Influence of fluorescent probe size and cytochrome b5 on drug-drug interactions in CYP2C9

7-Methoxy-4-trifluoromethylcoumarin (MFC) has been used extensively in high-throughput screens for the identification of potential CYP2C9 interactions. More recently, additional probes from Invitrogen have been used. Vivid 2C9 Green is the largest of the probes and has had limited prior characterization. The new series of probes differ significantly from MFC and were examined for their ability to identify interactions with 19 CYP2C9 substrates/inhibitors. The inhibition profiles depend largely on the physical differences between the fluorescent probe substrates. Cytochrome b5 (cyt b5) was also investigated for the ability to alter the inhibition profile of a given compound. The stoichiometric addition of cyt b5 caused an increase in V max of MFC and Vivid 2C9 Green 4.4 and 1.7 times, respectively. Furthermore, cyt b5 imposes a steric component to the active site as the inhibition profiles were significantly affected in incubations with MFC. The addition of cyt b5 had limited impact on the inhibition profiles generated with Vivid 2C9 Green. The K(m) of Vivid 2C9 Green increased from 1.2+/-0.2 micro M to 4.8+/-0.3 micro Mas a result of cyt b5 addition. These results illustrate that multiple substrate probes may be necessary for screening drug-drug interaction in CYP2C9 and that cyt b5 effects can impart steric restraints on the CYP2C9 active site.