Fragment-Based Discovery of Potent RNA-Competitive Inhibitors of the DEAH-Box RNA Helicase DHX8
Benjamin J. Read, Caroline Ewens, Federica Gigante, Jemima Thomas, Catarina Felisberto-Rodrigues, Silvia Alvarez Peres, Catherine Tighe, Edgar de las Heras Ruiz, Kai Schiemann, Andrew G. Malcolm, Peter Craig McAndrew, Mark Stubbs, Harshnira Patani, Helena dos Santos Costa, Katharine Stoodley, Lisa Pickard, Michael Busch, Emma Gunnell, Sara Silva, Alexandra Knopp, Stephen T. Hallett, Martin Augustin, Alfred Lammens, Michael Carter, Mirco Meniconi, Marco Ballarotto, Jon Ainsley, Paul Meister, Debarati Sethi, Rosemary Burke, Andrea Scarpino, Yann-Vaï Le Bihan, Ulrich Grädler, Julian Blagg, Paul Workman, Paul A. Clarke, Andreas Blum, Christina Esdar, Gurdip Bhalay, Rob L. M. van MontfortAbstract
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.