Development of a fluorescence-based, ultra high-throughput screening platform for nanoliter-scale cytochrome p450 microarrays.
Cytochrome P450 enzyme (CYP450s) assays are critical enzymes in early-stage lead discovery and optimization in drug development. Currently available fluorescence-based reaction assays provide a rapid and reliable method for monitoring CYP450 enzyme activity but are confined to medium-throughput well-plate systems. The authors present a high-throughput, integrated screening platform for CYP450 assays combining enzyme encapsulation techniques, microarraying methods, and wide-field imaging. Alginate-containing microarrays consisting of up to 1134 CYP450 reaction elements were fabricated on functionalized glass slides (reaction volumes 20 to 80 nL, total enzyme content in pg) and imaged to yield endpoint activity, stability, and kinetic data. A charge-coupled device imager acquired quantitative, high-resolution images of a 20x20 mm area/snapshot using custom-built wide-field optics with telecentric lenses and easily interchangeable filter sets. The imaging system offered a broad dynamic intensity range (linear over 3 orders of magnitude) and sensitivity down to fluorochrome quantities of <5 fmols, with read accuracy similar to a laser scanner or a fluorescence plate reader but with higher throughput. Rapid image acquisition enabled analysis of CYP450 kinetics. Fluorogenic assays with CYP3A4, CYP2C9, and CYP2D6 on the alginate microarrays exhibited Z' factors ranging from 0.75 to 0.85, sensitive detection of inhibitory compounds, and reactivity comparable to that in solution, thereby demonstrating the reliability and accuracy of the microarray platform. This system enables for the first time a significant miniaturization of CYP enzyme assays with significant conservation of assay reagents, greatly increased throughput, and no apparent loss of enzyme activity or assay sensitivity.
Sukumaran
Sumitra M
SM
Department of Chemical and Biological Engineering and Center for Biotechnology & Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, New York, USA.
Potsaid
Benjamin
B
Lee
Moo-Yeal
MY
Clark
Douglas S
DS
Dordick
Jonathan S
JS
D000464:Alginates / Q000378:metabolism; D002138:Calibration; D065607:Cytochrome P-450 Enzyme Inhibitors; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004795:Enzyme Stability / Q000187:drug effects; D019793:Fluorescein / Q000378:metabolism; D005453:Fluorescence; D020723:Glucuronic Acid / Q000378:metabolism; D006603:Hexuronic Acids / Q000378:metabolism; D006801:Humans; D020128:Inhibitory Concentration 50; D007700:Kinetics; D046228:Microarray Analysis / Q000379:methods*; D013379:Substrate Specificity / Q000187:drug effects
D000464:Alginates; D065607:Cytochrome P-450 Enzyme Inhibitors; D004791:Enzyme Inhibitors; D006603:Hexuronic Acids; D020723:Glucuronic Acid; D003577:Cytochrome P-450 Enzyme System; D019793:Fluorescein