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• W2335584316 abstract "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." @default.
• W2335584316 created "2016-06-24" @default.
• W2335584316 creator A5030506530 @default.
• W2335584316 creator A5050686879 @default.
• W2335584316 creator A5059569301 @default.
• W2335584316 date "2010-11-10" @default.
• W2335584316 modified "2023-09-27" @default.
• W2335584316 title "Magnetic “Fishing” Assay To Screen Small-Molecule Mixtures for Modulators of Protein−Protein Interactions" @default.
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• W2335584316 doi "https://doi.org/10.1021/ac102164d" @default.
• W2335584316 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/21067198" @default.
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