Co-immobilisation of enzymes acetylcholinesterase and ?-secretase1: study of conditions for screening ligands.

Author(s)

Adriana Ferreira Lopes Vilela

• Document type: Doctoral Thesis
• Press: Ribeirão Preto.
• Institution: Universidade de São Paulo (USP). Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto (PCARP/BC)
• Defense date: 2018-03-23

Examining board members

Carmen Lúcia Cardoso; Vanderlan da Silva Bolzani; Quezia Bezerra Cass; Marcela Cristina de Moraes

• Advisor: Carmen Lúcia Cardoso

Abstract

The enzymes, acetylcholinesterase (AChE) and ?-secretase1 (BACE1), are targets validated for the treatment of Alzheimer\'s Disease (AD) because they act in the central nervous system contributing to the disease advance. In this context, the interest in inhibitors of these enzymes and the search for selective and effective enzymatic assays is of crucial importance. This work describes the development of on-line methodologies, in fused silica capillaries, and off-line in agarose beads for screening ligands involving the AChE and BACE1 enzymes via immobilization and co-immobilization by covalent attachment. In the on-line methods the immobilization of BACE1 and co-immobilization of AChE and BACE1 enzymes in fused silica capillaries were performed via amino-glutaraldehyde bonding and the enzyme activities were monitored on-line employing high performance liquid chromatography and mass spectrometry (HPLC-MS) by measuring the masses corresponding to the products of the enzymatic cleavages. In these enabled the determination of kinetic parameters for immobilized enzymes such as; pH and temperature variations, kinetic constants (KMap) and validation with standard inhibitors. Also, the application in the screening of a collection of eight synthetic compounds with identification and characterizations of two compounds was carried out. In the off-line methods, the immobilizations of AChE and BACE1 on agarose supports were performed through different types of binding with nucleophilic groups previously attached to the support. The different active-agarose supports with immobilized AChE were studied using p-nitrophenyl propionate as substrate by measuring the absorbance of the product formed p-nitrophenol at 348 nm. The system with the immobilized enzyme optimal agarose-triethylamine-AChE (agarose-TEA-AChE) was used for validation of the method for screening ligands with the standard inhibitor being determined the kinetic and thermodynamic parameters of inhibition, such as; inhibitory power (IC50), inhibition constant (Ki) and dissociation constant (KD). For BACE1 enzyme, the enzymatic activity was monitored by fluorescence at ?em = 495 nm (?ex = 395 nm) using the substrate IV, and it was possible to select the best method for immobilization of BACE1. In both methods, both on-line and off-line, the immobilizations procedures were efficient reflecting on the activity of each enzyme after the process. The screening methods developed in this work are unprecedented for both enzymes and represent an improvement to the already consolidated screenings assays, because the co-immobilization contributed to the construction of an innovative screening model, being possible the evaluation of the specific interactions of a ligand against two biological targets in the same automated system. It is worth emphasizing that the AChE and BACE1 methods in two different matrices have provided models of assays in different format that can be applied according to the available analytical tools, from the simplest ones such as UV-Vis spectrophotometry, fluorescence, to the automated systems such as HPLC-MS. (AU)

• FAPESP's process: 16/02873-0 - Acetylcholinesterase and beta-secretase 1: a study of co-immobilization conditions for screening of ligands
• Grantee: Adriana Ferreira Lopes Vilela
• Support Opportunities: Scholarships in Brazil - Doctorate