Improvement of Workflows and Assay Reproducibility by The Introduction of "Assay-Ready" Culturing of MDCK Cells for Transport Studies.

OBJECTIVE: In previous studies, we established and validated three Madin Darby Canine Kidney MDCKII cell lines, recombinantly modified with zinc finger nuclease (ZFN) technology. Here, we investigated the applicability of seeding these three canine P-gp deficient MDCK_ZFN cell lines, directly from frozen cryopreserved stocks without previous cultivation for efflux transporter and permeability studies. This technique is referred to as "assay-ready" and allows for highly standardized conduction of cell-based assays and shorter cultivation cycles. METHODS: To obtain a rapid fitness of the cells for that purpose, a very gentle freezing and thawing protocol was applied. Assay-ready MDCK_ZFN cells were tested in bi-directional transport studies and compared to their traditionally cultured counterparts. Long-term performance robustness, human effective intestinal permeability (Peff) predictability and batch to batch variability were assessed. RESULTS: Efflux ratios (ER) and apparent permeability (Papp) results were highly comparable between assay-ready and standard cultured cell lines with R2 values of 0.96 or higher. Papp to Peff correlations obtained from passive permeability with non-transfected cells were comparable independent of the cultivation regime. Long-term evaluation revealed robust performance of assay-ready cells and reduced data variability of reference compounds in 75% of cases compared to standard cultured MDCK_ZFN cells. CONCLUSION: Assay-ready methodology for handling MDCK_ZFN cells allows more flexibility in assay planning and reduces performance fluctuations in assays caused by cell aging. Therefore, the assay-ready principle has proven superior over conventional cultivation for MDCK_ZFN cells and is considered as a key technology to optimize processes with other cellular systems.

Simple and Efficient Microsolid-Phase Extraction Tip-Based Sample Preparation Workflow to Enable Sensitive Proteomic Profiling of Limited Samples (200 to 10,000 Cells).

In-depth LC-MS-based proteomic profiling of limited biological and clinical samples, such as rare cells or tissue sections from laser capture microdissection or microneedle biopsies, has been problematic due, in large, to the inefficiency of sample preparation and attendant sample losses. To address this issue, we developed on-microsolid-phase extraction tip (OmSET)-based sample preparation for limited biological samples. OmSET is simple, efficient, reproducible, and scalable and is a widely accessible method for processing ∼200 to 10,000 cells. The developed method benefits from minimal sample processing volumes (1-3 µL) and conducting all sample processing steps on-membrane within a single microreactor. We first assessed the feasibility of using micro-SPE tips for nanogram-level amounts of tryptic peptides, minimized the number of required sample handling steps, and reduced the hands-on time. We then evaluated the capability of OmSET for quantitative analysis of low numbers of human monocytes. Reliable and reproducible label-free quantitation results were obtained with excellent correlations between protein abundances and the amounts of starting material (R2 = 0.93) and pairwise correlations between sample processing replicates (R2 = 0.95) along with the identification of approximately 300, 1800, and 2000 protein groups from injected ∼10, 100, and 500 cell equivalents, resulting from processing approximately 200, 2000, and 10,000 cells, respectively.

Capillary electrophoresis integrated immobilized enzyme reactor for kinetic and inhibition assays of ß-secretase as the Alzheimer's disease drug target.

Capillary electrophoresis integrated immobilized enzyme reactors are becoming an increasingly popular alternative for enzyme kinetic and inhibition assays thanks to their unique set of features including cost effectiveness, repeated use of the enzyme, minuscule sample consumption, rapid analysis time and easy automation. In this work we present the development and application of a capillary electrophoresis integrated immobilized enzyme reactor based on magnetic particles for kinetic and inhibition studies of ß-secretase, a key enzyme in the development of Alzheimer's disease and a promising drug target. We document the optimization of the immobilization procedure, characterization of immobilized ß-secretase, optimization of a mutually compatible incubation protocol and separation method as well as the production of the capillary electrophoresis integrated immobilized enzyme reactor. The applicability of the capillary electrophoresis integrated immobilized enzyme reactor was demonstrated by kinetic assay with an unlabelled substrate and by inhibition assays using three structurally different reference inhibitors. The resulting kinetic and inhibition parameters clearly support the applicability of the herein presented method as well as document the fundamental phenomena which need to be taken in account when comparing the results to other methods.

