to target enzymes or receptors allows separation from unbound components of TCMs. The bound ligands are subsequently dissociated and identified using liquid chromatography-mass spectrometry (LC–MS). Up to now, ligand fishing experiments have been carried out in different formats, including ultrafiltration [8], equilibrium dialysis [9], nanotubes [10], magnetic beads [11,12], zeolite [13] and hollow fibers [14]. These methods are mostly applied in an offline mode, which often is tedious and suffers from time-consuming analytical steps involving incubation, separation, dissociation, and analysis. Online ligand fishing may be more attractive, since the incubation, ligand-enzyme/receptor complex isolation, dissociation and HPLC–MS analysis can be carried out in a continuous, automated fashion, which can greatly enhance the screening efficiency [15].
Affinity based solid-phase extraction columns, which use enzyme-functionalized media for capturing potential ligands, have been employed for online ligand fishing. Jonker et al. used dynamic protein-affinity chromatography solid-phase extraction (DPAC-SPE) combined with LC–MS for screening and identifying estrogen receptor alpha (ERα) ligands in complex mixtures. However, this DPAC-SPE method can only be used for fishing of His-tagged proteins [16]. Recently, Peng et al. established online coupling of an affinity SPE column with LC–MS/MS for fishing xanthine oxidase (XO) inhibitors which allowed rapid isolation and identification of inhibitors from complex mixtures [17]. However, the efficient packing of affinity SPE columns, particularly in micro- and capillary format, can be difficult. Polymeric monoliths have shown to be a highly useful alternative support material to immobilize proteins for e.g. proteomics studies [18], ligand-protein binding studies, and ligand affinity ranking studies [[19], [20], [21]]. So far, the use of monolith-based immobilized capillary enzyme reactors (ICERs) for online ligand fishing, particularly in relation to TCM profiling, has not been reported.
When applying ligand fishing methods, due attention should be paid to the prevention of false positives caused by non-specific binding of compounds to the support material and/or non-functional sites of the enzyme [11,22]. Recently, Chen et al. developed an online comparative cell membrane chromatography (CMC) method by simultaneously using CMC columns packed with normal and pathological tissue-derived silica. This approach effectively increased the specificity of the screening results through visualized comparison of the chromatographic affinity behaviors between normal and pathological CMC columns [23].
Acetylcholinesterase (AChE) can terminate nerve impulse by hydrolyzing active neurotransmitter acetylcholine (ACh) in central nervous system (CNS) [24]. The inhibition of AChE from breaking down acetylcholine (ACh) is one of the most important therapeutic strategies in Alzheimer's disease treatment. Furthermore, AChE inhibitors can be used as insecticides to kill insects [25,26]. It is of importance to find new inhibitors that could modulate AChE activity. Some AChE-based immobilized enzyme reactors (AChE-IMERs) have already been developed for screening AChE inhibitors from pure compound library or assessing the overall inhibitory activity of natural products [[27], [28], [29], [30]]. For example, Bartolini et al. developed a human recombinant AChE micro-immobilized enzyem reactor (hrAChE-IMER) by immobilizing hrAChE on monolithic disk (12mm × 3mmi.d.) [31]. The prepared hrAChE-IMER allowed to screen potential hrAChE inhibitors rapidly from pure compound library, but it was not used as SPE column to directly fish ligands in natural products.
In this study, AChE-ICERs and control-ICERs were prepared through immobilizing AChE onto the surface of a poly (glycidyl methacrylate-co-ethylene dimethacrylate) (poly (GMA-co-EDMA)) monolithic support through a ring opening reaction between epoxy groups and amine groups. The resulting AChE-ICER and control-ICER were installed in parallel as SPE columns to establish a comparative online ligand fishing platform for rapid separation and identification of AChE ligands in TCMs (as shown in Fig. 1). With this system, ligands are first captured on the AChE-ICER, while inactive compounds are flushed to waste by washing buffer. For identification, the bound ligands are desorbed and eluted to LC–MS through valve switching. Parallel comparison is conducted by performing two subsequent analytical runs on the different SPE columns to eliminate false results caused by non-specific binding. The applicability of this comparative online ligand fishing platform was tested by screening AChE inhibitors from extracts of Corydalis yanhusuo. The activity of the found ligands was verified by an AChE inhibitory assay.
