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)-functionalized magnetic beads to recover an intact protein-protein complex through affinity capture of a polyhistidine-tagged primary target ("protein-complex fishing"). Protein-complex fishing, utilizing His(6)-tagged calmodulin (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, methylbenzethonium and pempidine tartrate. The ability to produce quantitative inhibition data is also shown through the development of dose-dependent response curves and the determination of inhibition constants (K(I)) for the novel compound methylbenzethonium (K(I) = 14-49 nM) and two known antagonists, calmidazolium (K(I) = 1.7-7.5 nM) and trifluoperazine (K(I) = 1.2-3.0 μM), with the latter two values being in close agreement with literature values.
Chemical Biology Graduate Program, Department of Chemistry and Chemical Biology, McMaster University, 1280 Main Street West, Hamilton, Ontario L8S 4M1, Canada.
Meghan J
MJ
McFadden
Murray S
MS
Junop
John D
JD
Brennan
D001558:Benzethonium / Q000031:analogs & derivatives / Q000737:chemistry; D002147:Calmodulin / Q000037:antagonists & inhibitors* / Q000378:metabolism; D006639:Histidine / Q000737:chemistry; D007700:Kinetics; D008280:Magnetics; D008555:Melitten / Q000037:antagonists & inhibitors* / Q000378:metabolism; D009532:Nickel / Q000737:chemistry; D009842:Oligopeptides / Q000737:chemistry; D010393:Pempidine / Q000737:chemistry; D011485:Protein Binding; D025941:Protein Interaction Mapping / Q000379:methods*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D014268:Trifluoperazine / Q000737:chemistry
D002147:Calmodulin; C471213:His-His-His-His-His-His; D009842:Oligopeptides; D001558:Benzethonium; D008555:Melitten; D014268:Trifluoperazine; D006639:Histidine; D009532:Nickel; D010393:Pempidine; C005106:methylbenzethonium chloride