synthesize derivatives with increased potency. Screening the 4288 compounds enabled us to identify two types of novel GGTI compounds: one group with a dihydropyrrole ring as its core scaffold and the other group with a tetrahydropyridine ring as its core scaffold [19,20]. In our previous study, we reported that derivatization of a carboxylic acid emanating from the dihydro- pyrrole ring of one of the GGTI compounds dramatically improves their cellular activity [20]. The improved GGTI, P61- A6, inhibits proliferation of a variety of human cancer cell lines, and causes G1 cell cycle arrest and induction of p21CIP1/WAF1 [20]. Our study with P61-A6 using a human pancreatic cancer xenograft model in mice showed that the GGTI exhibited prolonged blood circulation time and significant suppression of tumor growth [12]. In this study, we focused on P3-E5, a GGTI compound with a tetrahydropyridine ring [20], and modified it with an L-leucine methylester to replace the free acid. This led to the synthesis of GGTI P61-E7. Compound P61-E7 selectively inhibits protein geranylgeranlyation in cells with higher potency than P61-A6 and caused accumulation of both RhoA and RalA, which are known substrates of GGTase-I, in the cytosolic fractions. Furthermore, compared to P61-A6, P61-E7 exhibits improved potency to inhibit both cell proliferation and cell cycle progression and inducing p21CIP1/WAF1 level. In addition, compound P61-E7 increases p27Kip1 level and inhibits phosphorylation of p27Kip1 (T187). P61- E7 also blocks anchorage-independent growth and disrupts cell morphology of Panc-1. We also examined the mechanism of action of P61-E7 in pancreatic cancer cells. This is important because the cellular effects observed pointed to the involvement of RhoA, a geranylgeranylated small GTPase shown to mediate a number of cellular processes such as actin stress fiber organization, cell adhesion and cell proliferation, the significance of the inhibition of RhoA geranylgeranylation on GGTI-mediated cellular effects was evaluated. We showed that stable expression of farnesylated RhoA mutant (RhoA-F) prevents induction of p21CIP1/WAF1 and p27Kip1 by P61-E7 in Panc-1 and promotes partial resistance to the anti-proliferative effect of the GGTI compound. Further- more, stable expression of RhoA-F rescues Panc-1 cells from cell rounding and inhibition of focal adhesion formation following P61-E7 treatment. Taken together, our findings show that P61- E7 provides a novel addition to the range of GGTI compounds Novel Inhibitor of Geranylgeranyltransferase-I that are in development as anticancer drugs and that RhoA is an important target of the GGTI compound in Panc-1 pancreatic cancer cells. Materials and Methods Compounds The allenoate-derived compound library was synthesized as described in our previous publications [19,20]. Briefly, GGTI P61-E7 (Fig. 1A) was obtained by coupling P3-E5, a represen- tative tetrahydropyridine carboxylic acid hit compound [20], with L-leucine methyl ester, where the free acid is converted to a L-leucine amido methyl ester (Fig. 1B). A 20 mM stock solution of P61-E7 in dimethyl sulfoxide (DMSO) was kept at 220uC until use. GGTI-298 was purchased from Sigma-Aldrich (St. Louis, MO). Farnesyltransferase inhibitor (FTI) BMS-225975 [31] was kindly provided by Dr. Veeraswamy Manne (Bristol-Myers Squibb Co.). Plasmids We used 3xHA-RhoA pcDNA3.1 expression construct (UMR cDNA Resource Center, Rolla, MO) as template DNA for site- directed mutagenesis PCR driven by Pfu UltraH High-Fidelity DNA Polymerase (Aligent Technologies, Wilmington, DE) according to the manufacturer’s instructions with the following primers: Farnesylated RhoA-CLVS was generated using F (forward primer), 59AGAAAAAATCTGGTTGCCTTGTCT- CGTGACTCGAGTCTAG-39, and R (reverse primer), 59-C- TAGACTCGAGTCACGAGACAAGGCAACCAGATTTTTT- CT-39. Cell Lines and Cell Cultures MiaPaCa-2 (ATCC, Rockville, MD), NIH3T3 (ATCC, Rock- ville, MD) and Panc-1 (ATCC, Rockville, MD) cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM; Cellgro, Herndon, VA). Jurkat (ATCC, Rockville, MD) cells were maintained in RPMI 1640 medium (Cellgro, Herndon, VA). Both media were supplemented with 10% (v/v) fetal bovine serum (FBS; HyClone, Logan, UT) and 1% penicillin/1% streptomycin stock solution (Invitrogen, Carlsbad, CA). All cells were cultured at 37uC in a humidified incubator at 5% CO2. Figure 1. Structures of P61-A6 and P61-E7 and synthesis of P61-E7 from P3-E5. (A) Structures of compounds P61-A6 and P61-E7. (B) Treatment of P3-E5 with L-leucine methyl ester, O-benzotriazole-N,N,N9,N9-tetramethyluronium hexafluorophosphate (HBTU) and N,N-diisopropy- lethylamine (DIPEA) in dichloromethane provides P61-E7 in 95% isolated yield. doi:10.1371/journal.pone.0026135.g001 PLoS ONE | www.plosone.org 2 October 2011 | Volume 6 | Issue 10 | e26135