title: Characterization of the Raf kinase inhibitory protein (RKIP) binding pocket: NMR-based screening identifies small-molecule ligands.
abstract: BACKGROUND
  Raf kinase inhibitory protein (RKIP), also known as phoshaptidylethanolamine binding protein (PEBP), has been shown to inhibit Raf and thereby negatively regulate growth factor signaling by the Raf/MAP kinase pathway. RKIP has also been shown to suppress metastasis. We have previously demonstrated that RKIP/Raf interaction is regulated by two mechanisms: phosphorylation of RKIP at Ser-153, and occupation of RKIP's conserved ligand binding domain with a phospholipid (2-dihexanoyl-sn-glycero-3-phosphoethanolamine; DHPE). In addition to phospholipids, other ligands have been reported to bind this domain; however their binding properties remain uncharacterized.
  
  
  RESULTS
  In this study, we used high-resolution heteronuclear NMR spectroscopy to screen a chemical library and assay a number of potential RKIP ligands for binding to the protein. Surprisingly, many compounds previously postulated as RKIP ligands showed no detectable binding in near-physiological solution conditions even at millimolar concentrations. In contrast, we found three novel ligands for RKIP that specifically bind to the RKIP pocket. Interestingly, unlike the phospholipid, DHPE, these newly identified ligands did not affect RKIP binding to Raf-1 or RKIP phosphorylation. One out of the three ligands displayed off target biological effects, impairing EGF-induced MAPK and metabolic activity.
  
  
  CONCLUSIONS
  This work defines the binding properties of RKIP ligands under near physiological conditions, establishing RKIP's affinity for hydrophobic ligands and the importance of bulky aliphatic chains for inhibiting its function. The common structural elements of these compounds defines a minimal requirement for RKIP binding and thus they can be used as lead compounds for future design of RKIP ligands with therapeutic potential.
authors.affiliation: Ben May Department for Cancer Research, University of Chicago, Chicago, Illinois, United States of America.
authors.forename: Anne N
authors.initials: AN
authors.lastname: Shemon
authors.forename: Gary L
authors.initials: GL
authors.lastname: Heil
authors.forename: Alexey E
authors.initials: AE
authors.lastname: Granovsky
authors.forename: Mathew M
authors.initials: MM
authors.lastname: Clark
authors.forename: Dan
authors.initials: D
authors.lastname: McElheny
authors.forename: Alexander
authors.initials: A
authors.lastname: Chimon
authors.forename: Marsha R
authors.initials: MR
authors.lastname: Rosner
authors.forename: Shohei
authors.initials: S
authors.lastname: Koide
mesh_terms: D000818:Animals; D001665:Binding Sites; D001681:Biological Assay; D002470:Cell Survival / Q000187:drug effects; D004790:Enzyme Induction / Q000187:drug effects; D004815:Epidermal Growth Factor / Q000494:pharmacology; D006367:HeLa Cells; D006801:Humans; D006863:Hydrogen-Ion Concentration / Q000187:drug effects; D008024:Ligands; D009682:Magnetic Resonance Spectroscopy; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D049488:Phosphatidylethanolamine Binding Protein / Q000737:chemistry / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D011485:Protein Binding / Q000187:drug effects; D051381:Rats; D054852:Small Molecule Libraries / Q000032:analysis* / Q000378:metabolism; D048490:raf Kinases / Q000378:metabolism
chemical_list: D008024:Ligands; C511482:PEBP1 protein, human; D049488:Phosphatidylethanolamine Binding Protein; D054852:Small Molecule Libraries; D004815:Epidermal Growth Factor; D048490:raf Kinases; D020928:Mitogen-Activated Protein Kinases
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