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Efficient substrate screening and inhibitor testing of human CYP4Z1 using permeabilized recombinant fission yeast.

Authors
  • Yan, Qi1
  • Machalz, David2
  • Zöllner, Andy3
  • Sorensen, Erik J4
  • Wolber, Gerhard2
  • Bureik, Matthias5
  • 1 School of Pharmaceutical Science and Technology, Health Sciences Platform, Tianjin University, Tianjin 30072, China. , (China)
  • 2 Pharmaceutical and Medicinal Chemistry (Computer-Aided Drug Design), Institute of Pharmacy, Free University Berlin, Germany. , (Germany)
  • 3 Lab Logistics Group GmbH, D-53340 Meckenheim, Germany. , (Germany)
  • 4 School of Pharmaceutical Science and Technology, Health Sciences Platform, Tianjin University, Tianjin 30072, China; Department of Chemistry, Princeton University, Princeton, NJ 08544, USA. , (China)
  • 5 School of Pharmaceutical Science and Technology, Health Sciences Platform, Tianjin University, Tianjin 30072, China. Electronic address: [email protected] , (China)
Type
Published Article
Journal
Biochemical pharmacology
Publisher
New York, NY : Elsevier Science Inc
Publication Date
Dec 15, 2017
Volume
146
Pages
174–187
Identifiers
DOI: 10.1016/j.bcp.2017.09.011
PMID: 28951277
Source
Medline
Keywords
License
Unknown

Abstract

We have established a protocol for the preparation of permeabilized fission yeast cells (enzyme bags) that recombinantly express human cytochrome P450 enzymes (CYPs). A direct comparison of CYP3A4 activity gave an eightfold higher space-time yield for enzyme bag-catalyzed biotransformation as compared to whole-cell biotransformation, even though the total number of cells employed was lower by a factor of 150. Biotransformation of the luminogenic substrate Luciferin-H using CYP2C9-containing enzyme bags proceeded efficiently and stably for 24h. CYP4Z1 is of interest because it is strongly overexpressed both in breast cancer cells and in breast cancer metastases; however, current knowledge about its catalytic properties is very limited. Screening of CYP4Z1-containing enzyme bags with 15 luminogenic substrates enabled us to identify two new hydroxylations and eleven ether cleavage reactions that are catalyzed by CYP4Z1. By far the best substrate found in this study was Luciferin benzyl ether (Luciferin-BE). On the basis of the recently published crystal structure of CYP4B1 we created a new homology model of CYP4Z1 and performed molecular docking experiments, which indicate that all active substrates show a highly similar binding geometry compared to the endogenous substrates. The model predicts that Ser113, Ser222, Asn381, and Ser383 are key hydrogen bonding residues. We also identified five new inhibitors of CYP4Z1: miconazole, econazole, aminobenzotriazole, tolazoline, and 1-benzylimidazole respectively, with the last compound being the most potent giving an IC50 value of 180nM in our test system.

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