Interspecies differences in the metabolism of methotrexate: An insight into the active site differences between human and rabbit aldehyde oxidase

Kanika V Choughule; Carolyn A Joswig-Jones; Jeffrey P Jones

doi:10.1016/j.bcp.2015.05.010

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Interspecies differences in the metabolism of methotrexate: An insight into the active site differences between human and rabbit aldehyde oxidase

Journal article

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Interspecies differences in the metabolism of methotrexate: An insight into the active site differences between human and rabbit aldehyde oxidase

Kanika V Choughule, Carolyn A Joswig-Jones and Jeffrey P Jones

Biochemical pharmacology, Vol.96(3), pp.288-295

08/01/2015

DOI: https://doi.org/10.1016/j.bcp.2015.05.010

Handle:

https://hdl.handle.net/2376/109182

PMCID: PMC4574636

PMID: 26032640

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https://doi.org/10.1016/j.bcp.2015.05.010View

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Abstract

Liver - enzymology

Species Specificity

Humans

Molecular Sequence Data

Substrate Specificity

Macaca mulatta

Aldehyde Oxidase - chemistry

Antimetabolites, Antineoplastic - metabolism

Methotrexate - chemistry

Amino Acid Sequence

Rabbits

Catalytic Domain

Guinea Pigs

Methotrexate - metabolism

Mutagenesis, Site-Directed

Oxidation-Reduction

Rats

Antimetabolites, Antineoplastic - chemistry

Rats, Sprague-Dawley

Liver - chemistry

Sequence Homology, Amino Acid

Sequence Alignment

Animals

Aldehyde Oxidase - metabolism

Molecular Docking Simulation

Structural Homology, Protein

Kinetics

Several drug compounds have failed in clinical trials due to extensive biotransformation by aldehyde oxidase (AOX) (EC 1.2.3.1). One of the main reasons is the difficulty in scaling clearance for drugs metabolised by AOX, from preclinical species to human. Using methotrexate as a probe substrate, we evaluated AOX metabolism in liver cytosol from human and commonly used laboratory species namely guinea pig, monkey, rat and rabbit. We found that the metabolism of methotrexate in rabbit liver cytosol was several orders of magnitude higher than any of the other species tested. The results of protein quantitation revealed that the amount of AOX1 in human liver was similar to rabbit liver. To understand if the observed differences in activity were due to structural differences, we modelled rabbit AOX1 using the previously generated human AOX1 homology model. Molecular docking of methotrexate into the active site of the enzyme led to the identification of important residues that could potentially be involved in substrate binding and account for the observed differences. In order to study the impact of these residue changes on enzyme activity, we used site directed mutagenesis to construct mutant AOX1 cDNAs by substituting nucleotides of human AOX1 with relevant ones of rabbit AOX1. AOX1 mutant proteins were expressed in Escherichia coli. Differences in the kinetic properties of these mutants have been presented in this study.

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Title: Interspecies differences in the metabolism of methotrexate: An insight into the active site differences between human and rabbit aldehyde oxidase
Creators: Kanika V Choughule - Department of Chemistry, Washington State University, P.O. Box 644630, Pullman, WA 99164-4630, USA
Carolyn A Joswig-Jones - Department of Chemistry, Washington State University, P.O. Box 644630, Pullman, WA 99164-4630, USA
Jeffrey P Jones - Department of Chemistry, Washington State University, P.O. Box 644630, Pullman, WA 99164-4630, USA. Electronic address: jpj@wsu.edu
Publication Details: Biochemical pharmacology, Vol.96(3), pp.288-295
Academic Unit: Chemistry, Department of
Publisher: England
Grant note: R01 GM100874 / NIGMS NIH HHS GM100874 / NIGMS NIH HHS
Identifiers: 99900547016301842
Language: English
Resource Type: Journal article