Development of a liquid chromatographic system with fluorescent detection for beta-secretase immobilized enzyme reactor on-line enzymatic studies

A novel liquid chromatographic method has been developed for use in throughput screening of new inhibitors of human recombinant beta-amyloid precursor protein cleaving Enzyme (hrBACE1). The approach is based on the use of an immobilized Enzyme reactor (IMER) containing the target Enzyme (hrBACE1-IMER) and uses fluorescence detection. The bioreactor was prepared by immobilizing hrBACE1 on an ethylendiamine (EDA) monolithic disk (CIM) and a fluorogenic peptide (M-2420) containing the Beta-secretase site of the Swedish mutation of amyloid precursor protein (APP) was used as substrate. After injection into the hrBACE1-IMER system, M-2420 was enzymatically cleaved, giving rise to a fluorescent methoxycoumaryl-fragment (Rt=1.6min), which was separated from the substrate and selectively detected at lambda(exc)=320 and lambda(em)=420nm. Product and substrate were characterized by using a post monolithic C18 stationary phase coupled to an ion trap mass analyser. A calibration curve was constructed to determine the immobilized hrBACE1-IMER rate of catalysis and kinetic constants. Specificity of the enzymatic cleavage was confirmed by injecting the substrate on a blank CIM-EDA. The proposed method was validated by the determination of the inhibitory potency of five reference compounds with activities ranked over four order of magnitude (four peptidic inhibitors and a green tea polyphenol, (-)gallocatechin gallate). The obtained results were found in agreement with the data reported in literature, confirming the validity and the applicability of the hrBACE1-IMER as a tool for the fast screening of unknown inhibitors (more than 6 compounds per hour). Moreover, the hrBACE1-IMER showed high stability during the analysis, permitting its use for more than three months without affecting Enzyme activity.