Characterization of the Raf Kinase Inhibitory Protein (RKIP) Binding Pocket: NMR-Based Screening Identifies Small-Molecule Ligands Anne N. Shemon1., Gary L. Heil3., Alexey E. Granovsky1, Mathew M. Clark1,2, Dan McElheny3, Alexander Chimon 1 , Marsha R. Rosner1,2*", Shohei Koide3" 1 Ben May Department for Cancer Research, University of Chicago, Chicago, Illinois, United States of America, 2 Department of Neurobiology, Pharmacology and Physiology, University of Chicago, Chicago, Illinois, United States of America, 3 Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, Illinois, United States of America 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. Methods/Findings: 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/Significance: 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. Citation: Shemon AN, Heil GL, Granovsky AE, Clark MM, McElheny D, et al. (2010) Characterization of the Raf Kinase Inhibitory Protein (RKIP) Binding Pocket: NMR- Based Screening Identifies Small-Molecule Ligands. PLoS ONE 5(5): e10479. doi:10.1371/journal.pone.0010479 Editor: Paul Cobine, Auburn University, United States of America Received February 4, 2010; Accepted March 27, 2010; Published May 5, 2010 Copyright: 2010 Shemon et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by National Institutes of Health (NIH) grants GM087630 and CA112310 (to M.R.R.) and the Medical Research Council Australia (NHMRC) CJ Martin Fellowship (to A.N.S.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: m-rosner@uchicago.edu . These authors contributed equally to this work. " These authors also contributed equally to this work. Introduction The Raf-1 kinase inhibitory protein (RKIP) serves as a regulator of the MAP kinase signal transduction cascade through its interaction and inhibition of Raf-1 kinase activity [1,2]. Phos- phorylation of RKIP at Serine-153 by protein kinase C abolishes RKIP’s inhibition of Raf-1 [3] and converts it to an inhibitor of the G-protein coupled receptor kinase, GRK-2, thus facilitating cross-talk between the EGF and GPCR signaling pathways [4]. Furthermore, RKIP functions as a metastatic tumor suppressor in prostate [5] and breast cancers [6], sensitizes human prostate and breast cancer cells to drug induced apoptosis and regulates the integrity of the cell cycle via the spindle checkpoint [7,8] (reviewed in [9]). These reports highlight the significance of RKIP in mammalian cells as a vital regulator of mitosis, cell differentiation and apoptosis. Long before its function as a kinase inhibitor was identified, RKIP was isolated from the soluble fraction of bovine brain extracts through affinity chromatography to an immobilized dye (Cibacron Blue) [10]. This dye is generally considered a nucleotide analog, and thus the affinity of RKIP to the dye suggested a nucleotide-binding function for RKIP. In addition, gel filtration and lipid affinity chromatography were used to demonstrate binding of a number of organic anions, steroids and also the lipid membrane component, phosphatidylethanolamine [10,11]. Con- sequently, RKIP was originally named PEBP for its function as a PLoS ONE | www.plosone.org 1 May 2010 | Volume 5 | Issue 5 | e10479