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.
Mancini
Francesca
F
Department of Pharmaceutical Sciences, Via Belmeloro 6, University of Bologna, 40126 Bologna, Italy.
Andrisano
Vincenza
V
D053829:Amyloid Precursor Protein Secretases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D016282:Aspartic Acid Endopeptidases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D019149:Bioreactors; D002138:Calibration; D002853:Chromatography, Liquid / Q000379:methods*; D004795:Enzyme Stability; D004800:Enzymes, Immobilized / Q000378:metabolism*; D005240:Feasibility Studies; D006801:Humans; D020128:Inhibitory Concentration 50; D007700:Kinetics; D009862:Online Systems; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D012015:Reference Standards; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013056:Spectrophotometry, Ultraviolet / Q000379:methods; D013379:Substrate Specificity
D004800:Enzymes, Immobilized; D011994:Recombinant Proteins; D053829:Amyloid Precursor Protein Secretases; D016282:Aspartic Acid Endopeptidases; C509280:BACE1 protein, human