Application of capillary electrophoresis for beta-secretase inhibitor screening - development and comparison of two on-line methods
| Authors | |
|---|---|
| Year of publication | 2017 |
| MU Faculty or unit | |
| Citation | |
| Description | Alzheimer's disease (AD) is a progressive, degenerative disorder that affects the nervous tissue of the brain resulting in loss of patient's intellectual and social skills. Worldwide, it accounts for about 34 million dementia cases and its prevalence rapidly increases. Since beta-secretase (BACE) represents a rate-limiting enzyme of neurotoxic beta-amyloid peptide oligomers abnormally accumulated in the affected brain tissue, its specific inhibition appears to be a promising approach for slowing down or even stopping the progression of the disease. Capillary electrophoresis (CE) constitutes a favorable analytical technique for enzyme activity studies due to its high separation efficiency, high throughput via automation and very limited need of sample pretreatment. Furthermore, the assay can be carried out inside the capillary reducing the consumption of the enzyme and reactants to tens of nL per analysis. The mixing of the individually injected components of the reaction mixture representing the crucial part of these methods can be accomplished using two fundamental approaches - transverse diffusion of laminar flow profiles (TDLFP) and electrophoretically mediated microanalysis (EMMA), based on the diffusional and electro-driven merging, respectively. For these reasons, the main goals of this study was to i) develop CE methods based on the TDLFP and EMMA approaches for BACE inhibitor screenings and ii) compare these methodologies with respect to the determined validation criteria and inhibition data. The BACE reaction with decapeptide SEVNLDAEFR was monitored using UV-vis detection of the proteolytic product DAEFR. After the optimization procedures were finished, both methods were thoroughly validated and used for kinetic study and inhibition assays with two probe inhibitors - statine-containing tridecapeptide and LY2886721. The results obtained were found to be comparable with literature data established using different analytical techniques. The represented methods constitute a miniaturized and fully automated tool, which should be suitable for inhibition studies of BACE as a target of AD pharmacological treatment discovery in the early stages of the development of a new drug. |
| Related projects: |