Extending the Detection Limit in Fragment Screening of Proteins Using Reverse Micelle Encapsulation

Detection of very weak (K_d > 10 mM) interactions of proteins with small molecules has been elusive. This is particularly important for fragment-based drug discovery, where it is suspected that the majority of potentially useful fragments will be invisible to current screening methodologies. We describe an NMR approach that permits detection of protein-fragment interactions in the very low affinity range and extends the current detection limit of ∼10 mM up to ∼200 mM and beyond. Reverse micelle encapsulation is leveraged to effectively reach very high fragment and protein concentrations, a principle that is validated by binding model fragments to E. coli dihydrofolate reductase. The method is illustrated by target-detected screening of a small polar fragment library against interleukin-1β, which lacks a known ligand-binding pocket. Evaluation of binding by titration and structural context allows for validation of observed hits using rigorous structural and statistical criteria. The 21 curated hit molecules represent a remarkable hit rate of nearly 10% of the library. Analysis shows that fragment binding involves residues comprising two-thirds of the protein's surface. Current fragment screening methods rely on detection of relatively tight binding to ligand binding pockets. The method presented here illustrates a potential to faithfully discover starting points for development of small molecules that bind to a desired region of the protein, even if the targeted region is defined by a relatively flat surface.