Identification of RKIP Ligands Figure 4. DHPE but not compounds 98, 48, 26 nor locostatin decreases RKIP interactions with Raf-1 and PKC. (A) Representative western blot of RKIP association with Raf-1 in the presence of 1.5 mM Compounds 26, 48 or 98 as well as locostatin or DHPE. (B) PKC phosphorylation of RKIP in the presence of increasing concentrations of DHPE or compounds 98, 48, or 26 (Representative autoradiogram is shown). doi:10.1371/journal.pone.0010479.g004 One motivation in this screen was to identify a compound that affected RKIP function via pocket binding. From this study we established that these compounds, even up to millimolar concentrations did not affect RKIP association with Raf-1 and did not alter Ser-153 phosphorylation by PKC. These results confirm and extend previous findings that locostatin can similarly bind the RKIP pocket without altering association with Raf-1 or kinases such as PKC. Based on these findings, we postulated that MAPK should not be affected by drug binding. However, one out of the three compounds (compound 48) displayed off target effects by reducing MAPK activity as well as metabolic activity in HeLa cells. This effect was more pronounced in RKIP depleted cells suggesting a transient protective role for RKIP with respect to MAPK activation, against toxic drugs as previously observed for locostatin. In this study, we describe the importance of the microenviron- ment with respect to pH for ligand binding to the RKIP pocket. The observation that bulky lipids such as DHPE inhibit both Raf-1 and kinase association with RKIP suggests that interaction with residues in the vicinity of the pocket rather than binding within the PLoS ONE | www.plosone.org 7 May 2010 | Volume 5 | Issue 5 | e10479