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

Biochem Pharmacol. 2017 Dec 15;146:174-187. doi: 10.1016/j.bcp.2017.09.011. Epub 2017 Sep 23.

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.

Keywords: 1-Benzylimidazole (PubChem CID: 77918); Aminobenzotriazole (PubChem CID: 1367); Arachidonic acid (PubChem CID: 444899); Biotransformation; Econazole (PubChem CID: 3198); Eicosadienoic acid (PubChem CID: 6439848); Ether cleavage; Fission yeast; Human cytochrome P450; Hydroxylation; Lauric acid (PubChem CID: 3893); Miconazole (PubChem CID: 4189); Myristic acid (PubChem CID: 11005); Tolazoline (PubChem CID: 5504).

MeSH terms

  • Binding Sites
  • Biotransformation
  • Breast Neoplasms / enzymology
  • Breast Neoplasms / genetics
  • Breast Neoplasms / metabolism
  • Cytochrome P-450 CYP2C9 / metabolism
  • Cytochrome P-450 CYP3A / metabolism
  • Cytochrome P450 Family 4 / antagonists & inhibitors*
  • Cytochrome P450 Family 4 / genetics
  • Cytochrome P450 Family 4 / metabolism*
  • Enzyme Inhibitors / pharmacology*
  • Female
  • Gene Expression Regulation, Enzymologic
  • Gene Expression Regulation, Neoplastic
  • Humans
  • Microsomes, Liver / drug effects
  • Microsomes, Liver / enzymology
  • Models, Molecular
  • Octoxynol / pharmacology
  • Permeability
  • Protein Conformation
  • Schizosaccharomyces / genetics
  • Schizosaccharomyces / metabolism*
  • Substrate Specificity
  • Testosterone

Substances

  • Enzyme Inhibitors
  • Testosterone
  • Octoxynol
  • CYP2C9 protein, human
  • Cytochrome P-450 CYP2C9
  • CYP4Z1 protein, human
  • Cytochrome P-450 CYP3A
  • Cytochrome P450 Family 4
  • CYP3A4 protein, human