A Systematic Approach to the Discovery of Protein–Protein Interaction Stabilizers

Abstract

Dysregulation of protein–protein interactions (PPIs) commonly leads to disease. PPI stabilization has only recently been systematically explored for drug discovery despite being a powerful approach to selectively target intrinsically disordered proteins and hub proteins, like 14-3-3, with multiple interaction partners. Disulfide tethering is a site-directed fragment-based drug discovery (FBDD) methodology for identifying reversibly covalent small molecules. We explored the scope of disulfide tethering for the discovery of selective PPI stabilizers (molecular glues) using the hub protein 14-3-3σ. We screened complexes of 14-3-3 with 5 biologically and structurally diverse phosphopeptides derived from the 14-3-3 client proteins ERα, FOXO1, C-RAF, USP8, and SOS1. Stabilizing fragments were found for 4/5 client complexes. Structural elucidation of these complexes revealed the ability of some peptides to conformationally adapt to make productive interactions with the tethered fragments. We validated eight fragment stabilizers, six of which showed selectivity for one phosphopeptide client, and structurally characterized two nonselective hits and four fragments that selectively stabilized C-RAF or FOXO1. The most efficacious fragment increased 14-3-3σ/C-RAF phosphopeptide affinity by 430-fold. Disulfide tethering to the wildtype C38 in 14-3-3σ provided diverse structures for future optimization of 14-3-3/client stabilizers and highlighted a systematic method to discover molecular glues.

14-3-3ζ regulates adipogenesis by modulating chromatin accessibility during the early stages of adipocyte differentiation

“…14-3-3 proteins are not bona fide transcription factors as they do not contain DNA-binding domains (32). With the ability of 14-3-3 proteins to interact with metabolic transcription factors, such as FOXO1 and the CREB coactivators CRTCs (33, 34), it seemed plausible that 14-3-3ζ may influence the differentiation of preadipocytes by binding to and nucleating adipogenic transcriptional complexes. In fact, we previously showed that 14-3-3ζ interacts with C/EBP-β during adipogenesis(9).…”

Development of a NanoBRET assay for evaluation of 14-3-3σ molecular glues

“…Systematic technologies geared towards the identification of native PPI stabilizers [11][12][13] tend to be target-directed, compared to cell-based technologies for molecular glue degraders. [14][15][16] For instance, we have established biochemical fragment-based assays for the identification, validation, and optimization of PPI stabilizers.…”

“…Our workflow employs a mass spectrometry (MS)based site-directed disulfide tethering approach that has identified highly cooperative stabilizers for multiple 14-3-3/client complexes. 11,12,17 During chemical optimization, PPI stabilizers are evaluated using MS-and fluorescence anisotropy (FA)-based assays. These technologies measure stabilization of 14-3-3 and a peptide derived from the client protein, facilitating screening and x-ray crystallography.…”

Structure-Based Optimization of Covalent, Small-Molecule Stabilizers of the 14-3-3σ/ERα Protein–Protein Interaction from Nonselective Fragments

“…60,61 We recently expanded the disulfide-tethering study to develop 14-3-3σ/client glues for peptides with diverse shapes and binding modes. 62 The native Cys38 at the periphery of the peptide-binding groove on 14-3-3σ (Figure 1A) was targeted with a library consisting of ∼1600 disulfide fragments. Both client-selective and broadly stabilizing fragments were identified.…”