Systematic stabilization of specific protein-protein interactions

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Abstract Protein-protein interactions (PPIs) are ubiquitous in biology, and their dysregulation is closely associated with diseases, from cancer to neurodegeneration to rare genetic disorders. PPIs often form complex networks that include highly interacting ‘hub’ proteins. Methods to modulate single interactions would provide great insight into the functions of these hubs. Small-molecule probes and drug leads have focused on blocking PPIs; however, stabilizing PPIs could be just as important for drug discovery and could provide greater selectivity for chemical biology. However, there are few systematic methodologies to discover PPI stabilizers prospectively. This proposal focuses on the systematic discovery of selective small-molecule PPI stabilizers, using the hub protein 14-3-3 as a model system. 14-3-3s are seven highly homologous adaptor proteins that bind to serine and threonine sites on client proteins to alter their function and fate. Hundreds of proteins in signal transduction pathways, cell-cycle regulation, transcription regulation, and protein homeostasis are clients of 14-3-3. Given the importance of protein phosphorylation and the ubiquity of 14-3-3 as an effector of phosphorylation, it is surprisingly underappreciated. We propose that developing a tool kit of selective, cell-active stabilizers of native 14-3-3/client PPIs will stimulate biological study and may lead to new drugs. Based on the structural diversity of clients, we hypothesize that we can develop client-selective PPI stabilizers that bind to the composite 14-3- 3/client interface. These selective stabilizers should amplify the native biology of the 14-3-3/client complex. We will provide proof-of-concept for this approach through three aims: Aim 1. Screen for selective stabilizers of 14-3-3/phosphopeptide clients. We have previously discovered disulfide-bound fragments that stabilize 14-3- 3/phosphopeptide complexes. We will now screen six, structurally and biologically diverse 14-3-3/client complexes, using a native C38 residue found only on the 14-3-3 isoform. We hypothesize that client sequences with more open or flexible structures near the C38 will yield higher quality hits. Aim 2. Optimize 14-3-3/client stabilizers for cell-based activity. We have demonstrated the ability to convert disulfides to cell-active electrophilic warheads and to tune the selectivity of 14-3-3/client stabilizers. We will optimize C38-bound fragments with (or without) an electrophile with the goal of achieving target-selective PPI stabilization in cells for the 14-3-3 clients CRAF kinase, estrogen receptor  (ER), and the transcription factor FOXO1. Aim 3. Design PROTAC-based degraders of 14-3-3 clients. PROTACs are bifunctional molecules that induce proximity between a ubiquitin ligase and a target, leading to the target’s degradation. We will expand the targets accessible to PROTAC technology by using 14-3-3 as a scaffolding protein to link intrinsically disordered proteins (IDPs) to the ubiquitin ligase. We will first develop the technology for ER, where PROTACs are known, then translate our learning to IDPs, including FOXO1. Successful completion of these aims will provide approaches that are broadly applicable to the 14-3-3 network and are extendable to other native and nonnative PPIs.