Fragment-Based Discovery of Potent RNA-Competitive Inhibitors of the DEAH-Box RNA Helicase DHX8.

Human DHX8 is a spliceosomal DEAH-box RNA helicase involved in releasing mRNA from the spliceosome and crucial in ensuring splicing fidelity. DHX8 was identified as a promising therapeutic oncology target due to its role in regulating stress-adaptive gene expression, including HSF1-dependent transcription, while having broader transcriptional effects in cells under oncogenic stress. We report the discovery of novel RNA-competitive DHX8 inhibitors based on a 2-(phenethylthio)nicotinic acid scaffold, which were optimized using a structure-guided design approach, following a biophysical fragment screen. This yielded compound 53 with nanomolar biochemical potency, good in vitro PK, and activity in a cellular target engagement assay. Optimizing inhibitor binding between Arg647 and the nonconserved His693, coupled with extending into a pocket in the DHX8 Winged-Helix domain, was crucial for potency improvement. By binding in the Winged-Helix domain, these inhibitors restrict the helicase domain's conformational plasticity, stabilizing a closed, inactive conformation while sterically blocking ssRNA translocation.