Modulation of the 14-3-3σ/C-RAF "auto"inhibited complex by molecular glues.

Area of Science:

• Chemical Biology
• Drug Discovery
• Molecular Biology

Background:

• The MAPK signaling pathway, particularly C-RAF, is crucial in oncology and RASopathies, yet direct kinase inhibition has limitations.
• Modulating protein-protein interactions (PPIs), like that between C-RAF and 14-3-3, presents an underexplored therapeutic avenue.
• 14-3-3 binding to phospho-serine 259 of C-RAF regulates its activation, making this PPI a key regulatory point.

Purpose of the Study:

• To design and validate molecular glues that stabilize the native C-RAF/14-3-3 protein-protein interaction.
• To explore the potential of targeting intrinsically disordered regions via PPI stabilization.
• To provide chemical probes for studying the MAPK pathway and developing new therapeutics.

Main Methods:

• Fragment-merging approach to design molecular glue scaffolds targeting the 14-3-3/C-RAF complex.
• Biophysical assays to quantify complex stabilization and assess selectivity among RAF isoforms and 14-3-3 clients.
• Crystal structures of ternary complexes to confirm the glue-based mechanism of action.
• Cell-based assays to validate on-target engagement and downstream signaling effects.

Main Results:

• Synthesized molecular glues stabilized the 14-3-3/C-RAF complex up to 300-fold.
• Confirmed glue-based mechanism via crystal structures and demonstrated high selectivity for the target PPI.
• Cell assays showed on-target engagement, increased C-RAF pS259 phosphorylation, reduced RAF dimerization, and decreased ERK phosphorylation.

Conclusions:

• Molecular glues effectively stabilize the C-RAF/14-3-3 interaction, offering a novel strategy beyond direct kinase inhibition.
• This approach enables targeting previously inaccessible protein interfaces and provides valuable chemical probes.
• The developed molecular glues serve as starting points for drug discovery in oncology and RASopathies.