# Macrocyclic Molecular Glues for the 14‐3‐3/ChREBP Interaction: Affinity and Cooperativity in an Inverse Relationship

## TL;DR

This study shows how macrocyclic molecular glues can stabilize a key protein interaction, with a surprising inverse relationship between binding strength and cooperativity.

## Contribution

The study reveals a novel inverse relationship between macrocycle binding affinity and cooperativity in stabilizing a protein–protein interaction.

## Key findings

- Macrocycles with optimized linker lengths reduced entropic costs while maintaining key contacts with 14-3-3.
- An inverse relationship was found between macrocycle binding affinity to 14-3-3 and cooperativity in PPI stabilization.
- Ternary co-crystal structures provided molecular insights into the affinity and cooperativity interplay.

## Abstract

Molecular glues (MGs) stabilize protein–protein interactions (PPIs) by simultaneously binding two or more proteins at their composite interface. Macrocycles present attractive properties as MGs, including large contact surfaces to address the often flat and undefined composite PPI interfaces, but their structure‐based design has remained intangible. We have designed peptidomimetic macrocycles capable of enhancing the PPI between 14‐3‐3 and the carbohydrate response element binding protein (ChREBP), a regulatory transcription factor. Biophysical characterization of these MGs revealed the importance of optimized linker length, displaying a reduced entropic cost compared to the linear counterparts, while preserving key contacts with 14‐3‐3. Binding assays demonstrated that the macrocycles selectively and cooperatively stabilized the 14‐3‐3/ChREBP complex, with an intriguing inverse relationship between intrinsic binding affinity to 14‐3‐3 and cooperativity in PPI stabilization. Ternary co‐crystal structures of the macrocycles binding at the composite 14‐3‐3/ChREBP interface provided a molecular rationale for the affinity and cooperativity differences. Overall, this study highlights structural, kinetic, and thermodynamic features that guide effective macrocyclic MG design and brings forward the crucial interplay of affinity and cooperativity in stabilizing PPIs.

Macrocycles are effective molecular glues to stabilize the 14‐3‐3/ChREBP interaction. Linker length modulation uncovered an inverse correlation between the macrocycle binding affinity to 14‐3‐3 and its cooperativity in 14‐3‐3/ChREBP stabilization. Co‐crystal structures of these macrocycles in the composite 14‐3‐3/ChREBP binding pocket revealed structural clues behind this affinity and cooperativity interplay.