Section snippets
Chemicals and materials
Acetylcholinesterase from Electrophorus electricus (eelAChE) type VI-S, acetylthiocholine iodide (ATCh) and 5,5′-dithio-bis-(2-nitrobenzoic acid) (DTNB or Ellman's reagent) were purchased from Sigma-Aldrich (Shanghai, China). 3-(trimethoxysilyl)propyl methacrylate (γ-MAPS), 2,2′-azobisisobutyronitrile (AIBN), glycidyl methacrylate (GMA), ethylene dimethacrylate (EDMA), galantamine, 1,4-butanediol, 1-propanol and ammonium acetate were all purchased from Aladdin Chemicals (Shanghai, China).
Preparation of the poly (GMA-co-EDMA) monolith
Poly (GMA-co-EDMA) monolith has been commonly employed as the support of choice for immobilizing biological agents. This is not only because of advantages related to organic monoliths, such as simple preparation and high stability under diverse pH conditions (pH 2–12), but also due to presence of highly reactive epoxy groups [34,35]. Various binding agents, such as enzymes and receptors, can be easily introduced to this type of monolithic surface via a ring opening reaction with epoxy groups [36
Conclusions
In this research, a comparative online ligand fishing platform integrating both functional and denatured monolith-based AChE-ICERs with LC–MS is presented. The label-free ligand-fishing system successfully allowed screening and identification of AChE ligands from natural products in an automated manner. Polymeric monolith based AChE-ICERs with good physicochemical properties could be prepared straightforwardly. A comparison of the retention behavior of analytes on both functional and denatured
Acknowledgements
We gratefully appreciate the financial support from the National Natural Science Foundation of China (81673391), the Science and Technology Planning Project of Guangdong Province, China (2016A040403056) and the International Science and Technology Cooperation Program of Guangzhou, China (201807010022).
References (50)
Natural products in drug discovery
Drug Discov. Today
(2008)- et al.
New trends in LC protein ligand screening
J. Pharm. Biomed. Anal.
(2014) - et al.
Affinity selection-mass spectrometry screening techniques for small molecule drug discovery
Curr. Opin. Chem. Biol.
(2007) - et al.
Studies on the interactions between ginsenosides and liposome by equilibrium dialysis combined with ultrahigh performance liquid chromatography-tandem mass spectrometry
J. Chromatogr. B
(2013) - et al.
Fabrication of enzyme-immobilized halloysite nanotubes for affinity enrichment of lipase inhibitors from complex mixtures
J. Chromatogr. A
(2015) - et al.
Angiotensin converting enzyme immobilized on magnetic beads as a tool for ligand fishing
J. Pharm. Biomed. Anal.
(2017) - et al.
Hollow fiber based affinity selection combined with high performance liquid chromatography-mass spectroscopy for rapid screening lipase inhibitors from lotus leaf
Anal. Chim. Acta
(2013) - et al.
Screening of protein-ligand interactions using dynamic protein-affinity chromatography solid-phase extraction-liquid chromatography-mass spectrometry
J. Chromatogr. A
(2008) - et al.
A one-step preparation method of monolithic enzyme reactor for highly efficient sample preparation coupled to mass spectrometry-based proteomics studies
J. Chromatogr. A
(2015) - et al.
Use of peak decay analysis and affinity microcolumns containing silica monoliths for rapid determination of drug-protein dissociation rates
J. Chromatogr. A
(2011)
Evaluation of capillary chromatographic supports for immobilized human purine nucleoside phosphorylase in frontal affinity chromatography studies
J. Chromatogr. A
Sequential injection affinity chromatography utilizing an albumin immobilized monolithic column to study drug-protein interactions
J. Chromatogr. A
Screening for selective inhibitors of xanthine oxidase from Flos Chrysanthemum using ultrafiltration LC-MS combined with enzyme channel blocking
J. Chromatogr. B
A method to estimate acetylcholinesterase-active sites and turnover in insects
Anal. Biochem.
