Creation of a P450 array toward high-throughput analysis.
The rapid metabolism testing of many new chemical entities enables unsuitable candidates to be eliminated from consideration at an early stage of the drug discovery process. We have developed a P450 array toward high-throughput analysis of P450-mediated metabolic reaction. The microsomes containing expressed human P450 enzymes were immobilized on the microassay plate using sol-gel chemistry. A thin-film hydrogel containing microsomes was fabricated using aqueous silicate as a starting material. The TEM image clearly showed that the nanoclusters derived from the silicate formed branched chains, and microsomes were entrapped in the silica network. The different P450 isozymes were immobilized on the microassay plate, and the metabolites by each isozyme were visualized as fluorescent images, which creates opportunity for the inhibitor assays. This method offers several advantages over use of conventional enzyme preparations, including increased storage stability, ease of product isolation from the incubation mixture, and the ability to recover and reuse the enzyme. Because this methodology enabled the development of assay system using P450 that is unstable and involves other enzymes for its function, it can be applicable to various screening assays that require complicated reactions involving many biological components.
Sakai-Kato
Kumiko
K
Research Institute of Pharmaceutical Sciences, Musashino University, Nishitokyo-shi, Tokyo 202-8585, Japan. kumikato@musashino-u.ac.jp
Kato
Masaru
M
Homma
Hiroshi
H
Toyo'oka
Toshimasa
T
Utsunomiya-Tate
Naoko
N
D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D004800:Enzymes, Immobilized; D006801:Humans; D008861:Microsomes; D040081:Protein Array Analysis / Q000295:instrumentation* / Q000379:methods; D013379:Substrate Specificity
D004800:Enzymes, Immobilized; D003577:Cytochrome P-450 Enzyme System