Online screening of acetylcholinesterase inhibitors in natural products using monolith-based immobilized capillary enzyme reactors combined with liquid chromatography-mass spectrometry

Abstract

In order to develop a direct and reliable method for discovering lead compounds from traditional Chinese medicines (TCMs), a comparative online ligand fishing platform was developed using immobilized capillary enzyme reactors (ICERs) in combination with liquid chromatography-mass spectrometry (LC-MS). Methacrylate-based monolithic capillaries (400 μm I.D. × 10 cm) containing epoxy reactive groups were used as support to immobilize the target enzyme acetylcholinesterase (AChE). The activity and kinetic parameters of the AChE-ICER were investigated using micro-LC-UV. Subsequently, ligand fishing and identification from mixtures was carried out using the complete AChE-ICER-LC-MS platform. For efficient distinction of true actives from false positives, highly automated comparative analyses were run alternatingly using AChE-ICERs and negative control-ICERs, both online installed in the system. After washing unbound compounds to the waste, bound ligands were eluted from the AChE-ICER to a trapping loop using a denaturing solution. The trapped ligands were further separated and identified using LC-MS. Non-specific binding to the monolith support or non-functional sites of the immobilized enzyme was investigated by exposing analytes to the negative control-ICER. The specificity of the proposed approach was verified by analyzing a known AChE inhibitor in the presence of an inactive compound. The platform was applied to screen for AChE inhibitors in extracts of Corydalis yanhusuo. Eight compounds (columbamine, jatrorrhizine, coptisine, palmatine, berberine, dehydrocorydaline, tetrahydropalmatine and corydaline) with AChE binding affinity were detected and identified, and their AChE inhibitory activities were further verified by an in vitro enzymatic inhibition assay. Experimental results show that the proposed comparative online ligand fishing platform is suitable for rapid screening and mass-selective detection of AChE inhibitors in complex mixtures.

Recent advances in the preparation of adsorbent layers for thin‐layer chromatography combined with matrix‐assisted laser desorption/ionization mass‐spectrometric detection

“…Rigid porous cross‐linked polymers are successfully used in a very wide range of practical applications. The macroporous polymeric monoliths are known as stationary phases for adsorption modes of liquid chromatography , capillary and planar electro chromatography , microfluidics (lab‐on‐chip) , immobilized enzyme reactors , solid phase extraction . Synthetic monolithic materials are produced on the basis of different polymers, such as polystyrenes , polyacrylates , polyacrylamide , and others (Figure ).…”

In vitro and in silico approach to determine neuroprotective properties of iridoid glycosides from aerial parts of Scrophularia amplexicaulis by investigating their cholinesterase inhibition and anti-oxidant activities

“…Moreover, these inhibitors may have putative therapeutic effects on other neurotransmitterrelated neurological disorders, such as myasthenia gravis, Parkinson's disease, dementia, and ataxia [9]. Recent studies on other useful pathways in AD management and treatment may shed some light on the potential of butyrylcholinesterase (BChE) inhibitors [10]. Excessive BChE cortical level was shown to be directly proportional to increasing signs and symptoms of AD.…”

Solid-Supported Proteins in the Liquid Chromatography Domain to Probe Ligand-Target Interactions

“…Interactions between proteins and small molecules have also been well-explored (de Moraes et al, 2014a; Zheng et al, 2014; Hage, 2017). More recently, solid-supported proteins have been employed not only to assess these interactions, but mainly as a strategy to isolate small molecules from complex combinatory libraries (Forsberg and Brennan, 2014; Zhuo et al, 2016; Vanzolini et al, 2018a; Wang L. et al, 2018). These applications are based on the principle of specific and reversible affinity interactions with the immobilized target protein and correlate well with zonal and frontal chromatography.…”