Pharmacological inhibition of DEAD-Box RNA Helicase 3 attenuates stress granule assembly

Abstract

Stress granules (SGs) are non-membranous cytosolic protein-RNA aggregates that process mRNAs through stalled translation initiation in response to cellular stressors and in disease. DEAD-Box RNA helicase 3 (DDX3) is an active target of drug development for the treatment of viral infections, cancers, and neurodegenerative diseases. DDX3 plays a critical role in RNA metabolism, including SGs, but the role of DDX3 enzymatic activity in SG dynamics is not well understood. Here, we address this question by determining the effects of DDX3 inhibition on the dynamics of SG assembly and disassembly. We use two small molecule inhibitors of DDX3, RK33 and 16D, with distinct inhibitory mechanisms that target DDX3's ATPase activity and RNA helicase site, respectively. We find that both DDX3 inhibitors reduce the assembly of SGs, with a more pronounced reduction from RK-33. In contrast, both compounds only marginally affect the disassembly of SGs. RNA-mediated knockdown of DDX3 caused a similar reduction in SG assembly and minimal effect on SG disassembly. Collectively, these results reveal that the enzymatic activity of DDX3 is required for the assembly of SGs and pharmacological inhibition of DDX3 could be relevant for the treatment of SG-dependent pathologies.

Keywords: DDX3; DDX3X; DEAD-Box RNA helicase; RK-33; Stress granule.