UCSF

14-3-3 regulates the activity of thousands of client proteins in various biological pathways through physical occlusion, structural conformations, and scaffolding protein-protein interactions (PPIs). Utilizing the role of 14-3-3 to regulate client proteins, the Arkin lab has developed molecule glues (MGs) that stabilize native 14-3-3/client interactions, harnessing the negative regulatory interactions to “turn off” client proteins’ functions in disease. By targeting PPIs as opposed to singular proteins, selectivity can be enhanced through selective MGs that bind the unique composite interfaces for a PPI of interest. This also allows for unstructured proteins/regions that were previously difficult to target to gain new induced binding pockets for modulators for the development of novel therapeutics. The work presented in this dissertation showcases the work on exploring MGs for various 14-3-3/client interactions, with focus on the 14-3-3/ERα and 14-3-3/CRAF complexes. These client proteins represent crucial targets in cancer (ERα and CRAF) and in developmental RASopathies (CRAF) that have distinct functions and binding modes to 14-3-3. Capitalizing on the differences allowed for the development of selective MGs and assays to characterize the effects of stabilizing these 14-3-3/client interactions. Work along each step of the drug discovery and development for 14-3-3/client MGs is presented in this dissertation: screening and validation of stabilizing fragments, optimization of fragment hits into selective MGs, and characterization of cell-active MGs. The lessons learned in each chapter provide insight into 14- 3-3 biology in the regulation of transcription factors (ERα) and kinases (CRAF) as well as serving as building blocks to expand stabilization to other native or neomorphic PPIs.