Magnetic "Fishing" Assay To Screen Small-Molecule Mixtures for Modulators of Protein-Protein Interactions

Protein protein interactions are an intricate part of biological pathways and have become important targets for drug discovery Here we present a two stage magnetic bead assay to functionally screen small molecule mixtures for modulators of protein based interactions, with simultaneous affinity based isolation of active compounds and identification by mass spectrometry Proteins of interest interact in solution prior to the addition of Ni(II) functionahzed magnetic beads to recover an intactprotein-protein complex through affinity capture of a polyhistidine-tagged primary target ("protein-complex fishing") Protein complex fishing, utilizing His(6)-tagged calmodulm (CaM) as the primary (bait) protein and melittin (Mel) as the target, was used to screen a mass-encoded library of 1000 bioactive compounds (50 mixtures, 20 compounds each) and successfully identified three known antagonists, three naturally occurring phenolic compounds previously reported to disrupt CaM activated phosphodiesterase activity, and two newly identified modulators of the CaM Mel interaction, methylbenzethomum and pempidine tartrate The ability to produce quantitative Inhibition data 13 also shown through the development of dose-dependent response curves and the determination of inhibition constants (X) for the novel compound methylbenzethonium (K-1 = 14-49 nM) and two known antagonists, calmidazolium (K-1 = 1 7-7 5 nM) and trifluoperazine (K-1 = 1 2-3 0 mu M), with the latter two values being in close agreement with literature values