Evaluation of Rhesus Monkey and Guinea Pig Hepatic Cytosol Fractions as Models for Human Aldehyde Oxidase

Abstract

Aldehyde oxidase (AOX) is a cytosolic enzyme expressed across a wide range of species, including guinea pig and rhesus monkey. These species are believed to be the best preclinical models for studying human AOX-mediated metabolism. We compared AOX activity in rhesus monkeys, guinea pigs, and humans using phthalazine and N-[2-(dimethylamino)ethyl]acridone-4-carboxamide (DACA) as substrates and raloxifene as an inhibitor. Michaelis-Menten kinetics was observed for phthalazine oxidation in rhesus monkey, guinea pig, and human liver cytosol, whereas substrate inhibition was seen with DACA oxidase activity in all three livers. Raloxifene inhibited phthalazine and DACA oxidase activity uncompetitively in guinea pig, whereas mixed-mode inhibition was seen in rhesus monkey.Our analysis of the primary sequence alignment of rhesus monkey, guinea pig, and human aldehyde oxidase isoform 1 (AOX1) along with homology modeling has led to the identification of several amino acid residue differences within the active site and substrate entrance channel of AOX1. We speculate that some of these residues might be responsible for the differences observed in activity. Overall, our data indicate that rhesus monkeys and guinea pigs would overestimate intrinsic clearance in humans and would be unsuitable to use as animal models. Our study also showed that AOX metabolism in species is substrate-dependent and no single animal model can be reliably used to predict every drug response in humans.

Inhibition of Human Aldehyde Oxidase Activity by Diet-Derived Constituents: Structural Influence, Enzyme-Ligand Interactions, and Clinical Relevance

“…5). These interactions are consistent with previous molecular modeling studies involving the AO substrates zoniporide, DACA, and substituted phthalazines (Dastmalchi and Hamzeh-Mivehrod, 2005;Dalvie et al, 2012;Choughule et al, 2013), supporting the hypothesis that the inhibitors are competing with substrates within the active site. A recent report describing the X-ray crystal structure of quercetin bound to the active site of xanthine oxidase, which is highly homologous to AO, further supports this contention (Cao et al, 2014).…”

“…Previous QSAR studies using a series of flavonoid compounds involved rat AO (Hamzeh-Mivehroud et al, 2013; however, given the significant between-species variation in enzyme structure, inhibitory potency of compounds can vary widely (Sahi et al, 2008;Choughule et al, 2013). These results provide a first step toward developing in silico models to predict the human AO inhibitory activity of a xenobiotic solely The final aim of this study was to identify dietary constituents as potential perpetrators of clinically relevant AO-mediated interactions with conventional medications.…”

“…The homology model for human AO (AOX1) protein was developed as described previously (Choughule et al, 2013). In brief, Schrödinger Prime module was used to generate a homology model using the solved crystal structure (PDB ID 3ZYV) for mouse AOX3 (Coelho et al, 2012) as a template.…”

Inhibition of Xanthine Oxidase by the Aldehyde Oxidase Inhibitor Raloxifene: Implications for Identifying Molybdopterin Nitrite Reductases

“…Previous reports have indicated potent inhibition (K i = 1.0-51 nM, depending on the reducing substrate) properties of raloxifene for AO and thus this compound has been used to explore AO-mediated biochemistry including reduction [4,13,16]. However, there exists no detailed analysis regarding crossover inhibition of XO by raloxifene.…”

In Vitro Oxidative Metabolism of 6-Mercaptopurine in Human Liver: Insights into the Role of the Molybdoflavoenzymes Aldehyde Oxidase, Xanthine Oxidase, and Xanthine Dehydrogenase

“…One caveat to bear in mind is that all of these in vitro experiments were performed using AO/XO from nonhuman sources. Major species differences have already been reported for several compounds metabolized by AO and hence extrapolation of pharmacokinetic data from other mammalian species to humans is not advisable (Choughule et al, 2013;Dalvie et al, 2013). Moreover, mammalian xanthine oxidoreductase (XOR) exists in two interconvertible forms, xanthine oxidase (XO) and xanthine dehydrogenase (XDH).…”