Application of Capillary Electrophoresis for High-Throughput Screening of Drug Metabolism

2013 ◽  
pp. 309-318 ◽  
Author(s):  
Roman Řemínek ◽  
Jochen Pauwels ◽  
Xu Wang ◽  
Jos Hoogmartens ◽  
Zdeněk Glatz ◽  
...  
Keyword(s):  
Capillary Electrophoresis ◽  
Drug Metabolism ◽  
High Throughput ◽  
High Throughput Screening

scholarly journals A 96-multiplex capillary electrophoresis screening platform for product based evolution of P450 BM3

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Anna Gärtner ◽  
Anna Joëlle Ruff ◽  
Ulrich Schwaneberg
Keyword(s):  
Capillary Electrophoresis ◽  
High Throughput ◽  
High Throughput Screening ◽  
Product Formation ◽  
Resonance Spectroscopy ◽  
P450 Bm3 ◽  
Microtiter Plates ◽  
Main Challenge ◽  
Total Turnover ◽  
Screening Systems

Abstract The main challenge that prevents a broader application of directed enzyme evolution is the lack of high-throughput screening systems with universal product analytics. Most directed evolution campaigns employ screening systems based on colorimetric or fluorogenic surrogate substrates or universal quantification methods such as nuclear magnetic resonance spectroscopy or mass spectrometry, which have not been advanced to achieve a high-throughput. Capillary electrophoresis with a universal UV-based product detection is a promising analytical tool to quantify product formation. Usage of a multiplex system allows the simultaneous measurement with 96 capillaries. A 96-multiplexed capillary electrophoresis (MP-CE) enables a throughput that is comparable to traditional direct evolution campaigns employing 96-well microtiter plates. Here, we report for the first time the usage of a MP-CE system for directed P450 BM3 evolution towards increased product formation (oxidation of alpha-isophorone to 4-hydroxy-isophorone; highest reached total turnover number after evolution campaign: 7120 mol4-OH molP450−1). The MP-CE platform was 3.5-fold more efficient in identification of beneficial variants than the standard cofactor (NADPH) screening system.

scholarly journals High Throughput Screening of Pharmacokinetics and Metabolism in Drug Discovery (II) —Investigation on In vitro and In vivo Correlation in Drug Metabolism Screening—

2005 ◽  
Vol 125 (1) ◽  
pp. 131-139 ◽  
Author(s):  
Hiroshi KOMURA ◽  
Kenichi MATSUDA ◽  
Yukie SHIGEMOTO ◽  
Iichiro KAWAHARA ◽  
Rieko ANO ◽  
...  
Keyword(s):  
Drug Discovery ◽  
Drug Metabolism ◽  
High Throughput ◽  
High Throughput Screening

scholarly journals Development of a Miniaturized 384-Well High Throughput Screen for the Detection of Substrates of Cytochrome P450 2D6 and 3A4 Metabolism

2001 ◽  
Vol 6 (2) ◽  
pp. 91-99 ◽  
Author(s):  
Ilona Kariv ◽  
Mark P. Fereshteh ◽  
Kevin R. Oldenburg
Keyword(s):  
Drug Metabolism ◽  
High Throughput ◽  
High Throughput Screening ◽  
Biologically Active ◽  
In Vitro Assays ◽  
Main Concern ◽  
Assay Sensitivity ◽  
Cyp Enzymes ◽  
Assay Optimization

The identification of a large number of biologically active chemical entities during high throughput screening (HTS) necessitates the incorporation of new strategies to identify compounds with druglike properties early during the lead prioritization and development process. One of the major steps in lead prioritization is the assessment of drug metabolism mediated by the cytochrome P450 (CYP) enzymes to evaluate the potential drug-drug interactions. CYP2D6 and CYP3A4 comprise the main human CYP enzymes involved in drug metabolism. The recent availability of specific CYP cDNA expression systems and the development of specific fluorescent probes have accelerated the ability to develop robust in vitro assays in HTS format. The aim of this study was to optimize conditions for the CYP2D6 and CYP3A4 HTS assays and subsequently adapt those assays to a miniaturized 384-well format. Assay conversion to a miniaturized format presents certain difficulties, such as robustness of the signal and of compound delivery. Thus the assay optimization involved the comparison of different substrates to identify those most suitable for use in a miniaturized format. Because of current technical limitations in liquid dispensing of nanoliter volumes, assay sensitivity to organic solvents also provides a main concern during assay miniaturization. Therefore, compound activity from redissolved dry films and from DMSO stocks directly delivered into assay buffer was compared. The data indicate that compound activity was comparable in both formats. The data support the conclusion that CYP2D6 and CYP3A4 in vitro metabolism assays can be successfully performed in 384-well plate format and the substrate potencies, as evaluated by the IC50 values, determined.

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