Venue : Centre Broca
Manon Darribère
Team : Dynamic organization and function of synapses
IINS
Supervisor: Matthieu Sainlos
Titre
Design of protein-protein interaction stabilizers by directed evolution: application to synaptic interactions
Abstract
Protein–protein interactions (PPIs) are fundamental to cellular organization and molecular communication across virtually all biological systems. While extensive research has focused on inhibiting PPIs, comparatively few strategies exist for their stabilization. Yet, PPI stabilization represents a powerful approach, particularly valuable for targeting transient interactions and selectively modulating those mediated by hub domains. Despite promising examples involving small molecules, often derived from natural products, no systematic methodology currently exists for generating PPI stabilizers.
To address this gap, we developed a strategy combining protein engineering and directed evolution to generate protein–protein interaction stabilizers. This approach consists of two phases: a first phase specifically aimed at identifying orthosteric candidates, followed by a second phase focused on improving their affinity. In the first phase, synthetic libraries derived from the tenth human fibronectin type III domain (10Fn3) are screened by phage display against an artificial fusion of the targeted PPI partners. Conducted under tailored experimental conditions, these selections allow isolation of orthosteric binders capable of recognizing the neo-surface formed by the interaction of the two partners. In the second phase, these candidates serve as templates for the creation of focused libraries via random mutagenesis. The resulting libraries are screened under adapted conditions to enrich variants with increased affinity and enhanced potential to stabilize the native complex. Selected candidates are subsequently characterized using biophysical, structural, and cellular fluorescence imaging methods to confirm their binding and stabilizing activity toward the target complex.
We applied this strategy to PDZ domain interactions, particularly those of the scaffold protein PSD-95, which play a central role in synaptic signaling and organization. Using our directed evolution approach, we generated multiple selective stabilizers targeting PPIs mediated by PDZ domains of PSD-95. The successful development of these stabilizers demonstrates the robustness and versatility of our approach, providing valuable tools for fundamental research and a potential foundation for the future development of therapies targeting specific PPIs.
Jury
Agathe Urvoas, Luc Brunsveld, Françoise Ochsenbein, Arnaud Gautier, Gilles Guichard.