APPLICATION OF AN IMMOBILIZED ENZYME REACTOR BASED ON HUMAN RECOMBINANT BETA-SECRETASE (HRBACE-1-IMER) FOR THE FAST SCREENING OF INHIBITORS

The aspartic protease beta-secretase (BACE-1) is the rate-limiting enzyme for the generation of the amyloid protein (Aβ), a main and core component of Alzheimer’s disease (AD) senile plaques [1]. Amyloid precursor protein (APP) proteolysis and Aβ generation represent potential targets for AD therapy. Moreover, mice with inactivated genes encoding BACE1 have not showed anatomical or physiological abnormalities [2]. In the last decade, the BACE-1 role in AD pathogenysis was well established and the search for new non peptidic inhibitors has been intensified [3]. BACE-1 activity is usually detected in FRET assays, by using peptidic substrates coupled to donor-acceptor pairs. The in-solution assays have some drawbacks, including the long time assay and the high enzyme consumption. Immobilization of enzymes can be a valid solution to overcome these problems. An immobilized enzyme reactor (IMER) results an interesting tool for the fast screening of enzyme inhibitors. In a previous work, we developed and characterized a hrBACE-1-IMER on an ethylendiamine (EDA) CIM disk by using a bidimensional LC system [4]. Here, to speed and automatise the analysis, a novel LC methods was developed. A fluorogenic peptide was used as BACE-1 substrate; enzymatic activity was evaluated by measuring the peak area of the hydrolysis product eluted in less than 10 min. The specificity of the product formation was confirmed by injecting the substrate on a blank EDA-CIM. The method was validated by measuring enzyme inhibition from standard inhibitors and the results obtained were found in agreement with the data reported in literature, confirming the validity of the hrBACE-1-IMER as a tool for the fast screening of novel inhibitors.