ecoAO
- 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 ofAlthough 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.…
Author details: | Erickson M. ParagasORCiD, Sara C. Humphreys, Joshua Min, Carolyn A. Joswig-Jones, Silke LeimkühlerORCiDGND, Jeffrey P. Jones |
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DOI: | https://doi.org/10.1021/acsomega.7b01054 |
ISSN: | 2470-1343 |
Pubmed ID: | https://pubmed.ncbi.nlm.nih.gov/28884164 |
Title of parent work (English): | ACS OMEGA |
Subtitle (English): | a simple system for the study of human aldehyde oxidases role in drug metabolism |
Publisher: | American Chemical Society |
Place of publishing: | Washington |
Publication type: | Article |
Language: | English |
Date of first publication: | 2017/08/31 |
Publication year: | 2017 |
Release date: | 2022/04/11 |
Volume: | 2 |
Number of pages: | 8 |
First page: | 4820 |
Last Page: | 4827 |
Funding institution: | NIH [RR0631401 RR12948]; NSF [CHE-9115282, DBI-9604689]; Murdock Charitable Trust; [GM100874] |
Organizational units: | Mathematisch-Naturwissenschaftliche Fakultät / Institut für Biochemie und Biologie |
DDC classification: | 5 Naturwissenschaften und Mathematik / 57 Biowissenschaften; Biologie / 570 Biowissenschaften; Biologie |
Peer review: | Referiert |
Publishing method: | Open Access / Gold Open-Access |
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