Rapid scaffold-hopping for molecular glues: from fragments to cell-active probes targeting the 14-3-3/ERα complex

Molecular glues, small molecules that bind cooperatively at a protein-protein interface, have emerged as powerful modalities for the modulation of protein-protein interactions (PPIs) and “undruggable” targets. The systematic identification of new chemical matter with a molecular glue mechanism of action remains a significant challenge in drug discovery. Here, we present a scaffold hopping approach, using as a starting point our previously developed molecular glues for the native 14-3-3/estrogen receptor alpha (ERα) complex. The novel, computationally designed scaffold was based on the Groebke-Blackburn-Bienaymé multi-component reaction (MCR), leading to drug-like analogs with multiple points of variation, thus enabling the rapid derivatization and optimization of the scaffold. Structure-activity relationships (SAR) were developed using intact mass spectrometry and TR-FRET. Rational structure-guided optimization was facilitated by crystal structures of ternary complexes with the glues, 14-3-3 and phospho-peptides mimicking the highly disordered C-terminus of ERα. We measured the kinetics of 14-3-3/ERαpeptide binding by SPR, using a format in which a 14-3-3/molecular glue complex was immobilized on the SPR chip. The most potent compounds stabilized the complex by 100-fold and increased the residence time by 14-fold. Cellular stabilization of 14-3-3/ERα for the most potent analogs was confirmed using a NanoBRET assay with full-length proteins in live cells (EC50 = 2.7 – 5 µM). Our approach highlights the potential of MCR chemistry, combined with scaffold hopping, to drive the development and optimization of unprecedented molecular glue scaffolds.