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Characterization of the Raf kinase inhibitory protein (RKIP) binding pocket: NMR-based screening identifies small-molecule ligands.

Shemon AN, Heil GL, Granovsky AE, Clark MM, McElheny D, Chimon A, Rosner MR, Koide S - PLoS ONE (2010)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Ben May Department for Cancer Research, 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.

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A portion of RKIP HSQC spectra illustrating the difference in the DHPA-induced chemical shift perturbations of residue 182 at pH 7.4 and 6.0.(A) DHPA titration at pH 7.4 causes the peak to continuously shift, characteristic of fast exchange kinetics. (B) Titration at pH 6.0 causes the peak intensity to decrease with a concomitant appearance of a new peak, characteristic of slow exchange kinetics. The arrows indicate the trajectories of cross peak movements. Panel (A) shows data with DHPA concentrations of 0, 0.05 0.25, 0.5, 1.0, 2.5 and 7.5 mM, and (B) data with 0, 0.05, 0.25, 0.5, 1.0, 2.5 and 4.5 mM DHPA.
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pone-0010479-g002: A portion of RKIP HSQC spectra illustrating the difference in the DHPA-induced chemical shift perturbations of residue 182 at pH 7.4 and 6.0.(A) DHPA titration at pH 7.4 causes the peak to continuously shift, characteristic of fast exchange kinetics. (B) Titration at pH 6.0 causes the peak intensity to decrease with a concomitant appearance of a new peak, characteristic of slow exchange kinetics. The arrows indicate the trajectories of cross peak movements. Panel (A) shows data with DHPA concentrations of 0, 0.05 0.25, 0.5, 1.0, 2.5 and 7.5 mM, and (B) data with 0, 0.05, 0.25, 0.5, 1.0, 2.5 and 4.5 mM DHPA.

Mentions: Two of the four compounds exhibiting binding at pH 7.4, DHPG and DHPS, demonstrated similar patterns of chemical shift perturbations at pH 6.0 as previously described [21]. Titration of the phospholipids at pH 6.0 confirmed interactions with similar affinity for DHPG and DHPS to that observed at pH 7.4 (Table S1). In contrast, while PA binding at pH 6.0 perturbed a similar subset of residues as it did at pH 7.4, the trajectory of the peak shifts was different. As PA was titrated in, the HSQC peaks did not change their positions but their intensities decreased, and a new set of peaks appeared (Figure 2A). This behavior is characteristic of slow interconversion between the free and ligand-bound states [26]. Furthermore, the affinity of PA for RKIP was at least 3-fold higher at pH 6.0 than at pH 7.4 (Figure 2B). The mono-substituted phosphate moiety of PA has a pKa of 8.0 [29] indicating that its protonation state is sensitive to changes in pH between pH 7.4 and pH 6.0. In comparison, the pKa values of the other phospholipids examined in this study are expected to be much lower, as the pKa of the phosphate group of dimyristoyl-phosphatidylethanolamine has been calculated to be ∼1.7 [29]. The remarkable similarities between the peak shift patterns observed for DHPE, DHPG and DHPS at pH 6.0 and pH 7.4 are in stark contrast with the differences seen for PA binding at the two pH values, suggesting that the higher affinity for PA at pH 6.0 results from the protonation of the ligand and not from a pH-dependent effect on the RKIP binding pocket itself. Together, these results suggest that the compounds that showed no significant binding in our NMR assays (Table S1) have very weak affinity at best to RKIP in near physiological pH range.


Characterization of the Raf kinase inhibitory protein (RKIP) binding pocket: NMR-based screening identifies small-molecule ligands.

Shemon AN, Heil GL, Granovsky AE, Clark MM, McElheny D, Chimon A, Rosner MR, Koide S - PLoS ONE (2010)

A portion of RKIP HSQC spectra illustrating the difference in the DHPA-induced chemical shift perturbations of residue 182 at pH 7.4 and 6.0.(A) DHPA titration at pH 7.4 causes the peak to continuously shift, characteristic of fast exchange kinetics. (B) Titration at pH 6.0 causes the peak intensity to decrease with a concomitant appearance of a new peak, characteristic of slow exchange kinetics. The arrows indicate the trajectories of cross peak movements. Panel (A) shows data with DHPA concentrations of 0, 0.05 0.25, 0.5, 1.0, 2.5 and 7.5 mM, and (B) data with 0, 0.05, 0.25, 0.5, 1.0, 2.5 and 4.5 mM DHPA.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2864760&req=5

pone-0010479-g002: A portion of RKIP HSQC spectra illustrating the difference in the DHPA-induced chemical shift perturbations of residue 182 at pH 7.4 and 6.0.(A) DHPA titration at pH 7.4 causes the peak to continuously shift, characteristic of fast exchange kinetics. (B) Titration at pH 6.0 causes the peak intensity to decrease with a concomitant appearance of a new peak, characteristic of slow exchange kinetics. The arrows indicate the trajectories of cross peak movements. Panel (A) shows data with DHPA concentrations of 0, 0.05 0.25, 0.5, 1.0, 2.5 and 7.5 mM, and (B) data with 0, 0.05, 0.25, 0.5, 1.0, 2.5 and 4.5 mM DHPA.
Mentions: Two of the four compounds exhibiting binding at pH 7.4, DHPG and DHPS, demonstrated similar patterns of chemical shift perturbations at pH 6.0 as previously described [21]. Titration of the phospholipids at pH 6.0 confirmed interactions with similar affinity for DHPG and DHPS to that observed at pH 7.4 (Table S1). In contrast, while PA binding at pH 6.0 perturbed a similar subset of residues as it did at pH 7.4, the trajectory of the peak shifts was different. As PA was titrated in, the HSQC peaks did not change their positions but their intensities decreased, and a new set of peaks appeared (Figure 2A). This behavior is characteristic of slow interconversion between the free and ligand-bound states [26]. Furthermore, the affinity of PA for RKIP was at least 3-fold higher at pH 6.0 than at pH 7.4 (Figure 2B). The mono-substituted phosphate moiety of PA has a pKa of 8.0 [29] indicating that its protonation state is sensitive to changes in pH between pH 7.4 and pH 6.0. In comparison, the pKa values of the other phospholipids examined in this study are expected to be much lower, as the pKa of the phosphate group of dimyristoyl-phosphatidylethanolamine has been calculated to be ∼1.7 [29]. The remarkable similarities between the peak shift patterns observed for DHPE, DHPG and DHPS at pH 6.0 and pH 7.4 are in stark contrast with the differences seen for PA binding at the two pH values, suggesting that the higher affinity for PA at pH 6.0 results from the protonation of the ligand and not from a pH-dependent effect on the RKIP binding pocket itself. Together, these results suggest that the compounds that showed no significant binding in our NMR assays (Table S1) have very weak affinity at best to RKIP in near physiological pH range.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Ben May Department for Cancer Research, 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.

Show MeSH
Related in: MedlinePlus