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High-Throughput Screening to Identify Inhibitors of DEAD Box Helicase DDX41
Mariko Yoneyama-Hirozane1, Mitsuyo Kondo1, Shin-Ichi Matsumoto1
1Pharmaceutical Research Division, Takeda Pharmaceutical Company Limited, Fujisawa, Kanagawa, Japan.
Insights
Researchers identified small-molecule inhibitors targeting the DDX41 protein, which is implicated in myeloid neoplasms. These compounds selectively inhibit the R525H mutant form, offering potential therapeutic avenues for related leukemias.
Area of Science:
- Biochemistry
- Molecular Biology
- Oncology
Background:
- DDX41 (Asp-Glu-Ala-Asp) box protein is a DEXDc helicase family member with ATPase and nucleic acid translocase activities.
- Somatic and germline mutations in DDX41 are linked to myeloid neoplasms, including myelodysplastic syndrome and acute myeloid leukemia.
- The R525H mutation is specifically associated with hereditary myelodysplastic syndrome and acute myeloid leukemia.
Purpose of the Study:
- To characterize the biochemical properties of wild-type human DDX41 and its R525H mutant.
- To identify small-molecule inhibitors targeting the R525H mutant DDX41 protein.
Main Methods:
- Recombinant expression and purification of human DDX41 and its R525H mutant in E. coli.
- Biochemical assays to assess ATP hydrolysis dependent on double-stranded DNA (dsDNA).
- High-throughput screening using a dsDNA-dependent ATPase assay with R525H mutant proteins, followed by hit confirmation and counterscreening.
Main Results:
- Both wild-type and R525H DDX41 proteins exhibited ATP-dependent ATPase activity stimulated by dsDNA.
- High-throughput screening successfully identified several small-molecule compounds that inhibit DDX41.
- The identified inhibitors demonstrated selective inhibitory activity against DDX41.
Conclusions:
- The R525H mutation in DDX41 is biochemically active and can be targeted by small molecules.
- These DDX41-selective inhibitors represent potential therapeutic agents for myeloid neoplasms associated with DDX41 mutations.
Abstract:
The human DEAD (Asp-Glu-Ala-Asp) box protein DDX41, a member of the DEXDc helicase family, has nucleic acid-dependent ATPase and RNA and DNA translocase and unwinding activities. DDX41 is affected by somatic mutations in sporadic cases of myeloid neoplasms as well as in a biallelic fashion in 50% of patients with germline DDX41 mutations. The R525H mutation in DDX41 is thought to play important roles in the development of hereditary myelodysplastic syndrome and acute myelocytic leukemia. In this study, human DDX41 and its R525H mutant (R525H) were expressed in Escherichia coli and purified. The ATPase activities of the recombinant DDX41 and R525H proteins were dependent on both ATP and double-stranded DNA (dsDNA), such as poly(dG-dC) and poly(dA-dT). High-throughput screening was performed with a dsDNA-dependent ATPase assay using the human R525H proteins. After hit confirmation and counterscreening, several small-molecule inhibitors were successfully identified. These compounds show DDX41-selective inhibitory activities.
