Identification of RKIP Ligands Figure 5. 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 mM compounds (as indicated). EGF (10 ng/ml) was added in the final 5 minutes of incubation. (A) Whole cell lysates (30 mg) 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 6 SEM; n = 3 independent experiments). *p,0.05 indicates the significance of change relative to the DMSO control in the presence of EGF. doi:10.1371/journal.pone.0010479.g005 pocket is required for these protein-protein interactions. We also identified three compounds that bind to the RKIP binding pocket with no effect on Raf-1 association or RKIP phosphorylation. One out of the three compounds from this screen (compound 48) reduced MAPK activation in a dose dependent manner independent of RKIP, suggesting off target effects. The structure of these compounds which bind to RKIP should act as lead compounds for future drug design which could ultimately lead to intervention for diseases that develop from disregulation of the pathways involving RKIP and/or its targets. PLoS ONE | www.plosone.org 8 May 2010 | Volume 5 | Issue 5 | e10479