A capillary electrophoresis-mass spectrometry based method for the screening of β-secretase inhibitors as potential Alzheimer's disease therapeutics.
In this work a novel capillary electrophoresis-mass spectrometry (CE-MS) based method was developed and validated for the assay of β-secretase (BACE1) activity as a potential target for Alzheimer's disease (AD) treatment. In contrast with the typically used Förster resonance energy transfer (FRET) assays, an unlabelled decapeptide derived from the amyloid precursor protein BACE1 site with the "Swedish mutation" was used as the substrate. The CE usage enabled the enzymatic reaction to be carried out in as small a volume as 100μL in 60min with sufficient yields of proteolytic product, which was subsequently separated in a bare fused silica capillary using 12.5% acetic acid as a background electrolyte and detected by MS. The limits of detection and quantitation were estimated using the signal to noise ratio to be 5nM (S/N=3) and 15nM (S/N=10), respectively, both being well below the working range for kinetic and inhibition studies. Its applicability for the kinetic study of BACE1 was demonstrated using optimized enzyme assay conditions and the estimated kinetic parameter values were confirmed by classic CE-UV analyses. The method was finally used for the main purpose for which it was developed - to screen BACE1 inhibitors as potential AD therapeutics. The resulting kinetic and inhibition parameters values were compared to those published in the literature, which were almost exclusively obtained by FRET based assays. These comparisons brought up several issues that are further discussed below and favour the application of an unlabelled substrate. The proposed CE-MS based method offers a high-throughput capability for new drug development.
Department of Biochemistry, Faculty of Science, Masaryk University, Kamenice 5, 625 00, Brno, Czech Republic.
Jan
J
Schejbal
Department of Biochemistry, Faculty of Science, Masaryk University, Kamenice 5, 625 00, Brno, Czech Republic.
Lucie
L
Slezáčková
Department of Biochemistry, Faculty of Science, Masaryk University, Kamenice 5, 625 00, Brno, Czech Republic.
Roman
R
Řemínek
Department of Biochemistry, Faculty of Science, Masaryk University, Kamenice 5, 625 00, Brno, Czech Republic. Electronic address: glatz@chemi.muni.cz.
Zdeněk
Z
Glatz
D000544:Alzheimer Disease / Q000188:drug therapy*; D053829:Amyloid Precursor Protein Secretases / Q000037:antagonists & inhibitors*; D016282:Aspartic Acid Endopeptidases / Q000037:antagonists & inhibitors*; D004353:Drug Evaluation, Preclinical / Q000379:methods; D019075:Electrophoresis, Capillary / Q000379:methods*; D057809:HEK293 Cells; D006801:Humans; D007700:Kinetics; D013058:Mass Spectrometry / Q000379:methods*; D011480:Protease Inhibitors / Q000032:analysis* / Q000627:therapeutic use
D011480:Protease Inhibitors; D053829:Amyloid Precursor Protein Secretases; D016282:Aspartic Acid Endopeptidases; C509280:BACE1 protein, human
Alzheimer’s disease; Capillary electrophoresis; Inhibition; Kinetics; Mass spectrometry detection; β-secretase