No abstract

Cytoglobin (Cgb) was discovered a decade ago and is a fourth member of the group of hexacoordinated globin-folded proteins. Although some crystal structures have been reported and several functions have been proposed for Cgb, its physiological role remains uncertain. In this study, we measured cyanide binding to the ferric state of the wild-type (WT) Cgb, and found that the binding consisted of multiple steps. These results indicated that Cgb may be comprised of several forms, and the presence of monomers, dimers, and tetramers was subsequently confirmed by SDS-PAGE. Remarkably, each species contained two distinguishable forms, and, in the monomer, analyses of alternative cysteine states suggested the presence of an intramolecular disulfide bond (monomer SS form) and a structure with unpaired thiol groups (monomer SH form). These confirmed that forms were separated by gel-exclusion chromatography, and that the cyanide binding of the separated fractions was again measured; they showed different affinities for cyanide, with the monomer fraction showing the highest affinity. In addition, the ferrous state in each fraction showed distinct carbon monoxide (CO)-binding properties, and the affinities for cyanide and CO suggested a linear correlation. Furthermore, we also prepared several variants involving the two cysteine residues. The C38S and C83S variants showed a binding affinity for cyanide similar to the value for the monomer SH form, and hence the fraction with the highest affinity for exogenous ligands was designated as a monomer SS form. We concluded that polymerization could be a mechanism that triggers the exertion of various physiological functions of this protein and that an appropriate disulfide bond between the two cysteine residues was critical for regulating the binding affinity of Cgb, which can act as a ROS scavenger, for exogenous ligands.

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...

Peroxisome proliferator-activated receptor α (PPARα) is a ligand-activated transcription factor that belongs to the superfamily of nuclear hormone receptors. PPARα is mainly expressed in the liver, where it activates fatty acid oxidation and lipoprotein metabolism and improves plasma lipid profiles. Therefore, PPARα activators are often used to treat patients with dyslipidemia. To discover additional PPARα activators as potential compounds for use in hypolipidemic drugs, here we established human hepatoblastoma cell lines with luciferase reporter expression from the promoters containing peroxisome proliferator-responsive elements (PPREs) and tetracycline-regulated expression of full-length human PPARα to quantify the effects of chemical ligands on PPARα activity. Using the established cell-based PPARα-activator screening system to screen a library of >12,000 chemical compounds, we identified several hit compounds with basic chemical skeletons different from those of known PPARα agonists. One of the hit compounds, a 1H-pyrazolo[3,4-b]pyridine-4-carboxylic acid derivative we termed compound 3, selectively up-regulated PPARα transcriptional activity, leading to PPARα target gene expression both and Of note, the half-maximal effective concentrations of the hit compounds were lower than that of the known PPARα ligand fenofibrate. Finally, fenofibrate or compound 3 treatment of high fructose-fed rats having elevated plasma triglyceride levels for 14 days indicated that compound 3 reduces plasma triglyceride levels with similar efficiency as fenofibrate. These observations raise the possibility that 1H-pyrazolo[3,4-b]pyridine-4-carboxylic acid derivatives might be effective drug candidates for selective targeting of PPARα to manage dyslipidemia.

Cytochrome P450 reductase (CPR) is an important redox partner of microsomal CYPs. CPR is composed of a membrane anchor and a catalytic domain that contains FAD and flavin mononucleotide (FMN) as redox centers and mediates electron transfer to CYP. Although the CPR membrane anchor is believed to be requisite for interaction with CYP, its physiological role is still controversial. To clarify the role of the anchor, we constructed a mutant (Δ60-CPR) in which the N-terminal membrane anchor was truncated, and studied its effect on binding properties, electron transfer to CYP2C19, and drug metabolism. We found that Δ60-CPR could bind to and transfer electrons to CYP2C19 as efficiently as WT-CPR, even in the absence of lipid membrane. In accordance with this, Δ60-CPR could mediate metabolism of amitriptyline (AMT) and imipramine (IMP) in the absence of lipids, although activity was diminished. However, Δ60-CPR failed to metabolize omeprazole (OPZ) and lansoprazole (LPZ). To clarify the reason for this discrepancy in drug metabolism, we investigated the uncoupling reaction of the CYP catalytic cycle. By measuring the amount of H 2 O 2 by-product, we found that shunt pathways were markedly activated in the presence of OPZ/LPZ in the Δ60-CPR mutant. Because H 2 O 2 levels varied among the drugs, we conclude that the proton network in the distal pocket of CYP2C19 is perturbed differently by different drugs, and activated oxygen is degraded to become H 2 O 2 . Therefore, we propose a novel role for the membrane anchor as a suppressor of the uncoupling reaction in drug metabolism by CYP.