期刊
JOURNAL OF INORGANIC BIOCHEMISTRY
卷 117, 期 -, 页码 277-284出版社
ELSEVIER SCIENCE INC
DOI: 10.1016/j.jinorgbio.2012.05.017
关键词
Animal cytochrome P450; Chimeric protein; Immobilization; Electrode; Substrate turnover; HPLC
资金
- Region Piedmont CIPE (CYP-TECH project, Italy)
- Nanobiodesign s.r.l. (Italy)
In order to develop in vitro methods as an alternative to P450 animal testing in the drug discovery process, two main requisites are necessary: 1) gathering of data on animal homologues of the human P450 enzymes, currently very limited, and 2) bypassing the requirement for both the P450 reductase and the expensive cofactor NADPH. In this work, P450 2C20 from Macaca fascicularis, homologue of the human P450 2C8 has been taken as a model system to develop such an alternative in vitro method by two different approaches. In the first approach called molecular Lego, a soluble self-sufficient chimera was generated by fusing the P450 2C20 domain with the reductase domain of cytochrome P450 BM3 from Bacillus megaterium (P450 2C20/BMR). In the second approach, the need for the redox partner and also NADPH were both obviated by the direct immobilization of the P450 2C20 on glassy carbon and gold electrodes. Both systems were then compared to those obtained from the reconstituted P450 2C20 monooxygenase in presence of the human P450 reductase and NADPH using paclitaxel and amodiaquine, two typical drug substrates of the human P450 2C8. The K-M values calculated for the 2C20 and 2C20/BMR in solution and for 2C20 immobilized on electrodes modified with gold nanoparticles were 1.9 +/- 0.2, 5.9 +/- 2.3, 3.0 +/- 0.5 mu M for paclitaxel and 1.2 +/- 0.2, 1.6 +/- 0.2 and 1.4 +/- 0.2 mu M for amodiaquine, respectively. The data obtained not only show that the engineering of M. fasciculans did not affect its catalytic properties but also are consistent with K-M values measured for the microsomal human P450 2C8 and therefore show the feasibility of developing alternative in vitro animal tests. (C) 2012 Elsevier Inc. All rights reserved.
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