From Fleeting States to Therapeutics: New Perspectives in Protein-RNA Binding Dynamics and their Role in Drug Discovery

About This Grant

PROJECT SUMMARY The conventional approach to structural-knowledge driven drug discovery for protein targets often couples high-res- olution structural data (such as crystallography or electron microscopy) to identify potential binding sites with high- throughput computational docking simulations to optimize molecular design. While this approach, developed over decades, has been extremely productive for protein targets, it has limited applicability to unconventional targets like RNA and disordered proteins that are highly dynamic, sample rare and transient states that may contribute to bind- ing, and lack identifiable structure features that serve as high-aƯinity binding regions. These targets therefore lay outside the current druggable landscape. Progress extending this landscape requires the development of a compre- hensive understanding of the biophysics of currently undruggable biomolecules and new methodologies for identi- fying and characterizing unconventional targets. This proposal seeks to apply novel single-molecule spectroscopic methods, non-perturbative labeling, and computational modeling to help establish RNA stem loops, small RNA hair- pins that are critical regulatory molecules, as a viable target for peptide therapeutics. Using the trans-activation re- gion (TAR) RNA stem loop of the HIV1 virus as a model system, we seek to (Specific Aim 1) characterize the equilib- rium ensemble of the TAR molecule, (Specific Aim 2) resolve the binding mechanism between TAR and the native HIV1 polymerase as well as synthetic peptide mimics, and (Specific Aim 3) quantify state and nucleotide-resolved binding free energies to overcome the ensemble averaging limitation of conventional measurements of binding af- finities. Success of these aims is predicated on the unique information content of single-molecule spectroscopic data processed with multidimensional correlation function analyses, which has been shown to drastically expand the dynamic range of the spectroscopy (10-6-101s) and to be sensitive to ultra-rare conformations (<0.01% of an en- semble). Combining this new methodology with non-perturbative labeling provides a route to address the Specific Aims outlined above. Success in this project will provide a quantitative (thermodynamics and kinetics) mapping of protein-RNA interactions that can serve as the foundation for structure-knowledge based drug discovery. Further- more, comparing the binding mechanisms of HIV1 native polymerase protein with known cyclical peptides that are believed to mimic the binding will provide a productive road map for the further development and refinement of pep- tide therapeutics. In addition to what is learned about the specific system of study, the technical methodologies and scientific approach developed over the course of this project will be completely generalizable to any RNA stem loop system. We anticipate that the experimental technique will be extended to studying the binding of RNA to any pro- tein, peptide, or small molecule binding partner.