ecoAO: A Simple System for the Study of Human Aldehyde Oxidases Role in Drug Metabolism.

Although aldehyde oxidase (AO) is an important hepatic drug-metabolizing enzyme, it remains understudied and is consequently often overlooked in preclinical studies, an oversight that has resulted in the failure of multiple clinical trials. AO's preclusion to investigation stems from the following: (1) difficulties synthesizing metabolic standards due to the chemospecificity and regiospecificity of the enzyme and (2) significant inherent variability across existing in vitro systems including liver cytosol, S9 fractions, and primary hepatocytes, which lack specificity and generate discordant expression and activity profiles. Here, we describe a practical bacterial biotransformation system, ecoAO, addressing both issues simultaneously. ecoAO is a cell paste of MoCo-producing Escherichia coli strain TP1017 expressing human AO. It exhibits specific activity toward known substrates, zoniporide, 4-trans-(N,N-dimethylamino)cinnamaldehyde, O6-benzylguanine, and zaleplon; it also has utility as a biocatalyst, yielding milligram quantities of synthetically challenging metabolite standards such as 2-oxo-zoniporide. Moreover, ecoAO enables routine determination of kcat and V/K, which are essential parameters for accurate in vivo clearance predictions. Furthermore, ecoAO has potential as a preclinical in vitro screening tool for AO activity, as demonstrated by its metabolism of 3-aminoquinoline, a previously uncharacterized substrate. ecoAO promises to provide easy access to metabolites with the potential to improve pharmacokinetic clearance predictions and guide drug development.

The two faces of aldehyde oxidase: Oxidative and reductive transformations of 5-nitroquinoline.

Aldehyde oxidase (AOX) is a cytosolic enzyme responsible for the metabolism of some drugs and drug candidates. AOX catalyzes the oxidative hydroxylation of substrates including several aliphatic and aromatic aldehydes, and nitrogen-containing heterocyclic compounds. AOX is also reported to catalyze the reductive metabolism of nitro-compounds, N-oxides, sulfoxides, isoxazoles, isothiazoles, nitrite and hydroxamic acids. These reductive transformations are not well understood and are generally believed to only occur at low oxygen concentrations. In this study, we used 5-nitroquinoline (5NQ) as a substrate to further understand both the oxidative and the reductive transformations catalyzed by AOX. In vitro reaction of 5NQ with AOX under aerobic conditions generated the oxidized (2-oxo-5-nitroquinoline, 2-oxo-5NQ), the reduced (5-aminoquinoline, 5AQ) and the oxidized/reduced (2-oxo-5-aminoquinoline, 2-oxo-5AQ) metabolites. Interestingly, in human liver cytosol, co-incubation of 5NQ and known AOX oxidative substrates DACA and phthalazine significantly increased the yield of the reduced metabolite, while oxidized metabolites production decreased. These data indicate that 5NQ can be reduced at atmospheric oxygen concentrations and that the reductive transformation occurs at a second site that is kinetically distinct from the oxidative site.