Human Cytochrome P450 Enzymes in Drug and Fatty Acid Metabolism

Abstract

Human cytochrome P450 enzymes are membrane-embedded heme-containing monooxygenases responsible for a variety of physiological functions. The 57 human P450 enzymes share an overall canonical fold with important differences in their sequence and resulting structures leading to distinct activities. Many individual P450 enzymes play important roles in human health, including in drug metabolism and critical homeostatic pathways where they are common drug targets. Therefore understanding the structural nuances of each P450 enzyme is important. The work herein focuses on two P450 enzymes, CYP2W1 and CYP2J2, for which structural and functional information is limited. CYP2W1 is expressed in fetal tissue and then silenced in healthy adult tissues. Its endogenous substrates and role in fetal development is unknown. However, CYP2W1 protein expression reactivated in 30% of colorectal cancers and 50% of hepatocellular carcinoma can activate highly potent duocarmycin prodrugs, thus constituting a potential tool in targeted cancer therapies. However, another P450 enzyme expressed in healthy tissue can also activate those duocarmycin prodrugs. To limit off target effects, a duocarmycin prodrug selectively activated by CYP2W1 is required. In the absence of a CYP2W1 structure, prodrug design can be facilitated indirectly by determining common physiochemical characteristics of CYP2W1 ligands. A high throughput screen identified 694 CYP2W1 inhibitors used to generate a pharmacophore identifying common ligand features. However, without knowing ligand orientation within the active site, agreement in this model was poor. Thus, a high throughput binding screen was developed that includes orientation information for azole-containing compounds. Screening of 104 azoles identified 67 that coordinate the CYP2W1 active site heme iron for which binding affinities were then calculated. Resulting pharmacophores identified common aromatic rings, hydrophobic features, and hydrogen bond acceptors. Comparison of pharmacophores for tight binding ligands and for all CYP2W1 ligands suggested the positioning of the hydrogen bond acceptor may influence binding affinity. These results improved the understanding of the CYP2W1 pharmacophore that can now be used to inform CYP2W1-selective prodrug design and potentially to identify endogenous substrates. The second P450 enzyme examined herein is CYP2J2, which is involved in both xenobiotic and fatty acid metabolism in cardiac tissues. CYP2J2 metabolism of fatty acids such as arachidonic acid is linked to cardioprotective effects, and in the presence of CYP2J2 xenobiotic ligands, these cardioprotective effects are likely reduced. Thus it is critical that CYP2J2 active site features are better understood to inform therapeutic design to avoid CYP2J2 interaction. Pharmacophore modeling and X-ray crystallography were used to examine the CYP2J2 structure. The high throughput binding assay identified 44 CYP2J2 ligands used to generate pharmacophores, revealing that high affinity CYP2J2 ligands share a hydrophobic feature connected to an aromatic ring, while weaker affinity compounds with exhibit two hydrogen bond acceptors. Additionally, a CYP2J2 structure was determined, revealing the most open human P450 conformation observed to date. With the active site heme visible from the distal protein surface, this structure is useful in understanding ligand access to P450 active sites and is a first step in identifying active site residues. Future work determining a liganded CYP2J2 structure would provide insight into key contacts its residues make with ligands. In conclusion, a new high throughput binding assay facilitated the development of CYP2J2 and CYP2W1 pharmacophores which may assist in drug design. Future studies may also yield pharmacophores for P450 enzymes that are difficult to study through traditional structural biology methods.