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Journal of Biomolecular Screening
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1087057105274904v1
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Article

Development of a Novel Cytochrome P450 Bioaffinity Detection System Coupled Online to Gradient Reversed-Phase High-Performance Liquid Chromatography

Jeroen Kool1, Sebastiaan M. Van Liempd1, Rawi Ramautar1, Tim Schenk2, John H. N. Meerman1, Hubertus Irth2, Jan. N. M. Commandeur1, Nico P. E. Vermeulen1*

1 LACDR--Division of Molecular Toxicology, Department of Pharmacochemistry, Vrije Universiteit, Amsterdam, the Netherlands
2 Kiadis B.V., Groningen, the Netherlands

* To whom correspondence should be addressed. E-mail: npe.vermeulen{at}few.vu.nl.


  Abstract

A high-resolution screening platform, coupling online affinity detection for mammalian cytochrome P450s (Cyt P450s) to gradient reversed-phase high-performance liquid chromatography (HPLC), is described. To this end, the online Cyt P450 enzyme affinity detection (EAD) system was optimized for enzyme ({beta}-NF-induced rat liver microsomes), probe substrate (ethoxyresorufine), and organic modifier (methanol or acetonitrile). The optimized Cyt P450 EAD system has first been evaluated in a flow injection analysis (FIA) mode with 7 known ligands of Cyt P450 1A1/1A2 ({alpha}-naphthoflavone, {beta}-naphthoflavone, ellipticine, 9-hydroxy-ellipticine, fluvoxamine, caffein, and phenacetin). Subsequently, IC50 values were online in FIA-mode determined and compared with those obtained with standard microsomal assay conditions. The IC50 values obtained with the online Cyt P450 EAD system agreed well with the IC50 values obtained in the standard assays. For high-affinity ligands of Cyt P450 1A1/1A2, detection limits of 1 to 3 pmol injected (n = 3; signal to noise [S/N] = 3) were obtained. The individual inhibitory properties of ligands in mixtures of the ligands were subsequently investigated using an optimized Cyt P450 EAD system online coupled to gradient HPLC. Using the integrated online gradient HPLC Cyt P450 EAD platform, detection limits of 10 to 25 pmol injected (n = 1; S/N = 3) were obtained for high-affinity ligands. It is concluded that this novel screening technology offers new perspectives for rapid and sensitive screening of individual compounds in mixtures exhibiting affinity for liver microsomal Cyt P450s.

Key Words: cytochrome P450, online bioaffinity assay, high-resolution screening (HRS), fluorescence, enzyme affinity detection (EAD)

First published on June 24, 2005, doi:10.1177/1087057105274904

Journal of Biomolecular Screening 2005;10:427.

A more recent version of this article appeared on August 1, 2005

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