Development of A Continuous Fluorescence-Based Assay for N-Terminal Acetyltransferase D
1
Department of Medicinal Chemistry and Molecular Pharmacology, Institute for Drug Discovery, Center for Cancer Research, Purdue University, West Lafayette, IN 47907, USA
2
Chemical Genomics Facility, Institute for Drug Discovery, Purdue University, West Lafayette, IN 47907, USA
*
Author to whom correspondence should be addressed.
Int. J. Mol. Sci. 2021, 22(2), 594; https://doi.org/10.3390/ijms22020594
Received: 24 December 2020 / Revised: 5 January 2021 / Accepted: 5 January 2021 / Published: 8 January 2021
(This article belongs to the Special Issue Modifications of Protein Termini)
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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Keywords:
high-throughput screening; N-terminal acetyltransferase D; acetyltransferase inhibitors; fluorescence assay
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MDPI and ACS Style
Ho, Y.-H.; Chen, L.; Huang, R. Development of A Continuous Fluorescence-Based Assay for N-Terminal Acetyltransferase D. Int. J. Mol. Sci. 2021, 22, 594. https://doi.org/10.3390/ijms22020594
AMA Style
Ho Y-H, Chen L, Huang R. Development of A Continuous Fluorescence-Based Assay for N-Terminal Acetyltransferase D. International Journal of Molecular Sciences. 2021; 22(2):594. https://doi.org/10.3390/ijms22020594
Chicago/Turabian StyleHo, Yi-Hsun; Chen, Lan; Huang, Rong. 2021. "Development of A Continuous Fluorescence-Based Assay for N-Terminal Acetyltransferase D" Int. J. Mol. Sci. 22, no. 2: 594. https://doi.org/10.3390/ijms22020594
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