Mutual Influence of Human Cytochrome P450 Enzymes and UDP-Glucuronosyltransferases on Their Respective Activities in Recombinant Fission Yeast

Abstract

Cytochromes P450 (CYPs) and UDP-glucuronosyltransferases (UGTs) are the most important human drug metabolizing enzymes, but their mutual interactions are poorly understood. In this study, we recombinantly co-expressed of each one of the 19 human members of the UGT families 1 and 2 with either CYP2C9, CYP2D6, or CYP4Z1 in fission yeast. Using these strains, we monitored a total of 72 interactions: 57 cases where we tested the influence of UGT co-expression on CYP activity and 15 cases of the opposite approach. In the majority of cases (88%), UGT co-expression had a statistically significant (p < 0.05) effect on P450 activity (58% positive and 30% negative). Strong changes were observed in nine cases, including one case with an activity increase by a factor of 23 (CYP2C9 activity in the presence of UGT2A3) but also four cases with a complete loss of activity. When monitoring the effect of CYP co-expression on the activity of five UGTs, activity changes were generally not so pronounced and, if observed, always detrimental. UGT2B7 activity was not influenced by CYP co-expression, while the other UGTs were affected to varying degrees. These data suggest the notion that mutual influence of CYPs and UGTs on each other’s activity is a widespread phenomenon.

Mutual Modulation of the Activities of Human CYP2D6 and Four UGTs during the Metabolism of Propranolol

“…The previously published protocol for CYP-UGT activity assays with diploid fission yeasts was established using proluciferin probe substrates [ 19 ]. For monitoring of propranolol metabolism, this protocol needed to be optimized as this is a much more lipophilic substrate with a logD (7.4) value of 1.2 [ 36 ].…”

“…4-Hydroxypropranolol and 5-hydroxypropranolol were identified via LC-MS/MS comparison with reference standards in all samples ( Figure 4 B). The concentrations of 4-hydroxypropranolol and 5-hydroxypropranolol were calculated using a standard calibration curve and all the results were normalized using qPCR data of CYP2D6 expression in five diploid strains as reported in the previous study [ 19 ]. In comparison with control CYP2D6 samples, the production of 4-hydroxypropranolol using UGT1A7, UGT1A8 and UGT1A9 co-expression was significantly enhanced by a factor of 3.3, 2.1, or 2.8, respectively ( Figure 5 A).…”

“…Very recently, we have established a recombinant fission yeast system in which each one of the 19 human members of the UGT families 1 and 2 is co-expressed with either CYP2C9, CYP2D6, or CYP4Z1. A total of 72 interactions between CYPs and UGTs in these new strains were observed using proluciferin probe substrates [ 19 ]. With respect to CYP2D6, co-expression of eleven UGTs (including UGT1A7) led to a statistically significant activity decrease, while six (including UGT1A8, UGT1A9, and UGT2A1) caused an increase and the remaining two had no effect.…”

“…The construction of the diploid yeast strains was reported very recently [ 19 ]. All yeast strains used in this study are listed in Table 2 .…”

“…The fission yeast Schizosaccharomyces pombe has been successfully utilized for expressing human CYPs and UGTs [ 17 , 18 ]. Recently, diploid fission yeast strains were developed, enabling the simultaneous expression of human CYPs (along with human CPR) and UGTs [ 19 ]. A previous study demonstrated evidence of a proper recognition of human subcellular localization sequences of human CYPs in fission yeast systems [ 20 ].…”