J 2017

A capillary electrophoresis-mass spectrometry based method for the screening of beta-secretase inhibitors as potential Alzheimer's disease therapeutics

SCHEJBAL, Jan, Lucie SLEZÁČKOVÁ, Roman ŘEMÍNEK and Zdeněk GLATZ

Basic information

Original name

A capillary electrophoresis-mass spectrometry based method for the screening of beta-secretase inhibitors as potential Alzheimer's disease therapeutics

Authors

SCHEJBAL, Jan (203 Czech Republic, belonging to the institution), Lucie SLEZÁČKOVÁ (203 Czech Republic, belonging to the institution), Roman ŘEMÍNEK (203 Czech Republic, belonging to the institution) and Zdeněk GLATZ (203 Czech Republic, guarantor, belonging to the institution)

Edition

Journal of Chromatography A, Amsterdam, Netherlands, Elsevier, 2017, 0021-9673

Other information

Language

English

Type of outcome

Článek v odborném periodiku

Field of Study

10600 1.6 Biological sciences

Country of publisher

Netherlands

Confidentiality degree

není předmětem státního či obchodního tajemství

References:

Impact factor

Impact factor: 3.716

RIV identification code

RIV/00216224:14310/17:00095071

Organization unit

Faculty of Science

DOI

UT WoS

000395956700029

Keywords in English

Alzheimer's disease; beta-secretase; Kinetics; Inhibition; Capillary electrophoresis; Mass spectrometry detection

Tags

Tags

International impact, Reviewed
Změněno: 6/2/2019 11:42, prof. RNDr. Zdeněk Glatz, CSc.

Abstract

V originále

In this work a novel capillary electrophoresis-mass spectrometry (CE-MS) based method was developed and validated for the assay of p-secretase (BACE1) activity as a potential target for Alzheimer's disease (AD) treatment. In contrast with the typically used Forster 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 100 mu Lin 60 min 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 5 nM (S/N=3) and 15 nM (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.

Links

GA16-06106S, research and development project
Name: Vysoce efektivní systém založený na kapilární elektroforéze pro screening inhibitorů beta-sekretázy jako terapeutického cíle pro Alzheimerovu chorobu (Acronym: Alzheimer)
Investor: Czech Science Foundation
MUNI/A/1278/2016, interní kód MU
Name: Podpora biochemického výzkumu v roce 2017
Investor: Masaryk University, Category A