Identification and Characterization of Mechanism of Action of P61-E7, a Novel Phosphine Catalysis-Based Inhibitor of Geranylgeranyltransferase-I Lai N. Chan1,2, Hannah D. G. Fiji3, Masaru Watanabe1, Ohyun Kwon2,3, Fuyuhiko Tamanoi1,2* 1 Department of Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles, California, United States of America, 2 Molecular Biology Institute, University of California, Los Angeles, California, United States of America, 3 Department of Chemistry and Biochemistry, University of California, Los Angeles, California, United States of America Abstract Small molecule inhibitors of protein geranylgeranyltransferase-I (GGTase-I) provide a promising type of anticancer drugs. Here, we first report the identification of a novel tetrahydropyridine scaffold compound, P61-E7, and define effects of this compound on pancreatic cancer cells. P61-E7 was identified from a library of allenoate-derived compounds made through phosphine-catalyzed annulation reactions. P61-E7 inhibits protein geranylgeranylation and blocks membrane association of geranylgeranylated proteins. P61-E7 is effective at inhibiting both cell proliferation and cell cycle progression, and it induces high p21CIP1/WAF1 level in human cancer cells. P61-E7 also increases p27Kip1 protein level and inhibits phosphorylation of p27Kip1 on Thr187. We also report that P61-E7 treatment of Panc-1 cells causes cell rounding, disrupts actin cytoskeleton organization, abolishes focal adhesion assembly and inhibits anchorage independent growth. Because the cellular effects observed pointed to the involvement of RhoA, a geranylgeranylated small GTPase protein shown to influence a number of cellular processes including actin stress fiber organization, cell adhesion and cell proliferation, we have evaluated the significance of the inhibition of RhoA geranylgeranylation on the cellular effects of inhibitors of GGTase-I (GGTIs). Stable expression of farnesylated RhoA mutant (RhoA-F) results in partial resistance to the anti-proliferative effect of P61-E7 and prevents induction of p21CIP1/WAF1 and p27Kip1 by P61-E7 in Panc-1 cells. Moreover, stable expression of RhoA-F rescues Panc-1 cells from cell rounding and inhibition of focal adhesion formation caused by P61-E7. Taken together, these findings suggest that P61-E7 is a promising GGTI compound and that RhoA is an important target of P61-E7 in Panc-1 pancreatic cancer cells. Citation: Chan LN, Fiji HDG, Watanabe M, Kwon O, Tamanoi F (2011) Identification and Characterization of Mechanism of Action of P61-E7, a Novel Phosphine Catalysis-Based Inhibitor of Geranylgeranyltransferase-I. PLoS ONE 6(10): e26135. doi:10.1371/journal.pone.0026135 Editor: Pan-Chyr Yang, National Taiwan University Hospital, Taiwan Received July 20, 2011; Accepted September 20, 2011; Published October 18, 2011 Copyright: 2011 Chan et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was funded by National Institutes of Health (NIH) grants CA41996, GM071779, GM081282 and GM07185. The funders had no role in study design, data collection and analysis, decision to publish. or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: fuyut@microbio.ucla.edu Introduction Proteins such as the Rho family G-proteins are posttranslation- ally modified by the addition of a geranylgeranyl isoprenoid [1]. The isoprenoid modification is important for membrane associ- ation and functions of these proteins. Recent studies have highlighted the significance of protein geranylgeranylation in human cancers. First, it has been shown that a number of geranylgeranylated proteins play important roles in tumorigenesis and metastasis [2–6]. Second, characterization of GGTase-I- deficient cells showed that the inhibition of GGTase-I leads to proliferation inhibition and accumulation of p21CIP1/WAF1, pointing to the significance of GGTase-I in cell proliferation and cell cycle progression [7]. Furthermore, conditional knockout of the b-subunit of GGTase-I results in the inhibition of lung tumor growth and increased survival of mice expressing oncogenic K-ras [7]. Thus, inhibition of protein geranylgeranylation is a promising approach for developing anticancer drugs, and inhibitors of GGTase-I (GGTIs) are currently undergoing preclinical studies. The results obtained are consistent with the idea that GGTIs disrupt oncogenic and tumor survival pathways, inhibit prolifer- ation and anchorage-independent growth, and induce apoptosis [8–12]. A variety of approaches were taken to develop GGTI compounds. Peptidomimetic GGTI compounds were derived from the peptide bearing the C-terminal CAAL (cysteine followed by two aliphatic amino acids and the C-terminal residue is leucine or phenylalanine) motif, a sequence recognized by GGTase-I [11,13–16]. The first nonpeptidomimetic inhibitor, GGTI-DU40, was identified via high-throughput screening of a compound library [17]. More recently, quantitative structure-activity rela- tionship (QSAR) models for GGTIs have been developed and used to carry out virtual screen of more than 9 million commercially available compounds. This resulted in the identifi- cation of seven compounds with novel scaffolds [18]. Our approach [19,20] was to construct a library of allenoate-derived small molecules based on phosphine-catalyzed annulation reac- tions that produce diverse compounds such as dihydropyrroles [21,22], tetrahydropyridines [23–25], bicyclic succinimides, diox- anylidenes [26], a-pyrones [27], dihydropyrones [28,29], and cyclohexenes [30]. Initial GGTI compounds identified by screening a 171-compound pilot library were then used to PLoS ONE | www.plosone.org 1 October 2011 | Volume 6 | Issue 10 | e26135