Inhibitor Of Acetylcholinesterase Activity Detection And Screening Of New Methods

Acetylcholinesterase (AChE) is a serine hydrolase. Its main biological function is that it may rapidly hydrolyze the neurotransmitter acetylcholine in cholinergic synaptic cleft, which maintain the amount of acetylcholine to normal levels and ensure the delivery of the nerve impulses. So AChE plays an important role in the cholinergic system. Acetylcholinesterase activity assay has been widely applied to many fields. AChE is an important clinical biomarker of many diseases. AChE also serves as a biorecognition element in biosensors for detection of pesticide residues in food and in environment, as well as nerve gases in military field.Alzheimer’s disease (AD) is a progressive degenerative disease of the central nervous system. Acetylcholinesterase inhibitors are mainly used in the treatment of AD, which improve ACh content in the brain of AD patients. However, most of acetylcholinesterase inhibitors are synthetic, although they have been clinically used, it was found that they suffer low activity, unperfect selectivity and serious side effects. Therefore it is high desirable to screen novel acetylcholinesterase inhibitors, especially from plant with new structure and mechanism.We have developed a reliable and ultrasensitive assay of AChE activity and inhibitor screening using DNA-templated silver nanoclusters and coumarin460in this thesis, respectively. It contains the following three chapters:The first chapter gives a brief overview of the AChE structure, functions and its relationship with Alzheimer’s disease. We also summarize the research status of the AChE activity assay and AChE inhibitor screening.In chapter two, We developed a fluorescence turn-on assay using12polycytosine-templated silver nanoclusters (dC(12)-AgNCs), which is amenable to rapid, ultrasensitive assay of AChE activity and inhibitor screening. The fluorescent silver nanoclusters synthesized in the aqueous phase offer an attractive fluorescence and photophysical properties. We synthesized different DNA stabilized fluorescent silver nanoclusters and compared their fluorescence properties and sulfhydryl choline response.Finally, We selected dC(12)-AgNCs which have strong fluorescence intensity and TCh enhances the fluorescence of dC(12)-AgNCs.The detection mechanism is based on the concept, that is, AChE hydrolyzes the acetylthiocholine (ATCh) chloride to produce thiocholine (TCh). Subsequently, TCh sensitively and rapidly reacts with dC(12)-AgNCs via Ag-S bond forming and enhances the fluorescence of dC(12)-AgNCs. We studied the application for screening AChE inhibitors using tacrine and galantamine as models. The enhanced fluorescence pattern also inherited the high specificity in compassion to fluorescence quenching pattern because other quenchers or environmental stimuli can also lead to fluorescence reduction. The assay offers the benefits of extremely low background noise and high detection sensitivity. A detection limit of0.00005U/mL AChE was obtained. Furthermore, the simple "mix-and-detect" detection format and rapid chemical reactions were comparable to the widely used Ellman method and can be expanded to a range of important areas, such as neurobiology, toxicology, and pharmacology.The third chapter describes a new method for screening AChE inhibitors. The inhibitory effects of coumarin460on the activity of AChE were investigated by inhibition kinetics. The binding interaction and binding sites between coumarin460and AChE were also studied using fluorimetry and molecular docking, respectively. The results showed that coumarin460could potently inhibit the activity of AChE. The IC50value was determined to be89.8±0.7μM. Kinetic analyses showed that coumarin460was a reversible and mixed type AChE inhibitor. The inhibition constants KI and KIS were determined to be (7.17±0.30)×10-5and (5.18±0.28)×10-5mol·L-1, respectively. Fluorescence analysis showed that Forster-resonance-energy-transfer (FRET) occur between coumarin460and AChE. Coumarin460binds selectively to the AChE serine200site via covalent bond and forms hydrogen bonds with serine124. We present a new homogeneous competitive model of ligand affinities for screening AChE inhibitors based on quenching the fluorescence of Tryptophan/tyrosine residues in AChE via FRET using coumarin as the acceptor. The AChE donor contains both tyrosine and tryptophan, and coumarin possesses strong spectral overlap with AChE, a prerequisite for efficient FRET. Upon binding of coumarin to AChE, there is the quenching of AChE fluorescence at340nm and the enhancement of characteristic fluorescence at460nm for coumarin. When a candidate ligand binds stronger with AChE than coumarin, the characteristic fluorescence at460nm for coumarin will be quenched. We studied the application for screening AChE inhibitors using Huperzine A and galantamine as models. In this new homogeneous competitive assay, there is/are neither cost/time for labeling proteins nor problems associated with labeling, and conventional fluorospectrometers can be used to measure steady-state fluorescence with ease. Thus, it is expected to achieve high-throughput screening.