Functional Expression And Characterization Of Human CYP Enzymes

Cytochrome P450s(CYPs or P450s)are a structurally and functionally related superfamily of enzymes.In a reduced state and in complex with carbon monoxide the enzymes have a maximum UV absorption peak at 450 nm,which gave them their name.Many P450 s are responsible for the Phase I reactions in human drug metabolism and their substrates include a broad range of active pharmaceutical ingredients,environmental toxins,carcinogens,and food-derived chemicals.Other important functions of human CYPs are the biosynthesis,metabolism,and degradation of endogenous compounds,such as steroid hormones,fatty acids,and bile acids.This thesis is composed of three different projects.The first project focused on the identification of new substrates and inhibitors of human steroid 12?-hydroxylase CYP8B1,which is a crucial regulator of the balance of cholic acid(CA)and chenodeoxycholic acid(CDCA)in the liver.It was previously shown to catalyze the conversion of 7?-hydroxycholest-4-en-3-one,a CDCA precursor,to7?,12?-dihydroxycholest-4-en-3-one,which is an intermediate of CA biosynthesis.In this study it is demonstrated that CYP8B1 can also perform the 12?-hydroxylation of CDCA itself to CA.In addition,five derivatives of luciferin are shown to be metabolized by CYP8B1,and using one of these a rapid and convenient inhibitor test system was established.In this way,four new(but moderate)CYP8B1 inhibitors were identified,which are aminobenzotriazole,exemestane,ketoconazole,and letrozole.We expect that this new test system will facilitate the search for CYP8B1 inhibitors of high potency and selectivity,which have pharmacological potential for the treatment of metabolic diseases such as nonalcoholic fatty liver disease(NAFLD).In second part,the activity of three CYPs coexpressed with different redox system backgrounds to understand the interaction of CYP with its electron partners was investigated.In addition to the major microsomal electron donor,cytochrome P450reductase(CPR),there are other pathways for transferring electrons in P450 systems,which involve cytochrome b5(B5)and/or NADH-cytochrome b5 reductase(B5R).In these pathways,B5 is involved in the CPR-independent direct electron transfer of both electrons from B5 R to the CYP enzyme proper or in the transfer of the second electron to P450 from either B5 R or CPR.In addition,allosteric stimulation of P450 by B5 without electron transfer has also been reported.In this study,new strains of CYP3A4,CYP3A7,and CYP17A1,respectively,were constructed with three different reductase systems.In these strains,one bicistronic and two tricistronic expression constructs were used,which are CPR-B5,CPR-B5-B5 R and B5R-B5-CPR,respectively.It was found that CPR coexpressed with B5 and B5 R can increase the activity of CYP3A4 and CYP3A7 for luminogenic substrates as compared to the coexpression with CPR only.By contrast,CYP17A1 coexpressed with CPR only had the highest activity,and an expected 17,20-lyase activity was not obtained in the biotransformation performed with progesterone and pregnenolone as substrates.What's more,CPR-Q153 R coexpressed with CYP17A1 strain also showed a lower activity compared with the wild-type CPR strain.These data suggest that the interaction with B5 and/or B5 R can enhance the activity of some human CYP enzymes,such as those of the CYP3 A family,but not that of CYP17A1 and CPR-Q153 R was also not possible to improve the activity of CYP17A1.