Investigation of Indazole Unbinding Pathways in CYP2E1 by Molecular Dynamics Simulations

Human microsomal cytochrome P450 2E1 (CYP2E1) can oxidize not only low molecular weight xenobiotic compounds such as ethanol, but also many endogenous fatty acids. The crystal structure of CYP2E1 in complex with indazole reveals that the active site is deeply buried into the protein center. Thus, the unbinding pathways and associated unbinding mechanisms remain elusive. In this study, random acceleration molecular dynamics simulations combined with steered molecular dynamics and potential of mean force calculations were performed to identify the possible unbinding pathways in CYP2E1. The results show that channel 2c and 2a are most likely the unbinding channels of CYP2E1. The former channel is located between helices G and I and the B-C loop, and the latter resides between the region formed by the F-G loop, the B-C loop and the β1 sheet. Phe298 and Phe478 act as the gate keeper during indazole unbinding along channel 2c and 2a, respectively. Previous site-directed mutagenesis experiments also supported these findings

Differential activities of CYP1A isozymes in hepatic and intestinal microsomes of control and 3-methylcholanthrene-induced rats.

Differences in expression of CYP1A isoforms (CYP1A1 and CYP1A2) in liver and small intestine of male Wistar rats and their inducibility by 3-methylcholanthrene as well as the effect of different CYP1A1/1A2 expression on caffeine metabolism were investigated. In rat liver, CYP1A2 is the predominant isoform and CYP1A1 protein expression in liver is significantly increased after treatment by 3-methylcholanthrene. In contrast, only CYP1A1 was detected in control and 3-methylcholanthrene induced small intestine microsomes. Treatment with 3-methylcholanthrene (40 mg/kg intraperitoneally daily during 1, 2, 3 or 4 days) demonstrated that liver CYP1A1 is more sensitive for the induction effects than CYP1A2 and also that significant induction of CYP1A1 in rat small intestine only occurred after 3 to 4 days pretreatment. Caffeine metabolism and inhibition studies by furafylline, CYP1A1 antiserum and ketoconazole revealed that the differences in the expression of CYP1A1 and CYP1A2 in the two tissues led to significant changes in the contribution of the various isoenzymes involved in the biotransformation of caffeine. Whereas in liver paraxanthine formation was almost exclusively catalyzed by CYP1A2, in rat proximal intestine it was formed by CYP1A1. In addition, other CYP enzymes (most probably CYP3A) play a significant role in theobromine and theophylline formation from caffeine in rat intestine. Overall, this study shows different expression and inducibility of CYP1A1/1A2 by 3-methylcholanthrene in rat liver and small intestine. Furthermore in rat intestine cytochrome P450 isozymes such as CYP1A1 and CYP3A replace CYP1A2 in the caffeine metabolism

Studies on the inhibition of human cytochromes P450 by selenocysteine Se-conjugates

1. To investigate whether cytochrome P450 (P450) inhibition can contribute to the chemopreventive activity of selenocysteine Se-conjugates (SeCys conjugates), 21 SeCys conjugates were screened for their inhibitory potency towards seven of the most important human P450s. 2. The majority of the SeCys conjugates produced near complete inhibition of CYP1A1 at a concentration of 250μM. The most potent inhibitor, Se-benzyl-L-selenocysteine, displayed an I

Cytochrome P450 bio-affinity detection coupled to gradient HPLC: on-line screening of affinities to cytochrome P4501A2 and 2D6

Here we describe novel on-line human CYP1A2 and CYP2D6 Enzyme Affinity Detection (EAD) systems coupled to gradient HPLC. The use of the systems lies in the detection of individual inhibitory ligands in mixtures (e.g. metabolic mixtures or herbal extracts) towards two relevant drug metabolizing human CYPs. The systems can rapidly detect individual compounds in mixtures with affinities to CYP1A2 or 2D6. The HPLC-EAD systems were first evaluated and validated in flow injection analysis mode. IC50 values of known ligands for both CYPs, tested both in flow injection and in HPLC mode, were well comparable with those measured in microplate reader formats. Both EAD systems were also connected to gradient HPLC and used to screen known compound mixtures for the presence of CYP1A2 and 2D6 inhibitors. Finally, the on-line CYP2D6 EAD system was used to screen for the inhibitory activities of stereoisomers of a mixture of five methylenedioxy-alkylamphetamines (XTC analogs) on a chiral analytical column. © 2007 Elsevier B.V. All rights reserved