High-Throughput Screening to Identify Inhibitors of DEAD Box Helicase DDX41.

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 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.