Eukaryotic arylamine N-acetyltransferase investigation of substrate specificity by high-throughput screening

Akane Kawamura; James Graham; Adeel Mushtaq; Stefanos A. Tsiftsoglou; Gregory M. Vath; Patrick E. Hanna; Carston R. Wagner; Edith Sim

doi:10.1016/j.bcp.2004.09.014

Eukaryotic arylamine N-acetyltransferase investigation of substrate specificity by high-throughput screening

Akane Kawamura, James Graham, Adeel Mushtaq, Stefanos A. Tsiftsoglou, Gregory M. Vath, Patrick E. Hanna, Carston R. Wagner, Edith Sim

Medicinal Chemistry

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92 Scopus citations

Abstract

Arylamine N-acetyltransferases (NAT; EC 2.3.1.5) catalyse the transfer of acetyl groups from acetylCoA to xenobiotics, including drugs and carcinogens. The enzyme is found extensively in both eukaryotes and prokaryotes, yet the endogenous roles of NATs are still unclear. In order to study the properties of eukaryotic NATs, high-throughput substrate and inhibitor screens have been developed using pure soluble recombinant Syrian hamster NAT2 (shNAT2) protein. The assay can be used with a wide range of compounds and was used to determine substrate specificity of shNAT2. We describe the expression and characterisation of shNAT2 and also purified recombinant human NAT1 and NAT2, including the use of the assay to explore the substrate specificities of each of the enzymes. Hamster NAT2 has similar substrate specificity to human NAT1, acetylating para-aminobenzoate but not arylhydrazine and hydralazine compounds. The overlapping but distinct substrate-specific activity profiles of human NAT1 and NAT2 were clearly observed from the screen. Naturally occurring compounds were tested as substrates or inhibitors of shNAT2 and succinylCoA was found to be a potent inhibitor of shNAT2.

Original language	English (US)
Pages (from-to)	347-359
Number of pages	13
Journal	Biochemical Pharmacology
Volume	69
Issue number	2
DOIs	https://doi.org/10.1016/j.bcp.2004.09.014
State	Published - Jan 15 2005

Bibliographical note

Funding Information:
We thank Haiqing Wang (University of Minnesota, USA), Dr. Edward Brooke and Isaac Westwood (University of Oxford, UK) for helpful discussions and advice. We are also grateful to the Wellcome Trust for financial support and the Oxford University Kobe foundation for a scholarship (A.K.). Syrian hamster liver samples were kindly donated by Olivia Hibbitt (University of Oxford, UK).

Copyright:
Copyright 2017 Elsevier B.V., All rights reserved.

Keywords

AcetylCoA/CoA derivatives
Arylamine N-acetyltransferase
Arylamines
High-throughput screening
Recombinant protein
Xenobiotic metabolism

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title = "Eukaryotic arylamine N-acetyltransferase investigation of substrate specificity by high-throughput screening",

abstract = "Arylamine N-acetyltransferases (NAT; EC 2.3.1.5) catalyse the transfer of acetyl groups from acetylCoA to xenobiotics, including drugs and carcinogens. The enzyme is found extensively in both eukaryotes and prokaryotes, yet the endogenous roles of NATs are still unclear. In order to study the properties of eukaryotic NATs, high-throughput substrate and inhibitor screens have been developed using pure soluble recombinant Syrian hamster NAT2 (shNAT2) protein. The assay can be used with a wide range of compounds and was used to determine substrate specificity of shNAT2. We describe the expression and characterisation of shNAT2 and also purified recombinant human NAT1 and NAT2, including the use of the assay to explore the substrate specificities of each of the enzymes. Hamster NAT2 has similar substrate specificity to human NAT1, acetylating para-aminobenzoate but not arylhydrazine and hydralazine compounds. The overlapping but distinct substrate-specific activity profiles of human NAT1 and NAT2 were clearly observed from the screen. Naturally occurring compounds were tested as substrates or inhibitors of shNAT2 and succinylCoA was found to be a potent inhibitor of shNAT2.",

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note = "Funding Information: We thank Haiqing Wang (University of Minnesota, USA), Dr. Edward Brooke and Isaac Westwood (University of Oxford, UK) for helpful discussions and advice. We are also grateful to the Wellcome Trust for financial support and the Oxford University Kobe foundation for a scholarship (A.K.). Syrian hamster liver samples were kindly donated by Olivia Hibbitt (University of Oxford, UK). Copyright: Copyright 2017 Elsevier B.V., All rights reserved.",

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AU - Vath, Gregory M.

AU - Hanna, Patrick E.

AU - Wagner, Carston R.

AU - Sim, Edith

N1 - Funding Information: We thank Haiqing Wang (University of Minnesota, USA), Dr. Edward Brooke and Isaac Westwood (University of Oxford, UK) for helpful discussions and advice. We are also grateful to the Wellcome Trust for financial support and the Oxford University Kobe foundation for a scholarship (A.K.). Syrian hamster liver samples were kindly donated by Olivia Hibbitt (University of Oxford, UK). Copyright: Copyright 2017 Elsevier B.V., All rights reserved.

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N2 - Arylamine N-acetyltransferases (NAT; EC 2.3.1.5) catalyse the transfer of acetyl groups from acetylCoA to xenobiotics, including drugs and carcinogens. The enzyme is found extensively in both eukaryotes and prokaryotes, yet the endogenous roles of NATs are still unclear. In order to study the properties of eukaryotic NATs, high-throughput substrate and inhibitor screens have been developed using pure soluble recombinant Syrian hamster NAT2 (shNAT2) protein. The assay can be used with a wide range of compounds and was used to determine substrate specificity of shNAT2. We describe the expression and characterisation of shNAT2 and also purified recombinant human NAT1 and NAT2, including the use of the assay to explore the substrate specificities of each of the enzymes. Hamster NAT2 has similar substrate specificity to human NAT1, acetylating para-aminobenzoate but not arylhydrazine and hydralazine compounds. The overlapping but distinct substrate-specific activity profiles of human NAT1 and NAT2 were clearly observed from the screen. Naturally occurring compounds were tested as substrates or inhibitors of shNAT2 and succinylCoA was found to be a potent inhibitor of shNAT2.

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Eukaryotic arylamine N-acetyltransferase investigation of substrate specificity by high-throughput screening

Abstract

Bibliographical note

Keywords

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