Acetylcholinesterase capillary enzyme reactor for screening and characterization of selective inhibitors
J. Pharm. Biomed. Anal.
Monolithic micro-immobilized-enzyme reactor with human recombinant acetylcholinesterase for on-line inhibition studies
J. Chromatogr. A
A new and rapid colorimetric determination of acetylcholinesterase activity
Biochem. Pharmacol.
Effect of particle size on the rate of enzymatic hydrolysis of cellulose
Carbohydr. Polym.
Optimization of a trypsin-bioreactor coupled with high-performance liquid chromatography-electrospray ionization tandem mass spectrometry for quality control of biotechnological drugs
J. Chromatogr. A
New monolithic chromatographic supports for macromolecules immobilization: challenges and opportunities
J. Pharm. Biomed. Anal.
Highly sensitive detection of organophosphorus insecticides using magnetic microbeads and genetically engineered acetylcholinesterase
Biosens. Bioelectron.
Acetylcholinesterase inhibitors from plants
Phytomedicine
Qualitative and quantitative determination of ten alkaloids in traditional Chinese medicine Corydalis yanhusuo W.T. Wang by LC-MS/MS and LC-DAD
J. Pharm. Biomed. Anal.
Potent AChE and BChE inhibitors isolated from seeds of Peganum harmala Linn by a bioassay-guided fractionation
J. Ethnopharmacol.
Indole alkaloids from Ervatamia hainanensis with potent acetylcholinesterase inhibition activities
Bioorg. Med. Chem. Lett.
Cited by (56)
Advances in screening assays for identifying cholinesterase ligands
2023, TrAC - Trends in Analytical ChemistryThe composition, pharmacological effects, related mechanisms and drug delivery of alkaloids from Corydalis yanhusuo
2023, Biomedicine and PharmacotherapyAnalysis of natural products by liquid chromatography
2023, Liquid Chromatography: ApplicationsA novel strategy for screening angiotensin-converting enzyme inhibitors from natural products based on enzyme-immobilized ligand fishing combined with active-site blocking and directional enrichment
2022, Journal of Chromatography B: Analytical Technologies in the Biomedical and Life SciencesImmobilized-enzyme reactors integrated into analytical platforms: Recent advances and challenges
2021, TrAC - Trends in Analytical ChemistryCitation Excerpt :Open-tubular columns/open channels (OT), either with a porous layer (PLOT) or with chemical modification (i.e. nano-architectures [42]), provide low-pressure candidates for online integration and automation of IMERs [36,38,43,44]. OT-IMERs can be housed in microfluidic chips [53], capillaries [14,20,34,36,37,39–41,43], or parallel-channel capillaries (photonic crystal fibres) [42,44]. The end of an IMER capillary can be pulled to form an electrospray (ESI) needle to facilitate the coupling with mass-spectrometric (MS) detection [54].
Comprehensive screening and separation of cyclooxygenase-2 inhibitors from Pterocephalus hookeri by affinity solid-phase extraction coupled with preparative high-performance liquid chromatography
2021, Journal of Chromatography B: Analytical Technologies in the Biomedical and Life SciencesCitation Excerpt :Therefore, affinity solid-phase extraction (ASPE) column, filled with enzyme-functionalized silica gel, combined with HPLC could screen compounds with binding affinity effectively. Potential ligands of α-glucosidase [21], xanthine oxidase [22], and acetylcholinesterase [23] have successfully screened through affinity solid-phase ligand-fishing with HPLC-DAD-MS. Preparative HPLC, the most powerful and widely used separation technique [24], is the method for isolating pure and single components from complex extracts due to outstanding performance, separation reproducibility, and real-time detection. Therefore, in order to obtain potential COX-2 inhibitors to clarify structure and evaluate their COX-2 inhibitory activities, we combine preparative HPLC with the affinity solid-phase extraction screen method.