Identification of a Novel Laser Dye Substrate of Mammalian Cytochromes P450: Application in Rapid Kinetic Analysis,Inhibitor Screening,and Directed Evolution

Abstract

The author sought to develop a high-throughput activity screening assay to carry out rapid kinetic analysis, inhibitor screening, and directed evolution of cytochrome P450 2C enzymes. Initially, of the 9 fluorescent substrates and 10 P450 2C enzymes tested, several P450 2C enzymes showed > 1 nmol/min/nmol P450 activity in cumene hydroperoxide (CuOOH)—supported reaction with a laser dye, 7-dimethylamino-4-trifluoromethylcoumarin (C152). A high-throughput steady-state kinetic analysis of the human P450 2C8, 2C9, and 2C19 showed 1) kcat = 3 to 6 min—1, 2) Km, CuOOH = 100 to 200 µM, and 3) S50, C152 = 10 to 20 µM in the CuOOH system. In addition, P450 2C9 and 2C19 showed a very high kcat (27 and 38 min—1, respectively) in the nicotinamide adenine dinucleotide phosphate (NADPH)—supported reaction. Subsequently, when mammalian P450s from the other subfamilies were tested, P450 2B1dH, 2B4dH, 2B5dH, 3A4, and 3A5 exhibited a significant activity in both CuOOH and NADPH systems. Furthermore, a high-throughput activity screening assay using whole-cell suspensions of the human P450 2C8, 2C9, and 2C19 was optimized. Overall, the data suggested that C152 can be used as a model substrate for mammalian P450s in CuOOH-supported reaction to perform rapid kinetic analysis, inhibitor screening, and directed evolution. ( Journal of Biomolecular Screening 2007:677-682)

Keywords

cytochrome P450 coumarin 152 screening directed evolution P450 2C laser dye

References

Anzenbacher P. , Anzenbacherova F. : Cytochromes P450 and metabolism of xenobiotics. Cell Mol Life Sci 2001;58:737-747.

Johnson EF : Deciphering substrate recognition by drug-metabolizing cytochromes P450. Drug Metab Dispos 2003;31:1531-1540.

Kumar S. , Halpert JR : Use of directed evolution of mammalian cytochromes P450 for investigating the molecular basis of enzyme function and generating novel biocatalysts. Biochem Biophys Res Commun 2005;338:456-464.

Crespi CL : Higher-throughput screening with human cytochromes P450. Curr Opin Drug Des Dev 2005;2:15-19.

Kumar S. , Davydov DR , Halpert JR : Role of cytochrome b5 in modulating peroxide-supported CYP3A4 activity: evidence for a conformational transition and cytochrome P450 heterogeneity. Drug Metab Dispos 2005; 33:1131-1136.

Kumar S. , Chen CS , Waxman DJ , Halpert JR : Directed evolution of mammalian cytochrome P450 2B1: mutations outside of the active site enhance the metabolism of several substrates, including the anticancer prodrugs cyclophosphamide and ifosfamide. J Biol Chem 2005;280:19569-19575.

Kumar S. , Sun L. , Liu H. , Muralidhara BK , Halpert JR : Engineering mammalian cytochrome P450 2B1 by directed evolution for enhanced catalytic tolerance to temperature and dimethyl sulfoxide. Protein Eng Des Select 2006;34:1958-1965.

Kumar S. , Liu H. , Halpert JR : Engineering of cytochrome P450 3A4 for enhanced peroxide-mediated substrate oxidation using directed evolution and site-directed mutagenesis. Drug Metab Dispos 2006;19:547-554.

Richardson TH , Jung F. , Griffin KJ , Wester M. , Raucy JL , Kemper B. , et al: A universal approach to the expression of human and rabbit cytochrome P450s of the 2C subfamily in Escherichia coli . Arch Biochem Biophys 1995;323:87-96.

10.

Scott EE , Spatzenegger M. , Halpert JR : A truncation of 2B subfamily cytochromes P450 yields increased expression levels, increased solubility, and decreased aggregation while retaining function . Arch Biochem Biophys 2001;395:57-68.

11.

Donato MT , Jimenez N. , Castell JV , Gomez-Lechon MJ : Fluorescence-based assays for screening nine cytochrome P450 (P450) activities in intact cells expressing individual human P450 enzymes. Drug Metab Disp 2004; 32:699-706.

12.

Chakraborty A. , Seth D. , Setula P. , Sarkar N. : Photoinduced electron transfer in a protein-surfactant complex: probing the interaction of SDS and BSA. J Phys Chem B 2006;110:16607-16617.

13.

Ghosal A. , Hapangama N. , Yuan Y. , Lu X. , Horne D. , Patrick JE , et al: Rapid determinations of enzyme activities of recombinant human cytochromes P450, human liver microsomes and hepatocytes. Biopharm Drug Disp 2003;24:375-384.

14.

Domanski TL , Halpert JR : Analysis of mammalian cytochrome P450 structure and function by site-directed mutagenesis. Curr Drug Metab 2001;2:117-137.

15.

Atkins W. , Dough W. , Cook DL : Is there a toxicological advantage for non-hyperbolic kinetics in cytochrome P450 catalysis? J Bio Chem 2002;277:33258-33266.