An improved design to capture magnetic microparticles for capillary electrophoresis based immobilized microenzyme reactors.

In this paper, we demonstrate the effectiveness of a new 3D printed magnet holder that enables capture of magnetic microparticles in commercially available capillary electrophoresis equipment with a liquid or air based coolant system. The design as well as the method to capture magnetic microparticles inside the capillary are discussed. This setup was tested at temperature and pH values suitable for performing enzymatic reactions. To demonstrate its applicability in CE- immobilized microenzyme reactors (IMER) development, human flavin-containing monooxygenase 3 and bovine serum albumin were immobilized on amino functionalized magnetic microparticles using glutaraldehyde. These microparticles were subsequently used to perform in-line capillary electrophoresis with clozapine as a model substrate. This setup could be used further to establish CE-IMERs of other drug metabolic enzymes in a commercially available liquid based capillary coolant system. The CE-IMER setup was successful, although a subsequent decrease in enzyme activity was observed on repeated runs.

A label-free MALDI TOF MS-based method for studying the kinetics and inhibitor screening of the Alzheimer's disease drug target ß-secretase.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI TOF MS) is a well-established method with a unique set of qualities including sensitivity, minute sample consumption, and label-free detection, all of which are highly desired in enzyme assays. On the other hand, the application of MALDI TOF MS is usually limited by high concentrations of MS-incompatible compounds in the reaction mixture such as salts or organic solvents. Here, we introduce kinetic and inhibition studies of ß-secretase (BACE1), a key enzyme of the progression of Alzheimer's disease. Compatibility of the enzyme assay with MALDI TOF MS was achieved, providing both a complex protocol including a desalting step designed for rigorous kinetic studies and a simple mix-and-measure protocol designed for high-throughput inhibitor screening. In comparison with fluorescent or colorimetric assays, MALDI TOF MS represents a sensitive, fast, and label-free technique with minimal sample preparation. In contrast to other MS-based methodological approaches typically used in drug discovery processes, such as a direct injection MS or MS-coupled liquid chromatography or capillary electrophoresis, MALDI TOF MS enables direct analysis and is a highly suitable approach for high-throughput screening. The method's applicability is strongly supported by the high correlation of the acquired kinetic and inhibition parameters with data from the literature as well as from our previous research. Graphical abstract ᅟ.

Immobilized-enzyme reactors integrated with capillary electrophoresis for pharmaceutical research.

Enzymes play an essential role in many aspects of pharmaceutical research as drug targets, drug metabolizers, enzyme drugs and more. In this specific field, enzyme assays are required to meet a number of specific requirements, such as low cost, easy automation, and high reliability. The integration of an immobilized-enzyme reactor to capillary electrophoresis represents a unique approach to fulfilling these criteria by combining the benefits of enzyme immobilization, that is, increased stability and repeated use, as well as the minute sample consumption, short analysis time, and efficient analysis provided by capillary electrophoresis. In this review, we summarize, analyze, and discuss published works where pharmaceutically relevant enzymes were used to prepare capillary electrophoresis-integrated immobilized-enzyme reactors in an online manner. The presented assays are divided into three distinct groups based on the drug-enzyme relationship. The first, more extensively studied group employs enzymes that are considered to be therapeutic targets, the second group of assays present tools to assess drug metabolism and the third group assesses enzyme drugs. Furthermore, we examine various methods of enzyme immobilization and their implications for assay properties.

Screening of Beta-Secretase Inhibitors by Capillary Electrophoresis-Mass Spectrometry.

Alzheimer's disease is the most common cause of dementia, currently afflicting almost 40 million patients worldwide. According to the amyloid cascade hypothesis, the pathogenesis of the disease could be slowed down or even stopped by the inhibition of beta-secretase, making this aspartic acid protease a potentially important drug target site. Capillary electrophoresis is a promising technique for screening putative enzyme inhibitors due to highly effective separations, minuscule sample and other chemicals consumption, compatibility with a variety of detection techniques, and high throughput via automation. This chapter presents a method based on capillary electrophoresis coupled to mass spectrometry detection for kinetic and inhibition assays of the beta-secretase reaction with a decapeptide derived from an amyloid precursor protein.

