Frequency of geriatric diseases is increasing due to population ageing. One of the most common cause of dementia is Alzheimer's disease, which is a progressive neurodegenerative disease. Cognitive deterioration as a consequence of Alzheimer's disease is physically and mentally exhausting for caretakers and it often results in institutionalisation of the patient. Lack of cure and great socioeconomic burden of the disease make research of new medicine and diagnostic tools very important. The cholinergic hypothesis is the first attempt of explaining the pathogenesis of Alzheimer's disease, according to which cholinergic dysfunction causes decline in patients cognitive ability. The hypothesis led to the discovery of cholinesterase inhibitors. A new promising drug target is butyrylcholinesterase as its activity increases in later stages of Alzheimer's disease. On the contrary, activity of acetylcholinesterase decreases. Therefore, it is supposed that butyrylcholinesterase takes over the cholinesterase activity in advanced stages of the disease. Also, butyrycholinesterase is thought to be involved in formation of amyloid plaques, which are a pathophysiological hallmark of Alzheimer's disease. For the development of a simple and direct method for detecting butyrylcholinesterase on brain slices we tried to develop a selective fluorescently labeled butyrylcholinesterase inhibitor. Our attempt was based on the structure of an inhibitor 2 that was developed by researchers at the Faculty of Pharmacy. Firstly, we chemically modified its structure to allow introduction of derivatives of rhodamine B and NBD fluorophores by means of click chemistry. The introduction site of fluorophores was choosen on the basis of the crystal structure of the inhibitor 2 in complex with butyrylcholinesterase and well described structure-activity relationships. Fluorophores were attached by a hydrophilic linker in order to minimize nonspecific binding of the inhibitor to the lipophilic brain slice. Because the synthesis of inhibitor labeled with rhodamine B turned out to be troublesome, we managed to synthesise only an inhibitor 24 labeled with NBD. In vitro inhibitory potency of compound 24 against both cholinesterases was determined; compound is selective towards butyrylcholinesterase with half maximal inhibitory concentration of 214.16 nM. Attachment of the fluorophore caused a significant loss of binding capacity to the enzyme when compared to inhibitor 2 and as a consequence compound 24 was not appropriate for detection of butyrylcholinesterase on brain slices.
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