A Comparative Evaluation of Acetylcholinesterase Inhibition by AChE-ICER and in vitro Ellman's Modified Method of Simplified Analogs 3-O-Acetyl-N-Benzyl-Piperidine of Donepezil

Abstract

Resumo: Inibidores de Acetilcolinesterase (AChEI) ainda são os melhores fármacos usados para a terapêutica da doença de Alzheirmer (DA). Dentre os IAChEs disponíveis, o donepezil apresenta a maior meia-vida, os efeitos colaterais mais brandos, além de segurança e tolerância satisfatórias; portanto, este tem sido utilizado como medicamento de primeira escolha no tratamento de DA leve a moderada. A triagem bem sucedida de novos AChEI depende de métodos rápidos e eficientes. Os ensaios empregados mais frequentemente são baseados no uso do reagente de Ellman (ensaio colorimétrico) ou do reagente Fast Blue B, além de métodos radioquímicos, espectrométricos ou cromatográficos. Uma abordagem mais recente utiliza a enzima imobilizada em reator capilar, acoplada a um sistema cromatográfico, que é conhecido como ICER (do inglês immobilized capillary enzyme reactor). Como parte de um projeto que visa à identificação de novos IAChEs, este artigo descreve a avaliação comparativa da atividade inibitória da AChE de uma série de derivados 3-O-acetil-N-benzilpiperidínicos substituídos por duas diferentes abordagens: método de Ellman modificado in vitro e uma metodologia de ICER. Embora nenhum dos compostos da série tenha apresentado alta atividade inibitória, todos os ensaios revelaram resultados consistentes, demostrando que é possível aplicar diferentes metodologias confiáveis para a busca e idendificação de novos inibidores de AChE.Palavras-chave: Química Medicinal; análogos N-benzil-piperidinícos; inibidores de acetilcolinesterase; doença de Alzheimer; enzima imobilizada; enzima imobilizada em reator capilar. AbstractAcetylcholinesterase inhibitors (AChEI) remain the best pharmacotherapy for Alzheimer´s disease (AD). Among the currently available AChEI drugs, Donepezil has longer half-life, fewer clinical side interactions, and satisfactory safety and tolerability; therefore, it is the first choice medicine to treat mild to moderate AD. Successful screening for new AChEIs relies on effective and fast assays. The ost f e ue tly e ployed assays a e ased o the use of Ell a 's eage t olo i et i ethod o the Fast Blue B salt reagent, despite of other radiochemical, spectrometric, or chromatographic methods. A more recent and interesting approach is to immobilize the target enzyme in a chromatographic system, which is known as immobilized capillary enzyme reactors (ICERs). As part of a current project aiming to search for novel AChEIs, in this paper we have reported a comparative evaluation of AChE inhibitory activity of a series of substituted 3-O-acetyl-N-benzylpiperidine derivatives by two different approaches: in vitro Ell a 's odified ethod a d a ICER-based methodology. Although none of the compounds has shown high activity, all different assays revealed consistent results, showing that is possible to apply different reliable methodologies for the screening of new AChEIs.Keywords: N-benzyl-pipe idi e a alogs; A etyl holi este ase i hi itio ; Alzhei e 's Disease; e zy e i o ilizatio ; i o ilized capillary enzyme reactor.

Combined application of multiple biomarkers for early auxiliary diagnosis of silicosis

“…The modified Ellman method was used to determine the cholinesterases (ChEs) activity (Silva et al, 2015). The operation steps were as follows.…”

On-flow enzymatic inhibitor screening: The emerging success of liquid chromatography-based assays

“…Different solid supports can be employed for enzyme immobilization in the development of LC-based on-flow screening assays. Silica-based supports (de Moraes et al, 2012Vanzolini et al, 2013;da Silva et al, 2013;Hu et al, 2015;Silva et al, 2015;Vilela et al, 2015Vilela et al, , 2018Calil et al, 2016;Magalhães et al, 2016;Sarria et al, 2016;Ferreira Lopes Vilela and Cardoso, 2017;Cornelio et al, 2018;Lima et al, 2019;Medina et al, 2019;Qian et al, 2019;Seidl et al, 2019Seidl et al, , 2022Ferey et al, 2019;Chapla et al, 2020;Hou et al, 2020;de Castro et al, 2022) present a versatile and largely explored motif due presence of many hydroxyl groups on its surface. For example, those groups are readily reactive with 3aminopropyltriethoxysilane (APTES), furnishing an amine- rich surface that can further react with glutaraldehyde (GA), yielding a terminal reactive aldehyde surface susceptible reactions with nucleophilic groups present on the enzyme surface (Figure 2B).…”