A real-time PCR-based quantitative assay for 3-methylcytosine demethylase activity of ALKBH3

• Highly active recombinant ALKBH3 protein was produced from silkworms.
• A novel, high-throughput assay for ALKBH3 demethylase activity was developed.
• This assay enables us to screen for inhibitors of ALKBH3 demethylase.
• A prominent ALKBH3-targeted drug may be developed for the treatment of cancers.

Human AlkB homolog 3 (ALKBH3), a homolog of the Escherichia coli protein AlkB, demethylates 1-methyladenine and 3-methylcytosine (3-meC) in single-stranded DNA and RNA by oxidative demethylation. Immunohistochemical analyses on clinical cancer specimens and knockdown experiments using RNA interference in vitro and in vivo indicate that ALKBH3 is a promising molecular target for the treatment of prostate, pancreatic, and non-small cell lung cancer. Therefore, an inhibitor for ALKBH3 demethylase is expected to be a first-in-class molecular-targeted drug for cancer treatment. Here, we report the development of a novel, quantitative real-time PCR-based assay for ALKBH3 demethylase activity against 3-meC by highly active recombinant ALKBH3 protein using a silkworm expression system. This assay enables us to screen for inhibitors of ALKBH3 demethylase, which may result in the development of a novel molecular-targeted drug for cancer therapy.