The systematic stabilization of protein-protein interactions (PPI) has great potential as innovative drug discovery strategy to target novel and hard-to-drug protein classes. The current lack of chemical starting points and focused screening opportunities limits the identification of small molecule stabilizers that engage two proteins simultaneously. Starting from our previously described virtual screening strategy to identify inhibitors of 14-3-3 proteins, we report a conceptual molecular docking approach providing concrete entries for discovery and rational optimization of stabilizers for the interaction of 14-3-3 with the carbohydrate-response element-binding protein (ChREBP). X-ray crystallography reveals a distinct difference in the binding modes between weak and general inhibitors of 14-3-3 complexes and a specific, potent stabilizer of the 14-3-3/ChREBP complex. Structure-guided stabilizer optimization results in selective, up to 26-fold enhancement of the 14-3-3/ChREBP interaction. This study demonstrates the potential of rational design approaches for the development of selective PPI stabilizers starting from weak, promiscuous PPI inhibitors.
Protein-protein interactions (PPIs) are responsible for the proper function of biological processes and, when dysregulated, commonly lead 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 screening small molecules in a quantitative, high-throughput manner. We explore the scope of the disulfide tethering technology for the discovery of selective fragments as starting points for the development of potent small molecule PPI stabilizers and molecular glues using the hub protein 14-3-3s;. The complexes with 5 biologically and structurally diverse phospho-peptides, derived from the 14-3-3 client proteins ERa;, FOXO1, C-RAF, USP8, and SOS1, were screened for hit identification. Stabilizing fragments could be found for 4/5 client complexes with a diversified hit-rate and stabilizing efficacy for the different 14-3-3/client phospho-peptides. Extensive structural elucidation revealed the ability and adaptivity of the peptide to make productive interactions with the tethered fragments as key criterion for cooperative complex formation. We validated eight fragment stabilizers, six of which showed selectivity for one phospho-peptide client, and structurally characterized two nonselective hits and four fragments that selectively stabilized C-RAF or FOXO1. The most efficacious of these fragments increased 14-3-3s;/C-RAF phospho-peptide affinity by 430-fold. Disulfide tethering to the wildtype C38 in 14-3-3s; provided diverse structures for future optimization of 14-3-3/client stabilizers and highlighted a systematic method to discover molecular glues.
Molecular glues represent an evolution in drug discovery, however, targeted stabilization of protein complexes remains challenging, owing to a paucity of drug design rules. The functional mapping of hotspots has...
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.
Carbohydrate response element binding protein (ChREBP) is a glucose-responsive transcription factor with two splice isoforms (α and β). ChREBPα remains cytoplasmic by binding to 14-3-3. Increased glucose metabolism allows ChREBPα to enter the nucleus and induce ChREBPβ, a hyperactive isoform that is a key mediator of glucolipotoxicity. Using structure-guided stabilizer optimization, we have developed novel drugs that stabilize the protein-protein interaction between ChREBPα and 14-3-3, with a goal to prevent ChREBPα nuclear translocation, induction of ChREBPβ, and glucolipotoxicity. One hit compound, K391, has an EC50 of 0.3±0.1μM by fluorescent polarization assay and a Kd of 0.12μM by isotitration calorimetry. Using dual label analysis of the kinetics of cell growth and cell death, we found that K391 significantly reduced β-cell death by 31.8±3% and enabled proliferation in glucolipotoxic conditions in INS-1 cells (20mM glucose and 1mM palmitate). In response to glucose, 76.4±3.5 % of the cells stained for nuclear ChREBP. In the presence of K391, 39.75±5.3 % fewer cells displayed nuclear ChREBP in high glucose concentrations and 65.7±1.6% less in glucolipotoxic conditions, indicating that K391 retains ChREBPα in the cytoplasm and prevents transcription of ChREBPβ. Time course with an antibody against ChREBPα showed that ChREBPα transiently translocated to the nucleus at 30 min and then cleared out of the nucleus in high glucose, an effect blocked by K391 (N=3). In glucolipotoxic conditions, ChREBPα remained nuclear over time, an effect significantly attenuated by K391 (N=3). Txnip is an important mediator of glucose toxicity and a target gene of ChREBPβ. Importantly, K391 blocked the glucose response of the human Txnip promoter in a luciferase assay by 2±0.2 folds. We conclude that the K391 retains ChREBPα in the cytoplasm in high glucose and in glucolipotoxic conditions by stabilizing the ChREBPα and 14-3-3 interaction and thereby rescues β-cells from glucolipotoxicity. Disclosure L.S.Katz: None. I.L.Tse: None. E.Visser: None. S.Baumel-alterzon: None. M.Kaiser: None. L.M.Brunsveld: Consultant; Novartis, Ambagon Therapeutics, Research Support; Roche Diagnostics. M.Pennings: None. C.Ottmann: None. D.Scott: None. Funding National Institutes of Health (R01DK130300)
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