Efficient Substrate Screening And Inhibitor Testing Of Human CYP4Z1 Using Permeabilized Recombinant Fission Yeast

We have successfully established a protocol for the preparation of permeabilized fission yeast cells?enzyme bags?that recombinantly express human cytochrome P450enzymes?CYPs?.In comparison to the previous standard whole-cell biotransformation,the enzyme bag-catalyzed biotransformation can use much less amount of cells and within less reaction time to produce roughly eight times higher yield of product 6?-hydroxytestosterone,which was one metabolite of testosterone by CYP3A4 and was detected by the HPLC-MS.Biotransformation of the luminogenic substrate Luciferin-H using CYP2C9-containing enzyme bags proceeded efficiently and stably for 24 h.Moreover,the permeabilized fission yeast cells containing single human cytochrome P450 enzyme can be stored for a long time in 25%glycerol with1X PBS buffer,and the activity was not significantly decreased after one month compared with the freshly prepared enzyme bags.Then we focus on the human CYP4Z1,which is strongly overexpressed not only in breast cancer cells but also in breast cancer metastases.And current knowledge about its catalytic properties is very limited,up to now only four substrates were identified including two saturated fatty acids and two unsaturated fatty acids,and with only one reported inhibitor HET0016.To investigate its catalytic properties,we screened 15 luminogenic substrates with enzyme bag method and encouragingly 13 of them can be metabolized by CYP4Z1,and among these substrates we identified 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?.For the economic reason we chose Luciferin-4F12 as the probe substrate to screen the potential inhibitors of CYP4Z1.Finally,we identified five new inhibitors of CYP4Z1:miconazole,econazole,aminobenzotriazole,tolazoline,and 1-benzylimidazole respectively,and the last compound gave an IC₅₀ value of 180 nM in our test system which had much potent inhibitory effects than the reported inhibitor HET0016.Then based on these experimental data and the recently published crystal structure of CYP4B1,we constructed a new homology model of CYP4Z1 and performed molecular docking experiments,which indicated that all active substrates show a highly similar binding geometry compared to the endogenous substrates.It is the first time to characterize the active site of CYP4Z1 and hydrogen binding modes in the active site.In addition,the model also predicts that Ser113,Ser222,Asn381,and Ser383 are key hydrogen bonding residues within the active site.After that,in the recently performed mutagenesis assay,the activity of CYP4Z1 in each mutant did not have a significant decrease to the wild-type,which needs more mutagenesis data in the future to validate that prediction and then correct the model.Once the homology model is corrected by the experimental data,more compounds can be docked to the enzyme in visual and more substrates and inhibitors can be found to enrich the knowledge about the catalytic properties of CYP4Z1.And there will be more possibilities to develop a prodrug strategy of CYP4Z1 to cure the breast cancer in the near future.