Detailed Information on Publication Record
2017
Application of off-line and on-line capillary electrophoretic methods for kinetic and inhibition studies of beta-secretase activity
ŘEMÍNEK, Roman, Jan SCHEJBAL and Zdeněk GLATZBasic information
Original name
Application of off-line and on-line capillary electrophoretic methods for kinetic and inhibition studies of beta-secretase activity
Name in Czech
Použití off-line a on-line kapilárně elektroforetických metod pro kinetické a inhibiční studie beta-sekretasy
Authors
ŘEMÍNEK, Roman, Jan SCHEJBAL and Zdeněk GLATZ
Edition
24th International Symposium on Electro- and Liquid Phase- Separation Techniques - ITP 2017, 2017
Other information
Language
English
Type of outcome
Vyžádané přednášky
Field of Study
10600 1.6 Biological sciences
Country of publisher
Czech Republic
Confidentiality degree
není předmětem státního či obchodního tajemství
References:
Organization unit
Faculty of Science
Keywords (in Czech)
kapilární elektroforéza, Alzheimerova choroba, beta-sekretasa, kinetika, inhibice
Keywords in English
capillary electrophoresis, Alzheimer's disease, beta-secretase, kinetics, inhibition
Tags
International impact, Reviewed
Změněno: 26/1/2018 08:29, prof. RNDr. Zdeněk Glatz, CSc.
V originále
Alzheimer‘s disease (AD) represents an irreversible, progressive brain disorder currently accounting for about 34 million dementia cases worldwide. Number of new diagnoses, moreover, increases very year. Beta-secretase (BACE1), an aspartic-acid protease, plays a key role in development of neurotoxic amyloid plagues in patients‘ brain tissue. Recent studies thus suggest that specific inhibition of this rate limiting enzyme could slow down or even stop the progression of the disease. For these reasons the main aim of the presented study was to develop a set of capillary electrophoretic (CE) methods for studies of BACE1 activity. Two off-line methods based on CE-ESI-MS and CE-UV systems and two on-line methods based on principles of the electrophoretically mediated microanalysis and transverse diffusion of laminar flow profiles using UV detection were introduced. The BACE1 reaction with decapeptide SEVNLDAEFR was incubated in 50 mM sodium acetate (pH 4.25) buffer in a vial or inside the bare silica capillary, respectively, and the proteolytic products SEVNL and DAEFR were separated in less than 9 minutes using acetic acid as a BGE. Currently used BACE1 assays are almost exclusively based on Förster resonance energy transfer (FRET); however, drawbacks of this approach such as very low solubility of fluorescently labeled substrates and intermolecular quenching may lead to misleading results. Utilization of unlabeled substrate and CE should eliminate these weak points and provide miniaturized assays for highthroughput screenings of potential AD drugs. The CE procedures were optimized with respect to either MS compatibility (off-line) or reaction products yields and homogeneity of the reaction mixture inside the capillary (on-line). After the methods validation, kinetic and inhibition studies of BACE1 activity were performed. Three structurally different probe inhibitors were selected – donepezil, LY2886721, and statine-containing tridecapeptide KTEEISEVN-(Statine)-VAEF, and results obtained were compared with published data. Determined IC50 and inhibition constant values of LY2886721 and statine inhibitor were in a good agreement with literature. On the other hand no inhibitory effect of donepezil was observed suggesting false reported results given by presence of the intermolecular quenching. Furthermore, higher solubility of the unlabeled substrate enabled determination of the whole Michaelis-Menten curve and accurate determination of Michaelis constant value whose variability in the literature is vast. Four CE methods with MS or UV detection for kinetic and inhibition studies of BACE1 activity were developed. The results obtained proved both their practical applicability in the early stages of the development of new AD drugs and necessity of various analytical approaches for achievement of the reliable biochemical data.
In Czech
Byly zavedeny 4 metody pro kinetické a inhibiční studie beta-sekretasy, využívající kapilární elektroforézu s MS a UV-Vis detekcí.
Links
| GA16-06106S, research and development project |
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| MUNI/A/1278/2016, interní kód MU |
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