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Development of A Continuous Fluorescence-Based Assay for N-Terminal Acetyltransferase D

N-terminal acetylation catalyzed by N-terminal acetyltransferases (NATs) has various biological functions in protein regulation. N-terminal acetyltransferase D (NatD) is one of the most specific NAT with only histone H4 and H2A proteins as the known substrates. Dysregulation of NatD has been implica...

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Autores principales: Ho, Yi-Hsun, Chen, Lan, Huang, Rong
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7827481/
https://www.ncbi.nlm.nih.gov/pubmed/33435607
http://dx.doi.org/10.3390/ijms22020594
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author Ho, Yi-Hsun
Chen, Lan
Huang, Rong
author_facet Ho, Yi-Hsun
Chen, Lan
Huang, Rong
author_sort Ho, Yi-Hsun
collection PubMed
description N-terminal acetylation catalyzed by N-terminal acetyltransferases (NATs) has various biological functions in protein regulation. N-terminal acetyltransferase D (NatD) is one of the most specific NAT with only histone H4 and H2A proteins as the known substrates. Dysregulation of NatD has been implicated in colorectal and lung cancer progression, implying its therapeutic potential in cancers. However, there is no reported inhibitor for NatD yet. To facilitate the discovery of small-molecule NatD inhibitors, we report the development of a fluorescence-based acetyltransferase assay in 384-well high-throughput screening (HTS) format through monitoring the formation of coenzyme A. The fluorescent signal is generated from the adduct in the reaction between coenzyme A and fluorescent probe ThioGlo4. The assay exhibited a Z′-factor of 0.77 and a coefficient of variation of 6%, indicating it is a robust assay for HTS. A pilot screen of 1280 pharmacologically active compounds and subsequent validation identified two hits, confirming the application of this fluorescence assay in HTS.
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spelling pubmed-78274812021-01-25 Development of A Continuous Fluorescence-Based Assay for N-Terminal Acetyltransferase D Ho, Yi-Hsun Chen, Lan Huang, Rong Int J Mol Sci Article N-terminal acetylation catalyzed by N-terminal acetyltransferases (NATs) has various biological functions in protein regulation. N-terminal acetyltransferase D (NatD) is one of the most specific NAT with only histone H4 and H2A proteins as the known substrates. Dysregulation of NatD has been implicated in colorectal and lung cancer progression, implying its therapeutic potential in cancers. However, there is no reported inhibitor for NatD yet. To facilitate the discovery of small-molecule NatD inhibitors, we report the development of a fluorescence-based acetyltransferase assay in 384-well high-throughput screening (HTS) format through monitoring the formation of coenzyme A. The fluorescent signal is generated from the adduct in the reaction between coenzyme A and fluorescent probe ThioGlo4. The assay exhibited a Z′-factor of 0.77 and a coefficient of variation of 6%, indicating it is a robust assay for HTS. A pilot screen of 1280 pharmacologically active compounds and subsequent validation identified two hits, confirming the application of this fluorescence assay in HTS. MDPI 2021-01-08 /pmc/articles/PMC7827481/ /pubmed/33435607 http://dx.doi.org/10.3390/ijms22020594 Text en © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Ho, Yi-Hsun
Chen, Lan
Huang, Rong
Development of A Continuous Fluorescence-Based Assay for N-Terminal Acetyltransferase D
title Development of A Continuous Fluorescence-Based Assay for N-Terminal Acetyltransferase D
title_full Development of A Continuous Fluorescence-Based Assay for N-Terminal Acetyltransferase D
title_fullStr Development of A Continuous Fluorescence-Based Assay for N-Terminal Acetyltransferase D
title_full_unstemmed Development of A Continuous Fluorescence-Based Assay for N-Terminal Acetyltransferase D
title_short Development of A Continuous Fluorescence-Based Assay for N-Terminal Acetyltransferase D
title_sort development of a continuous fluorescence-based assay for n-terminal acetyltransferase d
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7827481/
https://www.ncbi.nlm.nih.gov/pubmed/33435607
http://dx.doi.org/10.3390/ijms22020594
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