Chemical approaches for targeting ribonucleoprotein assemblies

PROJECT SUMMARY RNA-protein interactions play a fundamental role in biology through complex processes regulating gene expression programs. Perturbation of native RNA-protein assemblies (RNP) alters gene expression and can result in diseases, from infection to cancer to neurodegeneration. This proposal focuses on the discovery of chemical modulators of RNA-protein assemblies through the systematic targeting of DEAD-box RNA-dependent ATPases. DEAD-box ATPases are the driving force of many RNP remodeling processes, including the functioning of RNA polymerase II, the spliceosome, the ribosome, and biomolecular RNA condensates. Unsurprisingly, mutations in DEAD-box genes are implicated in cancer, neurodegenerative diseases, and rare genetic diseases. Despite their biological importance, our understanding of the remodeling activities and functions remains rudimentary for most of the 37 DEAD-box proteins in the human genome. We propose that developing selective chemical modulators of DEAD- box proteins will propel biological studies of known and yet-to-be-discovered RNP and may lead to new drugs. As DEAD-box ATPases allosterically regulate RNA affinity through distal communication with the catalytic site, we hypothesize that we can generate selective inhibitors exploiting these allosteric mechanisms. We will provide proof- of-concept for this approach through three projects: 1. Identify allosteric compounds that act as glues of DEAD-box proteins in complex with RNA by screening the large St. Jude compound collection. 2. Optimize stabilizers of RNA- helicase interaction for cell-based activity. We will combine ligand discovery with genetic-based approaches to generate a chemical probe for studying DDX19 function in Ewing sarcoma. 3. Systematic discovery of targetable allosteric circuits in DEAD-box ATPases. We have identified ligands that bind DDX19 together with the nucleotide cofactor. We will extend this approach to five structurally similar, but biologically and mechanistically distinct, DEAD- box ATPases. Using protein NMR and structural analyses, we will identify chemically targetable allosteric circuits and analyze their functional roles across the DEAD-box protein family. This multidisciplinary program will combine structure-based design (synthetic chemistry and X-ray crystallography), protein dynamics (NMR), computational methods, biochemistry, and cell biology to determine how chemical modulators of RNA-protein assemblies can be generated. Successful completion of these projects will break new ground and could yield first-in-class chemical probes for fundamental biological processes, providing approaches that are broadly applicable to nucleic acid- dependent ATPases and extendable to other protein-RNA interactions.