Publication details

Capillary electrophoresis applications for study of β secretase as an Alzheimer’s disease target

Authors

SCHEJBAL Jan ŘEMÍNEK Roman SLEZÁČKOVÁ Lucie ŠEFRANÁ Šárka GLATZ Zdeněk

Year of publication 2018
Type Appeared in Conference without Proceedings
Citation
Description Advantages of capillary electrophoresis (CE) are well established in the field of pharmaceutical analysis. The CE benefits arise mainly from its separation column – a bare silica capillary, which is available at negligible costs and offers nanolitre sample volumes. On the other hand, establishment of CE for enzyme assays is still in process, despite unique combination of features with excellent applicability in the field of enzyme studies. Among many, close to physiological conditions, coupling with mass spectrometry (MS) and high throughput capability are the most noteworthy. In our project we decided to explore the benefits of CE to study the ß-secretase (BACE1) – a key enzyme and a drug target of Alzheimer’s disease (AD). Although the history of AD is more than 100 years long, no effective treatment has been found yet and it is estimated that AD will become one of the greatest challenges human society will face. Based on the amyloid hypothesis, which states that the specific inhibition of BACE1 will slow down or even stop the progression of AD, BACE1 became a prominent target in AD drug development. Regrettably only slow progress has been made, although several BACE1 inhibitor drugs are still under investigation in clinical trials. Thus, the need for a cheap, reliable and high-throughput screening method for characterisation of lead BACE1 inhibitors remains. During our project we developed and explored numerous CE-based methods to assess BACE1 kinetic behaviour and inhibitors. In the first BACE1 assessment, we deployed CE-MS to study kinetic and inhibition behaviour with previously rarely applied unlabelled substrate. In so-called off-line assay, where enzyme reaction and subsequent product separation and quantitation are performed separately, we were able to precisely tune both incubation and separation conditions to acquire optimal parameters and highly reliable data. These experiments brought several surprising results when the Michaelis-Menten constant was found in millimolar substrate concentration despite previous reports of micromolar values. Also, one previously identified BACE1 inhibitor exhibited zero inhibition potency in our assays. To further explore the advantages of CE, three fully automatable online assays were developed. In the online setup the incubation step is performed inside the capillary thus reducing the required amount of reactants as well as allows for the aforementioned automation. In one of our works we developed and comprehensively compared two homogenous online assays based on established mixing procedures of electrophoretically-mediated microanalysis and transverse diffusion of laminar flow profiles. This work proved the applicability of both methods for BACE1 assessment while identifying specific applications where one outperforms the other and vice versa. In our final work, we developed a CE-integrated immobilized enzyme reactor which not only bares the abovementioned advantages of online assays but also enables the reuse of immobilized enzyme which further reduces the cost of analysis. Last but not least all of our advanced methods provided kinetic and inhibition data in absolute agreement with the classical off-line method, which were also confirmed by a fundamentally different analytical method – the matrix assisted laser desorption/ionization MS. In conclusion, our researched brought numerous novel insights not only with respect to BACE1 kinetic and inhibition behaviour but also in the applications of CE for the enzyme assay development, which show excellent potential well worth further exploring.
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