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A Small-Molecule Inhibitor of Isoprenylcysteine Carboxyl Methyltransferase with Antitumor Activity in Cancer Cells

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A Small-Molecule Inhibitor of Isoprenylcysteine Carboxyl Methyltransferase with Antitumor Activity in Cancer Cells A small-molecule inhibitor of isoprenylcysteine carboxyl methyltransferase with antitumor activity in cancer cells Ann M. Winter-Vann*, Rudi A. Baron*, Waihay Wong*, June dela Cruz*, John D. York*, David M. Gooden†, Martin O. Bergo‡, Stephen G. Young§, Eric J. Toone†, and Patrick J. Casey*¶ Departments of *Pharmacology and Cancer Biology and †Chemistry, Duke University Medical Center, Durham, NC 27710; ‡Department of Internal Medicine, Sahlgrenska University Hospital, S-413 45 Gothenburg, Sweden; and §Department of Medicine, University of California, Los Angeles, CA 90095 Edited by John A. Glomset, University of Washington, Seattle, WA, and approved February 7, 2005 (received for review November 1, 2004) Many key regulatory proteins, including members of the Ras family proteins have also been implicated in oncogenesis and tumor of GTPases, are modified at their C terminus by a process termed progression, and these proteins most likely require processing via prenylation. This processing is initiated by the addition of an the prenylation pathway for function (2, 15). isoprenoid lipid, and the proteins are further modified by a pro- Both the membrane targeting and the transforming abilities of teolytic event and methylation of the C-terminal prenylcysteine. Ras require processing through the prenylation pathway (16, 17). Although the biological consequences of prenylation have been For this reason, the protein prenyltransferases, most notably characterized extensively, the contributions of prenylcysteine FTase, have been targets of major drug discovery programs for methylation to the functions of the modified proteins are not well the last decade (18, 19). Presently, several FTase inhibitors are understood. This reaction is catalyzed by the enzyme isoprenyl- being evaluated in clinical trials (15, 19). These experimental cysteine carboxyl methyltransferase (Icmt). Recent genetic disrup- agents have shown significant activity in a number of clinical tion studies have provided strong evidence that blocking Icmt trials, but the overall response rates in patients have been less activity has profound consequences on oncogenic transformation. than initially hoped. One possible explanation for this lack of Here, we report the identification of a selective small-molecule efficacy is the process of alternate prenylation that allows some inhibitor of Icmt, 2-[5-(3-methylphenyl)-1-octyl-1H-indol-3-yl]acet- FTase substrates to be modified by geranylgeranyltransferase amide (cysmethynil). Cysmethynil treatment results in inhibition of type I when FTase activity is limiting (20–22). Recent studies cell growth in an Icmt-dependent fashion, demonstrating mecha- using genetic disruption of Icmt have demonstrated that Ras nism-based activity of the compound. Treatment of cancer cells proteins are significantly mislocalized and tumorigenesis is with cysmethynil results in mislocalization of Ras and impaired markedly impaired in cells that lack Icmt (23, 24). After this epidermal growth factor signaling. In a human colon cancer cell discovery, CaaX protein methylation has gained attention as a line, cysmethynil treatment blocks anchorage-independent target in oncogenesis (25). growth, and this effect is reversed by overexpression of Icmt. These With emerging evidence for the importance of Icmt-catalyzed findings provide a compelling rationale for development of Icmt CaaX protein methylation in oncogenesis, there is a clear need inhibitors as another approach to anticancer drug development. for specific pharmacological agents to target this process. How- ever, the only such agents available to date have been analogs of cell transformation ͉ protein isoprenylation ͉ protein methylation ͉ Ras the substrate prenylcysteine or the product S-adenosylhomocys- signaling teine; all of these analogs have been reported to have pleiotropic effects (26–28). Here, we report the discovery of an indole-based C-terminal CaaX motif, where C is cysteine, the a’s are small-molecule inhibitor of Icmt. Treatment of cancer cells with Aaliphatic amino acids, and X can be any of a number of this compound that we have named 2-[5-(3-methylphenyl)-1- amino acids, targets a variety of eukaryotic proteins to a series octyl-1H-indol-3-yl]acetamide (cysmethynil), results in a de- of posttranslational modifications important for their localiza- crease in Ras carboxylmethylation, mislocalization of Ras, and tion and function (1, 2). This processing is initiated by the impaired signaling through Ras pathways. Cysmethynil treat- covalent attachment of a 15-carbon farnesyl or a 20-carbon ment blocks anchorage-independent growth in a human colon