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

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 a Zdeněk GLATZ

Základní údaje

Originální název

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

Autoři

SCHEJBAL, Jan (203 Česká republika, domácí), Lucie SLEZÁČKOVÁ (203 Česká republika, domácí), Roman ŘEMÍNEK (203 Česká republika, domácí) a Zdeněk GLATZ (203 Česká republika, garant, domácí)

Vydání

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

Další údaje

Jazyk

angličtina

Typ výsledku

Článek v odborném periodiku

Obor

10600 1.6 Biological sciences

Stát vydavatele

Nizozemské království

Utajení

není předmětem státního či obchodního tajemství

Odkazy

URL

Impakt faktor

Impact factor: 3.716

Kód RIV

RIV/00216224:14310/17:00095071

Organizační jednotka

Přírodovědecká fakulta

DOI

http://dx.doi.org/10.1016/j.chroma.2017.01.057

UT WoS

000395956700029

Klíčová slova anglicky

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

Štítky

NZ, rivok

Příznaky

Mezinárodní význam, Recenzováno

Změněno: 6. 2. 2019 11:42, prof. RNDr. Zdeněk Glatz, CSc.

Anotace

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.

Návaznosti

GA16-06106S, projekt VaV

Název: Vysoce efektivní systém založený na kapilární elektroforéze pro screening inhibitorů beta-sekretázy jako terapeutického cíle pro Alzheimerovu chorobu (Akronym: Alzheimer)

Investor: Grantová agentura ČR, Vysoce efektivní systém založený na kapilární elektroforéze pro screening inhibitorů beta-sekretázy jako terapeutického cíle pro Alzheimerovu chorobu

MUNI/A/1278/2016, interní kód MU

Název: Podpora biochemického výzkumu v roce 2017

Investor: Masarykova univerzita, Podpora biochemického výzkumu v roce 2017, DO R. 2020_Kategorie A - Specifický výzkum - Studentské výzkumné projekty

Citovat

SCHEJBAL, Jan, Lucie SLEZÁČKOVÁ, Roman ŘEMÍNEK a Zdeněk GLATZ. A capillary electrophoresis-mass spectrometry based method for the screening of beta-secretase inhibitors as potential Alzheimer's disease therapeutics. Journal of Chromatography A. Amsterdam, Netherlands: Elsevier, 2017, roč. 1487, 3 March 2017, s. 235-241. ISSN 0021-9673. Dostupné z: https://dx.doi.org/10.1016/j.chroma.2017.01.057.

@article{1392753,
   author = {Schejbal, Jan and Slezáčková, Lucie and Řemínek, Roman and Glatz, Zdeněk},
   article_location = {Amsterdam, Netherlands},
   article_number = {3 March 2017},
   doi = {http://dx.doi.org/10.1016/j.chroma.2017.01.057},
   keywords = {Alzheimer's disease; beta-secretase; Kinetics; Inhibition; Capillary electrophoresis; Mass spectrometry detection},
   language = {eng},
   issn = {0021-9673},
   journal = {Journal of Chromatography A},
   title = {A capillary electrophoresis-mass spectrometry based method for the screening of beta-secretase inhibitors as potential Alzheimer's disease therapeutics},
   url = {http://www.sciencedirect.com/science/article/pii/S0021967317301425?via%3Dihub},
   volume = {1487},
   year = {2017}
}

TY  - JOUR
ID  - 1392753
AU  - Schejbal, Jan - Slezáčková, Lucie - Řemínek, Roman - Glatz, Zdeněk
PY  - 2017
TI  - A capillary electrophoresis-mass spectrometry based method for the screening of beta-secretase inhibitors as potential Alzheimer's disease therapeutics
JF  - Journal of Chromatography A
VL  - 1487
IS  - 3 March 2017
SP  - 235-241
EP  - 235-241
PB  - Elsevier
SN  - 00219673
KW  - Alzheimer's disease
KW  - beta-secretase
KW  - Kinetics
KW  - Inhibition
KW  - Capillary electrophoresis
KW  - Mass spectrometry detection
UR  - http://www.sciencedirect.com/science/article/pii/S0021967317301425?via%3Dihub
L2  - http://www.sciencedirect.com/science/article/pii/S0021967317301425?via%3Dihub
N2  - 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.
ER  -

SCHEJBAL, Jan, Lucie SLEZÁČKOVÁ, Roman ŘEMÍNEK a Zdeněk GLATZ. A capillary electrophoresis-mass spectrometry based method for the screening of beta-secretase inhibitors as potential Alzheimer's disease therapeutics. \textit{Journal of Chromatography A}. Amsterdam, Netherlands: Elsevier, 2017, roč.~1487, 3 March 2017, s.~235-241. ISSN~0021-9673. Dostupné z: https://dx.doi.org/10.1016/j.chroma.2017.01.057.

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