Many promising drug candidates metabolized by aldehyde oxidase (AOX) fail during clinical trial owing to underestimation of their clearance. AOX is species-specific, which makes traditional allometric studies a poor choice for estimating human clearance. Other studies have suggested using half-life calculated by measuring substrate depletion to measure clearance. In this study, we proposed using numerical fitting to enzymatic pathways other than Michaelis-Menten (MM) to avoid missing the initial high turnover rate of product formation. Here, product formation over a 240-minute time course of six AOX substrates-, and zoniporide-have been provided to illustrate enzyme deactivation over time to help better understand why MM kinetics sometimes leads to underestimation of rate constants. Based on the data provided in this article, the total velocity for substrates becomes slower than the initial velocity by 3.1-, 6.5-, 2.9-, 32.2-, 2.7-, and 0.2-fold, respectively, in human expressed purified enzyme, whereas the K m remains constant. Also, our studies on the role of reactive oxygen species (ROS), such as superoxide and hydrogen peroxide, show that ROS did not significantly alter the change in enzyme activity over time. Providing a new electron acceptor, 5-nitroquinoline, did, however, alter the change in rate over time for mumerous compounds. The data also illustrate the difficulties in using substrate disappearance to estimate intrinsic clearance.

The metabolic stability of a drug is an important property that should be optimized during drug design and development. Nitrogen incorporation is hypothesized to increase the stability by coordination of nitrogen to the heme iron of cytochrome P450, a binding mode that is referred to as type II binding. However, we noticed that the type II binding compound 1 has less metabolic stability at subsaturating conditions than a closely related type I binding compound 3. Three kinetic models will be presented for type II binder metabolism; 1) Dead-end type II binding, 2) a rapid equilibrium between type I and II binding modes before reduction, and 3) a direct reduction of the type II coordinated heme. Data will be presented on reduction rates of iron, the off rates of substrate (using surface plasmon resonance) and the catalytic rate constants. These data argue against the dead-end, and rapid equilibrium models, leaving the direct reduction kinetic mechanism for metabolism of the type II binding compound 1.

One goal in drug design is to decrease clearance due to metabolism. It has been suggested that a compound's metabolic stability can be increased by incorporation of a sp 2 nitrogen into an aromatic ring. Nitrogen incorporation is hypothesized to increase metabolic stability by coordination of nitrogen to the heme iron (termed type II binding). However, questions regarding binding affinity, metabolic stability, and how metabolism of type II binders occurs remain unanswered. Herein, we use pyridinyl quinoline-4-carboxamide analogs to answer these questions. We show that type II binding can have a profound influence on binding affinity for CYP3A4, and the difference in binding affinity can be as high as 1,200 fold. We also find that type II binding compounds can be extensively metabolized, which is not consistent with the dead-end complex kinetic model assumed for type II binders. Two alternate kinetic mechanisms are presented to explain the results. The first involves a rapid equilibrium between the type II bound substrate and a metabolically oriented binding mode. The second involves direct reduction of the nitrogen-coordinated heme followed by oxygen binding.

ABSTRACT:Human aldehyde oxidase 1 (AOX1) has been subcloned into a vector suitable for expression in Escherichia coli, and the protein has been expressed. The resulting protein is active, with sulfur being incorporated in the molybdopterin cofactor. Expression levels are modest, but 1 liter of cells supplies enough protein for both biochemical and kinetic characterization. Partial purification is achieved by nickel affinity chromatography through the addition of six histidines to the amino-terminal end of the protein. Kinetic analysis, including kinetic isotope effects and comparison with xanthine oxidase, reveal similar mechanisms, with some subtle differences. This expression system will allow for the interrogation of human aldehyde oxidase structure/function relationships by site-directed mutagenesis and provide protein for characterizing the role of AOX1 in drug metabolism.Over the past decade there has been a growing awareness that metabolism can play a major role in drug development and toxicity (O'Brien and de Groot, 2005;Rettie and Jones, 2005). In particular, drug design has evolved in the direction of more metabolically stable molecules, often with the major metabolite being oxidation of an aromatic ring by cytochrome P450 enzymes. This leads to reasonable pharmacokinetics; however, the oxidation products can show toxicity. One method to slow aromatic oxidation is incorporation of a nitrogen to make a heteroaromatic ring (Dowers et al., 2004). The electronwithdrawing characteristics of nitrogen slow the electrophilic chemistry of the cytochrome P450 enzymes. The net result can lead to a change in the metabolic pathways to nucleophilic addition by the molybdenum iron-sulfur flavoproteins, xanthine oxidase (XO) and aldehyde oxidase (AO) (Obach and Walsky, 2005). Both XO and AO can oxidize aldehydes and nitrogen-containing aromatic compounds ( Fig. 1) (Beedham et al., 1990;Panoutsopoulos and Beedham, 2004). At present more than 40 drugs, nutritional supplements, and xenobiotics are metabolized to some extent by AO (Kitamura et al., 2006). Given that the changes in structural characteristics to decrease P450 metabolism have only been happening over the past 10 years we should see a significant increase in the importance of XO and AO in drug metabolism over the next decade as these drug design efforts result in drugs (Obach et al., 2004).XO has a reasonably well defined physiological role in the metabolism of purines (Garattini et al., 2008), whereas the physiological role of AO has only recently been defined in mammals. The role of these enzymes in plants has received more attention (Mendel, 2007). Knockdown experiments on AOX1 (the mouse ortholog of the only active human enzyme) in mice indicate that this enzyme plays a functional role in adipogenesis (Weigert et al., 2008), whereas knockout of aldehyde oxidase homolog 2 in mice causes a decrease in retinoid-dependent genes and alteration of the epidermis .One confounding feature in both drug metabolism and in understanding the physiological roles of AO e...

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