geranylgeranyl lipid to the cysteine of the CaaX motif, a reaction cancer cell line, and this effect is reversed by overexpression of catalyzed by protein farnesyltransferase (FTase) or protein Icmt. These findings, together with the findings from genetic geranylgeranyltransferase type I (3). After prenylation, the disruption of this enzyme, suggest that Icmt inhibitors may have C-terminal three amino acids (i.e., the -aaX) are removed by a significant therapeutic potential. specific CaaX protease termed Rce1 (4, 5) and the now C- terminal prenylcysteine is methylated by isoprenylcysteine car- Materials and Methods boxyl methyltransferase (Icmt; refs. 6–8). As polytopic mem- Materials. Farnesyl pyrophosphate was from Biomol, the chem- brane proteins localized to the endoplasmic reticulum, both ical library was from PPD Discovery (Research Triangle Park, Rce1 and Icmt are unusual in their respective classes (9). NC), streptavidin-Sepharose beads were from Amersham Phar- Proteins that terminate in a –CaaX motif regulate a number macia, puromycin and S-adenosylmethionine (AdoMet) were of pathways important in oncogenesis. The best studied example from Sigma, and S-(5Ј-adenosyl)-L-homocysteine was from is the central role of the Ras family of proteins in growth factor activation of the MAP kinase signaling cascade (10, 11). Con- stitutive activation of this pathway is transforming in a wide This paper was submitted directly (Track II) to the PNAS office. variety of cell types, and activating mutations in Ras have been Abbreviations: Icmt, isoprenylcysteine carboxyl methyltransferase; cysmethynil, 2-[5-(3- found in almost 30% of all cancers, including 50% of colon methylphenyl)-1-octyl-1H-indol-3-yl]acetamide; Rce1, CaaX protease; AdoMet, S-adeno- cancers and up to 90% of pancreatic cancers (12). In addition, sylmethionine; BFC, biotin-S-farnesyl-L-cysteine; MAPK, mitogen-activated protein kinase; many cancers contain alterations upstream of Ras, and the MDCK, Madin–Darby canine kidney; FTase, farnesyltransferase. resultant hyperactivation of Ras is thought to contribute to ¶To whom correspondence should be addressed. E-mail: [email protected]. tumorigenesis in these cancers as well (13, 14). Many other CaaX © 2005 by The National Academy of Sciences of the USA 4336–4341 ͉ PNAS ͉ March 22, 2005 ͉ vol. 102 ͉ no. 12 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0408107102 Downloaded by guest on September 30, 2021 Fluka. Epidermal growth factor was from EMD Bioscience. Background absorbance from blank wells containing only media [methyl-3H]methionine and [methyl-3H]AdoMet were from with compound or vehicle were subtracted from each test well. PerkinElmer. CellTiter 96 Aqueous One solution cell prolifer- ation assay was from Promega. pEGFP and pLPCX were from Localization of GFP Proteins in MDCK Cells. MDCK cells stably Clontech. Sf9 membranes containing recombinant Rce1 and expressing GFP-H-Ras, GFP-K-Ras, or GFP-N-Ras were grown Icmt, termed Rce1 membranes and Icmt membranes, respec- on 35-mm coverslips treated with poly-D-lysine. MDCK cells tively, were made in our laboratory as described (5). Farnesy- expressing Yes-GFP were prepared by transient transfection of lated K-Ras was made by in vitro modification of bacterially the Yes-GFP construct (30), using Superfect reagent (Qiagen, expressed K-Ras with purified FTase as described (5). Biotin- Valencia, CA) following the manufacturer’s instructions. Cells S-farnesyl-L-cysteine (BFC) was synthesized as detailed else- were grown in media containing 10% FBS to Ϸ25% confluence where (R.A.B. and P.J.C., unpublished work). Mouse embryonic and then treated with 1% DMSO or cysmethynil at the indicated fibroblast cell lines were established and maintained as described concentrations. Cells were imaged 72 h after drug treatment (24 (29). Madin–Darby canine kidney (MDCK) cells stably express- h for transiently transfected cells) on an Olympus inverted ing GFP-H-Ras, GFP-K-Ras and GFP-N-Ras were a generous microscope with an UltraView spinning-disk confocal gift of M. Philips (New York University Medical School, New (PerkinElmer LAS) and a krypton͞argon laser with a 488-nm York). line, attached to a cooled charge-coupled device camera (Hamamatsu). Initial image acquisition and manipulation was Icmt Assay: Screening. The small-molecule screen was performed performed with METAMORPH software (Universal Imaging, in 96-well multiscreen filtration plates (Millipore) by using a Downingtown, PA). Beckman Biomek FX robot. Briefly, Sf9 membranes containing Rce1 and Icmt were suspended in 100 mM Hepes, pH 7.4͞5mM Phosphoprotein Analysis. Cells were grown for three days in media MgCl2 (Buffer A) such that 40 ␮l of suspension had 1 ␮g of Rce1 containing 1% FBS with cysmethynil or vehicle as indicated. membrane protein and 0.2 ␮g of Icmt membrane protein; Half of the wells were then treated with EGF (10 ng͞ml) and half protein values
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