Biophysical Approaches to Target Protein-Protein Interactions of Epigenetic Complexes by Small-Molecule Inhibitors

Abstract

Innumerable biological systems rely on Protein-Protein Interactions (PPIs) to govern basal physiological processes. Deregulation of PPIs is implicated in various human diseases, and PPIs have thus been frequently promoted as pharmacological targets. Yet, physiochemical and structural characteristics of binding interfaces, as well as modest affinities for binding partners, have yielded tremendous challenges in targeting PPIs with small-molecule inhibitors. Here we demonstrate biophysics-guided methodologies to reveal the mechanistic and structural bases of a tumorigenic PPI, and to identify and optimize small-molecule inhibitors of PPIs implicated in oncogeneses by Fragment-based lead discovery (FBLD) campaigns. We targeted GAS41 and BCL6, implicated as oncogenic drivers of non-small cell lung cancers and diffuse large B-cell lymphomas, respectively. First, we present biophysical approaches to characterize a bivalent mode of recognition of site-specific acetylated-Histone H3 by histone reader protein GAS41. We demonstrate that the bivalent mode of recognition confers an improvement in affinity by an order of magnitude, and further solve the crystal structure of complex to reveal the molecular details of Histone H3 acetyl-lysine binding GAS41 YEATS (Yaf9, ENL, Af9, Taf14, Sas5) domain. Subsequently, we report the development of small-molecule inhibitors of GAS41 histone reader function, and of BCL6-co-repressor interaction. We highlight a rational design strategy in which the molecular details of macromolecular recognition are incorporated into optimization of small-molecule inhibitors. In addition, we develop NMR-based methodology to quantify and rank binding of compounds in early-stage FBLD projects. We used protein-NMR-based fragment screens against GAS41 YEATS and BCL6 BTB domains to identify hits, and we applied protein-NMR, x-ray crystallography, biophysical studies, and biochemical assays to lead design of compounds targeting GAS41 and BCL6 with nanomolar and mid-micromolar inhibitory activities, respectively, in vitro. In total, our work presents an integrated, iterative strategy to characterize and block PPIs that may be deemed intractable to conventional small-molecule inhibitor development approaches.