Optimization of a fluorescence polarization assay for developing novel covalent inhibitors of BCL6-BTB
Introduction
BCL6 is an oncoprotein and master transcription factor that represses DNA damage and repair pathways to allow cell differentiation. When dysregulated, BCL6 continues to suppress these pathways after differentiation, allowing DNA damage to accumulate. This damage can result in development of diffuse large B-cell lymphoma (DLBCL), the most common non-Hodgkin’s lymphoma in the United States. A functional BTB domain of BCL6 is essential for the survival of DLBCL cells, making BCL6 an attractive drug target. In this work, we describe optimization of a fluorescence polarization assay to develop novel covalent BTB inhibitors.
Methods
The binding site in BCL6-BTB is a shallow protein-protein interaction region (PPI), optimized for binding native peptide corepressors. PPIs are often difficult to drug, so we screened a library of novel covalent fragments and successfully identified a set of 2-carboxy-4-halopyridines that effectively inhibit binding of the peptide corepressor. To probe the covalent inhibition mechanism, we constructed several site-directed BTB mutants.To better facilitate optimization of inhibitors, we varied several parameters and experimental conditions of a fluorescent polarization assay to more rapidly detect and evaluate reversible and irreversible BTB inhibitors.
Results
Covalent fragments containing a 2-carboxy-4-halopyridine scaffold are identified as fragment-sized inhibitors of corepressor peptide binding to the BCL6-BTB domain. This type of inhibitor typically requires a second “activating” residue to catalyze covalent inhibition, and we successfully mutated and purified BCL6-BTB domains with changes to the targeted Cys 53 residue as well as surrounding residues that are candidates as the “activating” residue. Finally, to speed these studies, we optimized a fluorescent polarization assay for inhibitor characterization.
Conclusion
Difficult-to-drug PPIs in the BCL6-BTB domain are targetable by covalent halopyridine fragments that modify Cys 53. Residues near the binding site can be mutated and produce soluble proteins suitable for functional characterization. A fluorescence polarization can be optimized to speed inhibitor development. These findings support the use of covalent drug development for developing new DLBCL therapeutics.