Development of formaldehyde dehydrogenase-coupled assay and antibody-based assays for ALKBH5 activity evaluation.
J Pharm Biomed Anal
; 162: 9-15, 2019 Jan 05.
Article
in En
| MEDLINE
| ID: mdl-30219599
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.
Key words
Full text:
1
Collection:
01-internacional
Database:
MEDLINE
Main subject:
RNA, Messenger
/
Adenosine
/
Fluorescent Antibody Technique
/
Aldehyde Oxidoreductases
/
Drug Discovery
/
High-Throughput Screening Assays
/
AlkB Homolog 5, RNA Demethylase
/
Formaldehyde
Type of study:
Prognostic_studies
Limits:
Humans
Language:
En
Journal:
J Pharm Biomed Anal
Year:
2019
Type:
Article