Effect of Cytochrome P450 2C19 and 2C9 Amino Acid Residues 72 and 241 on Metabolism of Tricyclic Antidepressant Drugs

Abstract

Although cytochromes P450 2C9 (CYP2C9) and 2C19 (CYP2C19) have 91% amino acid identity, they have different substrate specificities. Previous studies have suggested that several amino acid residues may be involved in substrate specificity. In this study, we focused on the roles of two amino acids, residues 72 and 241. The amino acids in these positions have opposite charges in CYP2C9 and 2C19; the former has lysines in both positions (Lys72 and Lys241), and the latter has glutamic acids (Glu72 and Glu241). Reciprocal mutants for both CYP2C19 and 2C9 were produced, and their metabolic activities and spectroscopic properties were examined using three tricyclic antidepressant (TCA) drugs: amitriptyline, imipramine, and dothiepin. Although CYP2C19 wild-type (WT) had a high metabolic activity for all three drugs, the E72K mutation decreased enzymatic activity by 29-37%, while binding affinities were diminished 2.5-to 20-fold. On the other hand, low activity and low affinity of CYP2C9 WT were recovered notably by K72E mutation. The metabolic activities and binding affinities were minimally affected by CYP2C19 E241K and CYP2C9 K241E mutations. We could also show linear correlations between metabolic activities and binding affinities, and hence we conclude that amino acid residue 72 plays a key role in TCA drug metabolism by limiting the binding affinities of CYP2C19 and CYP2C9.Key words cytochrome P450 2C19; cytochrome P450 2C9; drug metabolism; antidepressant; dissociation constant Depression is a psychiatric condition that affects 120 million people worldwide, and can interfere with independence and productivity in essentially all aspects of daily life. Depression is also associated with a high risk of self-harm, and ultimately suicide. Despite the considerable advances in depression treatments, tricyclic antidepressants (TCAs) remain an important therapeutic option for depression. TCAs such as amitriptyline (AMI), imipramine (IMI), and dothiepin (DOT) are heterocyclic chemical compounds (Fig. 1). These drugs are characterized by substantial pre-systemic first-pass metabolism and their clearance is dependent primarily on hepatic cytochrome P450 (CYP) oxidative enzymes, in particular CYP2C19.1) Because the activity of some CYP isoforms is individually influenced by genetic variations, there is wide inter-individual variability in TCA pharmacokinetics and active metabolite production, 2,3) which complicates the clinical use of these drugs. Therefore, the understanding of TCA drug metabolism at the molecular level is essential for the safe use of these drugs.CYPs are a superfamily of heme-containing enzymes that are mainly responsible for oxidative transformation of xenobiotics in humans, animals, and plants. The CYP1, 2, and 3 families in mammals have a wide range of overlapping substrates and play a role in the metabolism of both drug and endogenous substrates through N-, O-, and S-dealkylation, aromatic and aliphatic hydroxylation, epoxidation; oxidative desulfurization, and sulfoxidation. 4,5) The CYP2C subfamily in...

Membrane Anchor of Cytochrome P450 Reductase Suppresses the Uncoupling of Cytochrome P450

“…29,38) To evaluate the drug metabolism activities of CPR, we used CYP2C19, which was expressed and purified as reported previously. 35,36) Reduction Activity of CPR for Cyt c The electron transfer activity of CPR was evaluated by reduction of cyt c at 37°C in 100 mM KPi buffer following procedures reported previously 39) with slight modifications. Briefly, a solution containing 50 µM ferric cyt c (Sigma, horse heart) and 0.1 nM CPR (WT or Δ60) was incubated for 3 min at 37°C, and NADPH solution in its regenerating system (350 µM NADPH, 880 µM glucose-6-phosphate, 880 µM MgCl 2 , and 2 units/mL glucose-6-phosphate dehydrogenase) was added to start the reaction.…”

“…We previously reported several kinetic parameters for the metabolism of AMT and IMP with purchased CPR (SigmaAldrich). 35,36) The drug metabolizing activities of WT-and Δ60-CPRs were estimated in a similar manner using UPLC (Acquity H-class, Waters) 36) in the absence or presence of 30 µg/mL 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC).…”

“…We reported previously that human CYP2C19 is fully metabolically active even when its N-terminal membrane anchor is truncated. 35,36) Truncated CYP2C19 is soluble, but can associate with the membrane, thus allowing us to assess the role of the CPR anchor with this CYP in the presence and absence of phospholipids. We constructed a truncated human CPR in which the N-terminal 60 amino acids are removed (Δ60-CPR), and investigated the effect of the truncation on the binding, electron transfer, and drug metabolizing properties of CYP2C19 to propose a novel function for the CPR membrane anchor.…”

Identification of universal selectivity‐determining positions in cytochrome P450 monooxygenases by systematic sequence‐based literature mining

“…Standard position 47 is located on β‐strand 1_2, which is part of a substrate access channel in many CYPs and was described in articles about 19 CYPs. In CYP2C9 and CYP2C19, mutations at the corresponding positions (K72 and R72, respectively) were described to have a significant effect on the enzymatic activity and binding affinity towards tricyclic antidepressant drugs . In CYP46A1, which shares 22% sequence identity with CYP2C9, substitutions of the corresponding residue L82 were described to influence binding affinity of the cyano‐ and fluoro‐containing drug bicalutamide .…”

Effect of Drug Combination on Omeprazole Metabolism by Cytochrome P450 2C19 in <i>Helicobacter pylori</i> Eradication Therapy

“…36) Recombinant human CYP reductase was prepared and its purity and activity were checked in accordance with a previously reported method. 37) Reduced nicotinamide adenine dinucleotide phosphate (NADPH) generating solution-containing 1 mM of β-nicotinamide-adenine dinucleotide phosphate (β-NADP + ), 2.5 mM of glucose-6-phosphate (G6P), and 2 U/mL of glucose-6-phosphate dehydrogenase (G6PDH) purchased from Oriental Yeast Co., Ltd. (Tokyo, Japan)-was prepared in 100 mM potassium phosphate buffer (KPi buffer, pH 7.4) with 2.5 mM MgCl 2 .…”