2017
Application of off-line and on-line capillary electrophoretic methods for kinetic and inhibition studies of beta-secretase activity
ŘEMÍNEK, Roman, Jan SCHEJBAL a Zdeněk GLATZZákladní údaje
Originální název
Application of off-line and on-line capillary electrophoretic methods for kinetic and inhibition studies of beta-secretase activity
Název česky
Použití off-line a on-line kapilárně elektroforetických metod pro kinetické a inhibiční studie beta-sekretasy
Autoři
ŘEMÍNEK, Roman, Jan SCHEJBAL a Zdeněk GLATZ
Vydání
24th International Symposium on Electro- and Liquid Phase- Separation Techniques - ITP 2017, 2017
Další údaje
Jazyk
angličtina
Typ výsledku
Vyžádané přednášky
Obor
10600 1.6 Biological sciences
Stát vydavatele
Česká republika
Utajení
není předmětem státního či obchodního tajemství
Odkazy
Organizační jednotka
Přírodovědecká fakulta
Klíčová slova česky
kapilární elektroforéza, Alzheimerova choroba, beta-sekretasa, kinetika, inhibice
Klíčová slova anglicky
capillary electrophoresis, Alzheimer's disease, beta-secretase, kinetics, inhibition
Příznaky
Mezinárodní význam, Recenzováno
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.
Česky
Byly zavedeny 4 metody pro kinetické a inhibiční studie beta-sekretasy, využívající kapilární elektroforézu s MS a UV-Vis detekcí.
Návaznosti
| GA16-06106S, projekt VaV |
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| MUNI/A/1278/2016, interní kód MU |
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