Characterization of the Raf kinase inhibitory protein (RKIP) binding pocket: NMR-based screening identifies small-molecule ligands.
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.
Shemon
Anne N
AN
Ben May Department for Cancer Research, University of Chicago, Chicago, Illinois, United States of America.
Heil
Gary L
GL
Granovsky
Alexey E
AE
Clark
Mathew M
MM
McElheny
Dan
D
Chimon
Alexander
A
Rosner
Marsha R
MR
Koide
Shohei
S
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
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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