Efeitos da uleina em alvos relacionados com doença de Alzheimer e estudos enzimáticos on-line com enzima beta-secretase imobilizada

Abstract

Resumo: A doença de Alzheimer, uma doença progressiva, multifatorial, degenerativa do cérebro, é a causa mais comum de demência em idosos. Clinicamente, é caracterizada pela perda de memória progressiva e déficit em diferentes domínios cognitivos associados com a perda de marcadores pré-sinápticos do sistema colinérgico nas áreas do cérebro relacionadas com a memória e aprendizagem e com a presença de depósitos de placas amiloides e emaranhados neurofibrilares no cérebro de aflitos indivíduos. Uma abordagem nova para enfrentar a natureza multifatorial da doença pode ser representada pela descoberta de compostos únicos capazes de modular simultaneamente diferentes alvos envolvidos na cascata neurodegenerativa. Portanto, a capacidade inibitória do alcaloide uleína extraídos da casca do caule da planta medicinal brasileira Himatanthus lancifolius Woodson (Müll. Arg.); Apocynaceae contra conhecido alvos validados da doença de Alzheimer, incluindo as enzimas acetilcolinesterase humana, butirilcolinesterase de soro humano, beta-secretase humana e a agregação espontânea do peptídeo beta-amiloide foi realizada resultando em uma atividade inibidora marcante, dentro da gama nanomolar, contra a enzima beta-secretase humana, com um valor de IC50 de 180 (± 22) nM. Além disso, a uleína foi capaz de inibir ambas as colinesterases com valores de IC50 de 279,0 (± 4,5) ?M e 24,0 (± 1,5) ?M, respectivamente. Finalmente, a uleína também foi capaz de inibir significativamente a agregação espontânea do peptídeo beta-amiloide (40 % na proporção 5:1). Um método cromatográfico rápido, reprodutível e executável com detecção por fluorescência para o ensaio de inibição da enzima beta-secretase recombinante humana utilizando um substrato fluorogênico conhecido como substrato IV foi desenvolvido e aplicado para a avaliação de atividade enzima hrBACE1 imobilizada e sua inibição por um composto natural o qual apresentava propriedades auto-fluorescentes. O método provou ser sensível e permitiu a detecção seletiva do produto de reação enzimática e estudos de inibição. Palavras-chaves: Doença de Alzheimer, uleína, aceticholinesterase, butirilcolinesterase, betasecretase, peptídeo beta-amiloide, inibidores de agregação, IMER

Abstract: Alzheimer's disease, a progressive, multifactorial, degenerative disorder of the brain, is the most common cause of dementia among the elderly. It is clinically characterized by loss of memory and progressive deficit in different cognitive domains associated with a loss of the presynaptic markers of the cholinergic system in the brain areas related to memory and learning, and with the presence of amyloid deposits and neurofibrillary tangles in the brain of afflicted individuals. An emerging disease modifying approach to face the multifactorial nature of AD may be represented by the discovery of single compounds which may simultaneously modulate different targets involved in the neurodegenerative AD cascade. Therefore the inhibitory capacity of the indole alkaloid uleine extracted from the stem bark of the Brazilian medicinal plant Himatanthus lancifolius (Müll. Arg.) Woodson; Apocynaceae against known Alzheimer's disease validated targets, including human acetylcholinesterase, butyrylcholinesterase from human serum, human beta-secretase and spontaneous ?- amyloid(1-42) aggregation was performed resulting on a remarkable inhibitory activity, within the nanomolar range, against human beta-secretase, with an IC50 value of 180 (±22) nM. In addition, uleine was able to inhibit both cholinesterases showing IC50 values of 279.0 (±4.5) ?M and 24.0 (±1.5) ?M, respectively. Finally, uleine was also capable of significantly inhibit amyloid self-aggregation (40% at the 5x concentration of ?-amyloid(1-42). In addition, High throughput screening techniques are required for the fast hit inhibitors selection in the early discovery process. However, in inhibitors screening campaign, the most frequently used methoxycoumarin based peptide substrate (M-2420) is not widely applicable when aromatic or heterocyclic compounds of natural source show auto-fluorescence interferences. Here, in order to overcome these drawbacks, we propose the use of a highly selective 4-(4-dimethylaminophenylazo)benzoic acid/5-[(2-aminoethyl)amino]naphthalene-1- sulfonic acid based peptide substrate (Substrate IV), whose cleavage product is devoid of spectroscopic interference. The human beta-secretase immobilized enzyme reactor was prepared and characterized in terms of units of immobilized of beta-secretase. Substrate IV cleavage was kinetically characterized on-line in terms of KM and vmax, in a classical Michaelis and Menten study. The online kinetic constants were found consistent with those obtained with the in solution standard method. In order to further validate the use of Substrate IV for inhibition studies, the inhibitory potency of a well-known beta-secretase peptide inhibitor was determined. A further application with uleine as BACE1 inhibitor was carried out to prove the system suitability for natural compounds. Keywords: Alzheimer's disease, uleine, acetycholinesterase, butyrylcholinesterase, ?- secretase, amyloid-? peptide, inhibitors, aggregation, IMER