Development of formaldehyde dehydrogenase-coupled assay and antibody-based assays for ALKBH5 activity evaluation

Dan-Dan Shen; Feng-Zhi Suo; Qi-Meng Song; Jiao Chang; Ting Zhang; Jing-Jing Hong; Yi-Chao Zheng; Hong-Min Liu

doi:10.1016/j.jpba.2018.09.018

Development of formaldehyde dehydrogenase-coupled assay and antibody-based assays for ALKBH5 activity evaluation

J Pharm Biomed Anal. 2019 Jan 5:162:9-15. doi: 10.1016/j.jpba.2018.09.018. Epub 2018 Sep 7.

Authors

Dan-Dan Shen¹, Feng-Zhi Suo¹, Qi-Meng Song¹, Jiao Chang¹, Ting Zhang¹, Jing-Jing Hong¹, Yi-Chao Zheng², Hong-Min Liu³

Affiliations

¹ Key Laboratory of Advanced Pharmaceutical Technology, Ministry of Education of China, Co-innovation Center of Henan Province for New Drug R & D and Preclinical Safety, School of Pharmaceutical Science, Institute of Drug Discovery and Development, Zhengzhou University, 100 Kexue Avenue, Zhengzhou, Henan 450001, China.
² Key Laboratory of Advanced Pharmaceutical Technology, Ministry of Education of China, Co-innovation Center of Henan Province for New Drug R & D and Preclinical Safety, School of Pharmaceutical Science, Institute of Drug Discovery and Development, Zhengzhou University, 100 Kexue Avenue, Zhengzhou, Henan 450001, China; National Center for International Research of Micro-nano Molding Technology of Henan Province, Zhengzhou University, 100 Kexue Avenue, Zhengzho, Henan 450001, China. Electronic address: yichaozheng@zzu.edu.cn.
³ Key Laboratory of Advanced Pharmaceutical Technology, Ministry of Education of China, Co-innovation Center of Henan Province for New Drug R & D and Preclinical Safety, School of Pharmaceutical Science, Institute of Drug Discovery and Development, Zhengzhou University, 100 Kexue Avenue, Zhengzhou, Henan 450001, China. Electronic address: liuhm@zzu.edu.cn.

PMID: 30219599
DOI: 10.1016/j.jpba.2018.09.018

Abstract

N6-methyladenosine (m⁶A) is the most prevalent internal modification of eukaryotic messenger RNA (mRNA). Until now, two RNA demethylases have been identified, including FTO (fat mass and obesity-associated protein) and ALKBH5 (α-ketoglutarate-dependent dioxygenase alkB homologue 5). As a mammalian m⁶A demethylase, ALKBH5 significantly affects mRNA export and RNA metabolism as well as the assembly of mRNA processing factors in nuclear speckles, and ALKBH5 may play a significant role in these biological processes. Nevertheless, no modulator of ALKBH5 has been reported. The reason for that may be the lack of in vitro assays for ALKBH5 inhibitor screening. Herein, we describe the development of two homogeneous assays for ALKBH5 using N6-methyladenosine as substrate with different principles. Using ALKBH5 recombinant, we developed a formaldehyde dehydrogenase coupled fluorescence based assay and an antibody based assay for the activity evaluation of ALKBH5. These robust coupled assays are suitable for screening ALKBH5 inhibitors in 384-well format (Z' factors of 0.74), facilitating the discovery of modulators in the quest for the regulation of biological processes.

Keywords: ALKBH5; AlphaScreen; Formaldehyde dehydrogenase coupled assay; m(6)A demethylase.

MeSH terms

Adenosine / analogs & derivatives*
Adenosine / metabolism
Aldehyde Oxidoreductases / antagonists & inhibitors
Aldehyde Oxidoreductases / metabolism*
AlkB Homolog 5, RNA Demethylase / antagonists & inhibitors
AlkB Homolog 5, RNA Demethylase / metabolism*
Demethylation
Drug Discovery / methods*
Enzyme Inhibitors / pharmacology
Fluorescent Antibody Technique*
Formaldehyde / metabolism*
High-Throughput Screening Assays / methods*
Humans
Kinetics
RNA, Messenger / metabolism*
Reproducibility of Results
Spectrometry, Fluorescence

Substances

Enzyme Inhibitors
RNA, Messenger
Formaldehyde
N-methyladenosine
ALKBH5 protein, human
AlkB Homolog 5, RNA Demethylase
Aldehyde Oxidoreductases
formaldehyde dehydrogenase, glutathione-independent
Adenosine