# Modulation of the 14-3-3σ/C-RAF “Auto”inhibited Complex by Molecular Glues

## TL;DR

Scientists designed molecular glues that stabilize a protein complex involved in cancer and developmental disorders, offering new drug discovery opportunities.

## Contribution

A fragment-merging approach was used to design molecular glues that stabilize the 14-3-3/C-RAFpS259 complex with high selectivity and efficacy.

## Key findings

- Molecular glues stabilized the 14-3-3/C-RAFpS259 complex up to 300-fold.
- Compounds showed excellent selectivity among 80 14-3-3 clients and reduced RAF dimerization and ERK phosphorylation in cells.
- The glues enabled chemical biology studies of an intrinsically disordered C-RAF site previously untargeted.

## Abstract

Molecular glues, compounds that bind cooperatively at
protein–protein
interfaces (PPIs), are revolutionizing chemical biology and drug discovery,
allowing the modulation of traditional “undruggable”
targets. Here, we focus on a native regulatory PPI between the scaffolding
protein 14-3-3 and C-RAF, a key component of the MAPK signaling pathway.
Extensive drug discovery efforts have focused on the MAPK pathway
due to its central role in oncology and developmental disorders (RASopathies).
However, the modulation of its protein complexes is underexplored.
C-RAF activity is regulated on multiple levels including dimerization,
phosphorylation, and complex formation with 14-3-3, which prevents
C-RAF activation by binding to a C-RAF sequence centered on phospho-serine
259. We used a fragment-merging approach to design molecular glues
that bound to the composite surface of this 14-3-3/C-RAFpS259 complex.
Molecular glues stabilized the inhibitory complex up to 300-fold;
their glue-based mechanism of action was confirmed by crystallography
and biophysical studies. Selectivity among the other RAF isoforms
and other RAF phosphorylation sites was evaluated. The best compounds
showed excellent selectivity among a broad panel of 80 14-3-3 clients.
Cellular assays demonstrated on-target engagement, enhanced phosphorylation
levels of C-RAFpS259, and reduced levels of RAF dimerization and ERK
phosphorylation. Overall, this approach enabled chemical biology studies
for a C-RAF site that was intrinsically disordered prior to 14-3-3
binding and had not been targeted previously. These molecular glues
will be useful chemical probes and starting points for drug discovery
efforts to modulate native PPI stabilization in the MAPK pathway with
applications in oncology and RASopathies.