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).
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