Development and comprehensive comparison of two on-line capillary electrophoretic methods for ß-secretase inhibitor screening.

Alzheimer's disease is the most common cause of dementia, afflicting over 34 million patients worldwide. Since ß-secretase is a rate-limiting enzyme of the production of neurotoxic ß-amyloid peptide oligomers abnormally accumulated in the affected brain tissue, its specific inhibition appears to be a promising approach to slowing down or even stopping the progression of the disease. Hence two on-line capillary electrophoretic methods for studies of ß-secretase activity based on the principles of transverse diffusion of laminar flow profiles and electrophoretically mediated microanalysis were developed, both using a simple unlabeled peptide substrate and UV detection. The optimized procedures were thoroughly validated and applied for determining the enzyme's kinetic parameters and the inhibition characteristics of two potent probe inhibitors. The resulting values were found to be comparable to literature data obtained with other analytical techniques. The suitability of the employed methodologies for different experimental designs is discussed on the basis of a statistical evaluation of the experimental data. The presented methods constitute a miniaturized and fully automated tool, which should be suitable for kinetic and inhibition studies of ß-secretase as a target for Alzheimer's disease drug discovery in the early stages of the development of a new drug.

A capillary electrophoresis-mass spectrometry based method for the screening of ß-secretase inhibitors as potential Alzheimer's disease therapeutics.

In this work a novel capillary electrophoresis-mass spectrometry (CE-MS) based method was developed and validated for the assay of ß-secretase (BACE1) activity as a potential target for Alzheimer's disease (AD) treatment. In contrast with the typically used Förster resonance energy transfer (FRET) assays, an unlabelled decapeptide derived from the amyloid precursor protein BACE1 site with the "Swedish mutation" was used as the substrate. The CE usage enabled the enzymatic reaction to be carried out in as small a volume as 100µL in 60min with sufficient yields of proteolytic product, which was subsequently separated in a bare fused silica capillary using 12.5% acetic acid as a background electrolyte and detected by MS. The limits of detection and quantitation were estimated using the signal to noise ratio to be 5nM (S/N=3) and 15nM (S/N=10), respectively, both being well below the working range for kinetic and inhibition studies. Its applicability for the kinetic study of BACE1 was demonstrated using optimized enzyme assay conditions and the estimated kinetic parameter values were confirmed by classic CE-UV analyses. The method was finally used for the main purpose for which it was developed - to screen BACE1 inhibitors as potential AD therapeutics. The resulting kinetic and inhibition parameters values were compared to those published in the literature, which were almost exclusively obtained by FRET based assays. These comparisons brought up several issues that are further discussed below and favour the application of an unlabelled substrate. The proposed CE-MS based method offers a high-throughput capability for new drug development.

On-line coupling of immobilized cytochrome P450 microreactor and capillary electrophoresis: A promising tool for drug development.

In this work, the combination of an immobilized enzyme microreactor (IMER) based on the clinically important isoform cytochrome P450 2C9 (CYP2C9) with capillary electrophoresis (CE) is presented. The CYP2C9 was attached to magnetic SiMAG-carboxyl microparticles using the carbodiimide method. The formation of an IMER in the inlet part of the separation capillary was ensured by two permanent magnets fixed in a cassette from the CE apparatus in the repulsive arrangement. The resulting on-line system provides an integration of enzyme reaction mixing and incubation, reaction products separation, detection and quantification into a single fully automated procedure with the possibility of repetitive use of the enzyme and minuscule amounts of reactant consumption. The on-line kinetic and inhibition studies of CYP2C9's reaction with diclofenac as a model substrate and sulfaphenazole as a model inhibitor were conducted in order to demonstrate its practical applicability. Values of the apparent Michalis-Menten constant, apparent maximum reaction velocity, Hill coefficient, apparent inhibition constant and half-maximal inhibition concentration were determined on the basis of the calculation of the effective substrate and inhibitor concentrations inside the capillary IMER using a model described by the Hagen-Poisseulle law and a novel enhanced model that reflects the influence of the reactants' diffusion during the injection process.