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

Result code in IS VaVaI

<a href="https://www.isvavai.cz/riv?ss=detail&h=RIV%2F00216224%3A14310%2F17%3A00095071" target="_blank" >RIV/00216224:14310/17:00095071 - isvavai.cz</a>

Result on the web

<a href="http://www.sciencedirect.com/science/article/pii/S0021967317301425?via%3Dihub" target="_blank" >http://www.sciencedirect.com/science/article/pii/S0021967317301425?via%3Dihub</a>

DOI - Digital Object Identifier

<a href="http://dx.doi.org/10.1016/j.chroma.2017.01.057" target="_blank" >10.1016/j.chroma.2017.01.057</a>

Result language

angličtina

Original language name

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

Original language description

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.

Czech name

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Czech description

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Type

J_imp - Article in a specialist periodical, which is included in the Web of Science database

CEP classification

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OECD FORD branch

10600 - Biological sciences

Project

<a href="/en/project/GA16-06106S" target="_blank" >GA16-06106S: Capillary electrophoresis based high-throughput system for beta-secretase inhibitor screening as a therapeutic target of Alzheimer's disease</a><br>

Continuities

P - Projekt vyzkumu a vyvoje financovany z verejnych zdroju (s odkazem do CEP)<br>S - Specificky vyzkum na vysokych skolach

Publication year

2017

Confidentiality

S - Úplné a pravdivé údaje o projektu nepodléhají ochraně podle zvláštních právních předpisů

Name of the periodical

Journal of Chromatography A

ISSN

0021-9673

e-ISSN

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Volume of the periodical

1487

Issue of the periodical within the volume

3 March 2017

Country of publishing house

NL - THE KINGDOM OF THE NETHERLANDS

Number of pages

7

Pages from-to

235-241

UT code for WoS article

000395956700029

EID of the result in the Scopus database

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