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

Abstract

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.

Time Course of Aldehyde Oxidase and Why It Is Nonlinear

“…This can be explained if we consider the effect of substrate inhibition. It appears that O6BG does not show any substrate inhibition (Barr et al, 2015), whereas the reported K I value for DACA was 91 mM (Paragas et al, 2017b). We hypothesize that the second substrate molecule would bind to the reduction site and therefore make the reduction happen even more slowly, the same effect we saw by using a reductive substrate 5NQ, and that is why the k 5 value for DACA is lower than that of O6BG.…”

“…N-(2-dimethylamino)ethyl)acridine-4-carboxamide (DACA) was synthesized as described previously (Paragas et al, 2017b). O 6 -benzylguanine (O6BG) and zaleplon were purchased from Toronto Research Chemicals (North York, ON), zoniporide and dantrolene from Cayman Chemical Company (Ann Arbor, MI), BIBX1382 from Tocris Bioscience (Bristol, UK), and internal standards (phenacetin and 2-methly-4(3H)-quinazolinone), phthalazine, protocatechuic acid, protocatechuate 3,4dioxygenase (Pseudomonas spp.…”

“…), glucose, catalase (bovine liver), and SOD (bovine erythrocytes) were purchased from Sigma-Aldrich (St. Louis, MO). The standard metabolites were synthesized and purified as previously described (Paragas et al, 2017b) except 1-phthalazinone, which was purchased from Sigma-Aldrich. 5-Nitroquinoline was purchased from TCI America (Portland, OR).…”

“…1410012), was purchased from XenoTech (Lenexa, KS). Expression and purification of HAO were performed according to the method previously described (Alfaro et al, 2009;Paragas et al, 2017b). The total amount of AOX in HLC and HAO was quantified by liquid chromatography-tandem mass spectrometry (LC-MS/MS) .…”

“…2). These models were tested on two of our substrates, which were reported to exhibit substrate inhibition, DACA and phthalazine (Obach et al, 2004;Paragas et al, 2017b). The fits to these models were either almost identical to the MAM or could not fit the data points well (Supplemental Fig.…”