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Updated: Jun 29, 2025
Continuous Fluorescence-Based Endonuclease-Coupled DNA Methylation Assay to Screen for DNA Methyltransferase Inhibitors
Published on: August 5, 2022
A covalent compound selectively inhibits RNA demethylase ALKBH5 rather than FTO
Gan-Qiang Lai1,2, Yali Li1, Heping Zhu3
1State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences Shanghai 201203 China yangcg@simm.ac.cn.
Abstract:
N 6-Methyladenosine (m6A) is the most prevalent mRNA modification and is required for gene regulation in eukaryotes. ALKBH5, an m6A demethylase, is a promising target, particularly for anticancer drug discovery. However, the development of selective and potent inhibitors of ALKBH5 rather than FTO remains challenging. Herein, we used a targeted covalent inhibition strategy and identified a covalent inhibitor, TD19, which selectively inhibits ALKBH5 compared with FTO demethylase in protein-based and tumor cell-based assays. TD19 irreversibly modifies the residues C100 and C267, preventing ALKBH5 from binding to m6A-containing RNA. Moreover, TD19 displays good anticancer efficacy in acute myeloid leukemia and glioblastoma multiforme cell lines. Thus, the ALKBH5 inhibitor developed in this study, which selectively targets ALKBH5 compared with FTO, can potentially be used as a probe for investigating the biological functions of RNA demethylase and as a lead compound in anticancer research.
Insights
Researchers developed TD19, a selective inhibitor for ALKBH5 (an m6A demethylase), crucial for gene regulation. This compound shows promise as an anticancer agent and research tool, targeting cancer cells effectively.
Area of Science:
- Biochemistry
- Molecular Biology
- Oncology
Background:
- N6-Methyladenosine (m6A) is the most abundant mRNA modification regulating eukaryotic gene expression.
- ALKBH5, an m6A demethylase, is a potential anticancer drug target, but selective inhibitors are challenging to develop.
- Distinguishing between ALKBH5 and FTO (another m6A demethylase) is critical for targeted therapy.
Purpose of the Study:
- To develop a selective and potent inhibitor for ALKBH5.
- To investigate the potential of ALKBH5 inhibitors in anticancer drug discovery.
- To explore the utility of selective ALKBH5 inhibitors as biological probes.
Main Methods:
- Employed a targeted covalent inhibition strategy.
- Identified and characterized the covalent inhibitor TD19.
- Utilized protein-based and tumor cell-based assays to assess selectivity and efficacy.
Main Results:
- TD19 selectively inhibits ALKBH5 over FTO.
- TD19 irreversibly modifies ALKBH5 at C100 and C267, blocking m6A RNA binding.
- TD19 demonstrated anticancer efficacy in acute myeloid leukemia and glioblastoma cell lines.
Conclusions:
- A novel, selective ALKBH5 covalent inhibitor, TD19, has been developed.
- TD19 offers a potential therapeutic lead for anticancer treatments.
- TD19 can serve as a valuable tool for studying RNA demethylase functions.
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