Publication details

Capillary electrophoresis-integrated immobilized enzyme reactor for screening of β-secretase inhibitors – potential anti-Alzheimer’s disease drugs

Authors

SCHEJBAL Jan ŠEFRANÁ Šárka GLATZ Zdeněk

Year of publication 2017
Type Conference abstract
Citation
Description The Alzheimer’s disease (AD) is considered one of the greatest challenges of modern society. Despite the AD is known for more than a century, many aspects of its pathogenesis remain hidden together with an effective treatment. On the other hand, number of promising drug targets, including ß secretase (BACE1), were identified in last two decades and the search for an effective drug is underway. BACE1 is the rate limiting enzyme in the process of creating amyloid-ß, which in elevated concentrations aggregates and forms toxic oligomers and amyloid plaques – one of the AD hallmarks. Thus, specific inhibition of BACE1 is believed to slow down or even stop the AD progression. As the current drug development is based on testing of vast compounds libraries, fast, cost-effective, reliable and high-throughput analytical methods are demanded. Capillary electrophoresis (CE) is a unique analytical method which provides fast analysis and high resolution with minimal sample requirements. Its on line hyphenation to immobilized enzyme reactor (IMER) extends these benefits thanks to reuse of the target enzyme and easy automation. In this contribution, we present current stage of CE-IMER development for screening of BACE1 inhibitors. A protocol for BACE1 immobilization on carboxyl moieties-coated magnetic microparticles by standard carbodiimide chemistry was optimized. Enzyme-baring particles were thoroughly characterised in terms of enzymatic activity and stability and subsequently used to form an IMER inside a capillary by capturing in external magnetic field which was provided by two neodymium magnets. The capillary thus serves as both reaction and separation vessel. Such direct integration provides easy automation, but requires simultaneous optimization of incubation and separation parameters, which will provide sufficient separation performance while preserving the enzyme activity. Furthermore, two different detection techniques, namely UV-Vis spectroscopy and mass spectrometry (MS), are used and thus certain limitations required by CE-MS hyphenation must be considered as well, e.g. separation solution based on volatile compounds and maintaining electric currents below 50 µA. Applied strategies for such complex optimization is described and briefly discussed.
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