Development Of A Cell-based Activity Assay And Identification Of A New Potent Inhibitor Of Human CYP4Z1

Breast cancer is the most common invasive cancer in women.The human cytochrome P450 enzyme CYP4Z1 is a potential target for the treatment of breast cancer and possibly also for other types of malignancies（such as ovarian cancer）.CYP4Z1 is of interest because it is strongly overexpressed both in breast cancer cells and in breast cancer metastases.The aim of this project was to establish an overexpression system of CYP4Z1 in breast cancer cells in order to serve as a cell culture test system.Therefore,the first part of this project was a stable transfection of MCF-7 cells with an expression construct for CYP4Z1.In this expression construct,expression of the CYP4Z1 c DNA is driven by the strong constitutive cytomegalovirus（CMV）promoter.The resulting new cell line Clone-1 was characterized by RT-q PCR.The second part was to use the new cell line to perform CYP4Z1 activity assays with the proluciferin substrates Luciferin 6’-benzyl ether（Luciferin-BE）and Luciferin3’-fluoro benzyl ether（Luciferin-3FBE）.For ease of detection,a protocol that employs proluciferin substrates was preferred over reactions that need to be monitored by HPLC or LC-MS.These experiments demonstrated that Clone-1 has a27-fold higher activity than untransfected MCF-7 cells with the substrate Luciferin-BE,as well as 10-fold higher activity than untransfected MCF-7 cells with the substrate Luciferin-3FBE.Next,the test system was used to verify the effect of the best known CYP4Z1 inhibitor,1-benzylimidazole.Its inhibitory effect on the conversion of both test substrates was confirmed and for the metabolization of Luciferin-BE an IC₅₀value 5.92μM was determined.Using a homology model an in silico screen was performed by our cooperation partners to search for CYP4Z1inhibitors.When using the new Clone-1-based test system to evaluate the inhibitory potency of nine new inhibitors,one compound（inhibitor 9）was identified that displayed an IC₅₀value of 66.6 n M,which is two orders of magnitude lower than that of 1-benzylimidazole.Together,these experiments show that a robust and reliable cell culture test system for CYP4Z1 was established,which complements the previously exisiting activity assays based on recombinant CYP4Z1 expression in fission yeast.The protocols,tools and data obtained in this project are expected to contribute to the development of a CYP4Z1-based prodrug strategy to cure breast cancer in the near future.