Through their unique oxidative chemistry, cytochrome P450 monooxygenases (CYPs) catalyze the elimination of most drugs and toxins from the human body. Protein–protein interactions play a critical role in this process. Historically, the study of CYP–protein interactions has focused on their electron transfer partners and allosteric mediators, cytochrome P450 reductase and cytochrome b5. However, CYPs can bind other proteins that also affect CYP function. Some examples include the progesterone receptor membrane component 1, damage resistance protein 1, human and bovine serum albumin, and intestinal fatty acid binding protein, in addition to other CYP isoforms. Furthermore, disruption of these interactions can lead to altered paths of metabolism and the production of toxic metabolites. In this review, we summarize the available evidence for CYP protein–protein interactions from the literature and offer a discussion of the potential impact of future studies aimed at characterizing noncanonical protein–protein interactions with CYP enzymes.

The human organic cation transporter 1 (OCT1) is a polyspecific transporter involved in the uptake of positively charged and neutral small molecules in the liver. To date, few endogenous compounds have been identified as OCT1 substrates; more importantly, the effect of drugs on endogenous substrate transport has not been examined. In this study, we established monoamine neurotransmitters as substrates for OCT1, specifically characterizing serotonin transport in human embryonic kidney 293 cells. Kinetic analysis yielded a K_m of 197 micomolar and a V_max of 561 pmol/mg protein/minute for serotonin. Furthermore, we demonstrated that serotonin uptake was inhibited by diphenhydramine, fluoxetine, imatinib, and verapamil, with IC₅₀ values in the low micromolar range. These results were recapitulated in primary human hepatocytes, suggesting that OCT1 plays a significant role in hepatic elimination of serotonin and that xenobiotics may alter the elimination of endogenous compounds as a result of interactions at the transporter level.

The metabolism of testosterone to 6β-hydroxytestosterone (6β-OH-T) is a commonly used assay to evaluate human CYP3A enzyme activities. However, previous reports have indicated that CYP3A7 also produces 2α-hydroxytestosterone (2α-OH-T) and that a 2α-OH-T/6β-OH-T ratio may be a unique endogenous biomarker of the activity of the enzyme. Until now, the full metabolite and kinetic profile for testosterone hydroxylation by CYP3A7 has not been fully examined. To this end, we performed a complete kinetic analysis of the 6β-OH-T, 2α-OH-T, and 2β-hydroxytestosterone metabolites for recombinant Supersome CYP3A4, CYP3A5, and CYP3A7 enzymes and monitored metabolism in fetal and adult human liver microsomes for comparison. In general, a decrease in the velocity of the reaction was observed between CYP3A4 and the two other enzymes, with CYP3A7 showing the lowest metabolic capacity. Interestingly, we found that the 2α-OH-T/6β-OH-T ratio varied with substrate concentration when testosterone was incubated with CYP3A7, suggesting that this ratio would likely not function well as a biomarker for CYP3A7 activity. In silico docking studies revealed at least two different binding modes for testosterone between CYP3A4 and CYP3A7. In CYP3A4, the most energetically favorable docking mode places testosterone in a position with the methyl groups directed toward the heme iron, which is more favorable for oxidation at C6β, whereas for CYP3A7 the testosterone methyl groups are positioned away from the heme, which is more favorable for an oxidation event at C2α. In conclusion, our data indicate an alternative binding mode for testosterone in CYP3A7 that favors the 2α-hydroxylation, suggesting significant structural differences in its active site compared with CYP3A4/5.

Cytochrome P450 enzymes are versatile catalysts involved in a wide variety of biological processes from hormonal regulation and antibiotic synthesis to drug metabolism. A hallmark of their versatility is their promiscuous nature, allowing them to recognize a wide variety of chemically diverse substrates. However, the molecular details of this promiscuity have remained elusive. Here, we have utilized two-dimensional heteronuclear single quantum coherence NMR spectroscopy to examine a series of mutants site-specific labeled with the unnatural amino acid, [(13)C]p-methoxyphenylalanine, in conjunction with all-atom molecular dynamics simulations to examine substrate and inhibitor binding to CYP119, a P450 from Sulfolobus acidocaldarius. The results suggest that tight binding hydrophobic ligands tend to lock the enzyme into a single conformational substate, whereas weak binding low affinity ligands bind loosely in the active site, resulting in a distribution of localized conformers. Furthermore, the molecular dynamics simulations suggest that the ligand-free enzyme samples ligand-bound conformations of the enzyme and, therefore, that ligand binding may proceed largely through a process of conformational selection rather than induced fit.