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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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Compound 48 reduces EGF-induced MAPK activity.HeLa cells expressing wild-type or depleted RKIP were serum starved overnight and then pre-treated for 30 min with 20 µM compounds (as indicated). EGF (10 ng/ml) was added in the final 5 minutes of incubation. (A) Whole cell lysates (30 µg) were separated on SDS-PAGE gels (12.5%), transferred to nitrocellulose and analysed by immunoblotting with anti-phospho ERK (Top panel: representative Western blot) and anti-total ERK antibodies (Bottom panel: representative Western blot). (B) ERK phosphorylation was assessed by normalizing phospho-ERK levels to total ERK levels as depicted in bar graphs (Mean ± SEM; n = 3 independent experiments). *p<0.05 indicates the significance of change relative to the DMSO control in the presence of EGF.
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pone-0010479-g005: Compound 48 reduces EGF-induced MAPK activity.HeLa cells expressing wild-type or depleted RKIP were serum starved overnight and then pre-treated for 30 min with 20 µM compounds (as indicated). EGF (10 ng/ml) was added in the final 5 minutes of incubation. (A) Whole cell lysates (30 µg) were separated on SDS-PAGE gels (12.5%), transferred to nitrocellulose and analysed by immunoblotting with anti-phospho ERK (Top panel: representative Western blot) and anti-total ERK antibodies (Bottom panel: representative Western blot). (B) ERK phosphorylation was assessed by normalizing phospho-ERK levels to total ERK levels as depicted in bar graphs (Mean ± SEM; n = 3 independent experiments). *p<0.05 indicates the significance of change relative to the DMSO control in the presence of EGF.

Mentions: We have previously shown that EGF-induced MAPK activity is augmented in HeLa cells depleted of RKIP [1]. If RKIP binding to Raf-1 requires pocket binding, then compounds that bind the RKIP pocket would interfere with downstream signaling to MAPK. However, we showed that locostatin binds to the RKIP pocket with no effect on Raf-1 association nor RKIP phosphorylation by PKC and did not change the EGF-induced MAPK activity in HeLa cells in the presence or absence of RKIP [27]. Similarly, two of the compounds (98 and 26) from our screen mimicked the lack of effect by locostatin on EGF-induced MAPK activity (Figure 5). In addition, we observed compound 48 at 20 µM had no effect on wild-type HeLa cells; however compound 48 reduced MAPK activity by 34±11% (mean ± SEM, n = 3) in RKIP depleted cells (Figure 5). To further examine the effect of compound 48, we compared the dose response of compound 26 as a control to that of compound 48 (Figure 6 and 7). As predicted, compound 26 at all concentrations tested had no effect on EGF-induced MAPK activity in HeLa cells in the presence or absence of RKIP (Figure 6). In contrast, the inhibitory effect was dose-dependent for compound 48 with respect to RKIP depleted cells but dose-independent in wild-type RKIP cells (compare Figure7A with 7B).


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)

Compound 48 reduces EGF-induced MAPK activity.HeLa cells expressing wild-type or depleted RKIP were serum starved overnight and then pre-treated for 30 min with 20 µM compounds (as indicated). EGF (10 ng/ml) was added in the final 5 minutes of incubation. (A) Whole cell lysates (30 µg) were separated on SDS-PAGE gels (12.5%), transferred to nitrocellulose and analysed by immunoblotting with anti-phospho ERK (Top panel: representative Western blot) and anti-total ERK antibodies (Bottom panel: representative Western blot). (B) ERK phosphorylation was assessed by normalizing phospho-ERK levels to total ERK levels as depicted in bar graphs (Mean ± SEM; n = 3 independent experiments). *p<0.05 indicates the significance of change relative to the DMSO control in the presence of EGF.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2864760&req=5

pone-0010479-g005: Compound 48 reduces EGF-induced MAPK activity.HeLa cells expressing wild-type or depleted RKIP were serum starved overnight and then pre-treated for 30 min with 20 µM compounds (as indicated). EGF (10 ng/ml) was added in the final 5 minutes of incubation. (A) Whole cell lysates (30 µg) were separated on SDS-PAGE gels (12.5%), transferred to nitrocellulose and analysed by immunoblotting with anti-phospho ERK (Top panel: representative Western blot) and anti-total ERK antibodies (Bottom panel: representative Western blot). (B) ERK phosphorylation was assessed by normalizing phospho-ERK levels to total ERK levels as depicted in bar graphs (Mean ± SEM; n = 3 independent experiments). *p<0.05 indicates the significance of change relative to the DMSO control in the presence of EGF.
Mentions: We have previously shown that EGF-induced MAPK activity is augmented in HeLa cells depleted of RKIP [1]. If RKIP binding to Raf-1 requires pocket binding, then compounds that bind the RKIP pocket would interfere with downstream signaling to MAPK. However, we showed that locostatin binds to the RKIP pocket with no effect on Raf-1 association nor RKIP phosphorylation by PKC and did not change the EGF-induced MAPK activity in HeLa cells in the presence or absence of RKIP [27]. Similarly, two of the compounds (98 and 26) from our screen mimicked the lack of effect by locostatin on EGF-induced MAPK activity (Figure 5). In addition, we observed compound 48 at 20 µM had no effect on wild-type HeLa cells; however compound 48 reduced MAPK activity by 34±11% (mean ± SEM, n = 3) in RKIP depleted cells (Figure 5). To further examine the effect of compound 48, we compared the dose response of compound 26 as a control to that of compound 48 (Figure 6 and 7). As predicted, compound 26 at all concentrations tested had no effect on EGF-induced MAPK activity in HeLa cells in the presence or absence of RKIP (Figure 6). In contrast, the inhibitory effect was dose-dependent for compound 48 with respect to RKIP depleted cells but dose-independent in wild-type RKIP cells (compare Figure7A with 7B).

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