Determinants of Cytochrome P450 2C8 Substrate Binding

Abstract

Although a crystal structure and a pharmacophore model are available for cytochrome P450 2C8, the role of protein flexibility and specific ligand-protein interactions that govern substrate binding are poorly understood. X-ray crystal structures of P450 2C8 complexed with montelukast (2.8 Å ), troglitazone (2.7 Å ), felodipine (2.3 Å ), and 9-cis-retinoic acid (2.6 Å ) were determined to examine ligand-protein interactions for these chemically diverse compounds. Montelukast is a relatively large anionic inhibitor that exhibits a tripartite structure and complements the size and shape of the active-site cavity. The inhibitor troglitazone occupies the upper portion of the active-site cavity, leaving a substantial part of the cavity unoccupied. The smaller neutral felodipine molecule is sequestered with its dichlorophenyl group positioned close to the heme iron, and water molecules fill the distal portion of the cavity. The structure of the 9-cis-retinoic acid complex reveals that two substrate molecules bind simultaneously in the active site of P450 2C8. A second molecule of 9-cis-retinoic acid is located above the proximal molecule and can restrain the position of the latter for more efficient oxygenation. Solution binding studies do not discriminate between cooperative and noncooperative models for multiple substrate binding. The complexes with structurally distinct ligands further demonstrate the conformational adaptability of active site-constituting residues, especially Arg-241, that can reorient in the active-site cavity to stabilize a negatively charged functional group and define two spatially distinct binding sites for anionic moieties of substrates.Cytochrome P450 2C8 is one of the principal drug-metabolizing P450 4 monooxygenases expressed in human liver. It is the predominant hepatic P450 catalyzing the 6␣-hydroxylation of paclitaxel (1) and the epoxidation of arachidonic acid (2, 3). Additionally, P450 2C8 contributes extensively to the metabolism of drugs such as pioglitazone, rosiglitazone, troglitazone, amodiaquine, amiodarone, and cerivastatin (4) as well as to the oxidation of retinoic acid (5-8). A screen of 209 commonly used drugs and related compounds identified several potent inhibitors of P450 2C8, including montelukast, candesartan cilexetil, mometasone furoate, clotrimazole, and felodipine (9). In addition, the glucuronide of gemfibrozil is a potent inhibitor of P450 2C8 (10, 11), and this inhibition is thought to underlie a drug-drug interaction between gemfibrozil and cerivastatin that can increase risk of rhabdomyolysis. Interestingly, glucuronides of 17␤-estradiol (12) and diclofenac (13) are also P450 2C8 substrates.A pharmacophore model for substrates of P450 2C8 was proposed by Mansuy and co-workers (14) based on the observation that many substrates are large organic anions at physiologic pH and exhibit sites of oxidation that are located ϳ13 Å from the anionic group. Additionally, it was noted that several polar moieties are often present at intermediate distances between the site o...

Structures of Human Cytochrome P-450 2E1

“…5). CYP2C8 has two access channels, one on each side of the BЈ helix (57). The channel in CYP2C9 exits between helices F and I and the C-terminal ␤ 4 sheet system (37).…”

Role of Protein-Protein Interactions in Metabolism: Genetics, Structure, Function

“…As observed with CYP126A1, the CYP2C8 homodimer was formed around interactions at the hydrophobic F/G loop interface. The presence of the dimer was also confirmed in solution as well as under the original crystallization conditions used (Schoch et al, 2008). Remarkably, two molecules of the substrate palmitic acid were found to be bound in the dimer interface, illustrating the potential physiological relevance of dimer formation.…”

“…However, the dimer is disrupted when the inhibitor ketoconazole is bound to the protein, leading to conversion to the monomer (Chenge et al, 2017). Similarly, CYP2C8 has been shown to crystalize as a dimer (Schoch et al, 2008). As observed with CYP126A1, the CYP2C8 homodimer was formed around interactions at the hydrophobic F/G loop interface.…”

Computational modelling of the binding of arachidonic acid to the human monooxygenase CYP2J2

“…In the structure of the inhibitor troglitazone bound to human CYP2C8 (PDB ID: 2vn0 [47]), the inhibitor's position overlaps with the region occupied by the carboxylic acid of AA and interacts with Ser103, the residue corresponding to Arg117 in the sequence alignment of the two enzymes (Supplementary Figure 6a). In the same structure, Thr364, which corresponds to Leu378 in CYP2J2, shows no interactions with the co-crystallised inhibitor.…”