little inhibition was observed with FTase and RabGGTase (Figure S1). In this study, we derivatized the carboxyl group of P3-E5 to generate compounds that exhibit increased cellular activity. From a series of modified compounds, we identified P61-E7 that was synthesized by coupling carboxylic acid of P3-E5 with L-leucine methyl ester to yield an amido ester P61-E7 (Figure 1B). The reaction carried out under the influence of O-benzotriazole- N,N,N9,N9-tetramethyluronium hexafluorophosphate (HBTU) and N,N-diisopropylethylamine (DIPEA) in chloromethane [33] was efficient with 95% yield. P61-E7 demonstrated better cellular GGTI activity than P3-E5; GI50 values with Jurkat cells were 3.5 mM and 20 mM for P61-E7 and P3-E5, respectively. GGTI P61-E7 Exhibits Improved Ability to Inhibit Geranylgeranylation in Cultured Mammalian Cells When Compared to GGTI P61-A6 P61-E7 exhibits increased potency to inhibit protein geranylger- anylation in cells (Figure 2A and 2B). In the first assay, we assessed inhibition of protein geranylgeranylation by using an antibody that specifically detects unprenylated form of Rap1 [34]. Rap1 is a Ras- like GTPase that plays a role in cadherin-based cell adhesion [34], and is a known substrate of GGTase-I. Panc-1 (Figure 2A) and NIH3T3 cells (Figure 2B) were treated with DMSO, P61-A6, or P61-E7 for 48 hours. Treatment with either P61-A6 or P61-E7 led to the accumulation of unprenylated Rap1 in a dose-dependent manner in both Panc-1 (Figure 2A) and NIH3T3 (Figure 2B) cells. P61-E7 treatment resulted in the inhibition of protein geranylger- anylation at much lower concentrations when compared to P61-A6 Novel Inhibitor of Geranylgeranyltransferase-I in both cell lines tested (Figure 2A and 2B). For example, at 5 mM, P61-A6 increased the level of unprenylated Rap1 in Panc-1 cells by 2.6-fold when compared to DMSO, while 5 mM of P61-E7 caused the level of unprenylated Rap1 in Panc-1 cells to increase by 12.5- fold (Figure 2A). Likewise, at 5 mM, P61-A6 treatment did not significantly change the level of unprenyalted Rap1 in NIH3T3 cells compared to DMSO treatment, while 5 mM of P61-E7 resulted in 21-fold increase in unprenylated Rap1 level (Figure 2B). All these findings reflected a significant improvement in the potency of P61- E7 in inhibiting protein geranylgeranylation in cells. Next, the specificity of P61-E7 was measured by determining its ability to inhibit farnesylation of HDJ2 which is a cochaperone of the heat-shock cognate protein 70 (hsc70) involved in protein folding and traffic [35] and a known substrate of farnesyltransfer- ase (FTase). NIH3T3 cells were treated with various concentra- tions of P61-E7 (5, 10, and 20 mM) or BMS-225975, an FTase inhibitor (FTI). While treatment with BMS-225975 slowed the mobility of HDJ2 on SDS-polyacrylamide gel, no such mobility shift was observed with P61-E7 treatment even when cells were treated with 20 mM of P61-E7 (Figure 2C). These findings indicated that P61-E7 selectively inhibits protein geranylgeranyla- tion but not farnesylation in cells. Treatment with P61-E7 results in increase of cytosolic localization of geranylgeranylated proteins Because prenylation is required for geranylgeranylated proteins to be associated with cellular membranes, we assessed whether P61-E7 treatment inhibits membrane association of proteins such Figure 2. GGTI P61-E7 is a more potent inhibitor of geranylgeranylation compared with P61-A6. (A and B), P61-E7 or P61-A6 treatment inhibits Rap1 geranylgeranylation in Panc-1 (A) and NIH3T3 (B) cells. Whole cell lysates from cells treated with DMSO, P61-A6, or P61-E7 for 48 h were prepared and resolved on SDS-PAGE for immunoblotting analysis using antibodies against the unprenylated form of Rap1 (U-Rap1, upper panels, A and B), or HDJ2 (loading control, lower panels, A and B). Data shown are representative of three independent experiments. The U-Rap1 and HDJ2 bands were quantified using ImageJ. Intensities of U-Rap1 bands were normalized to their respective loading control, and the results are given above the images as fold change compared to the DMSO control. (C) Whole cell lysates from NIH3T3 cells treated with DMSO, P61-A6, P61-E7, or FTI BMS- 225975 for 48 h were prepared and resolved on SDS-PAGE for immunoblotting analysis using antibodies against the unprenylated form of Rap1 (U- Rap1, upper panel), or HDJ2 (lower panel). Data shown are representative of three independent experiments. doi:10.1371/journal.pone.0026135.g002 PLoS ONE | www.plosone.org 4 October 2011 | Volume 6 | Issue 10 | e26135