Date Published: June 19, 2017
Publisher: Springer Vienna
Author(s): Woo Seok Yang, Seung-Gu Yeo, Sungjae Yang, Kyung-Hee Kim, Byong Chul Yoo, Jae Youl Cho.
Among the enzymes involved in the post-translational modification of Ras, isoprenyl carboxyl methyltransferase (ICMT) has been explored by a number of researchers as a significant enzyme controlling the activation of Ras. Indeed, inhibition of ICMT exhibited promising anti-cancer activity against various cancer cell lines. This paper reviews patents and research articles published between 2009 and 2016 that reported inhibitors of ICMT as potential chemotherapeutic agents targeting Ras-induced growth factor signaling. Since ICMT inhibitors can modulate Ras signaling pathway, it might be possible to develop a new class of anti-cancer drugs targeting Ras-related cancers. Researchers have discovered indole-based small-molecular ICMT inhibitors through high-throughput screening. Researchers at Duke University identified a prototypical inhibitor, cysmethynil. At Singapore University, Ramanujulu and his colleagues patented more potent compounds by optimizing cysmethynil. In addition, Rodriguez and Stevenson at Universidad Complutense De Madrid and Cancer Therapeutics CRC PTY Ltd., respectively, have developed inhibitors based on formulas other than the indole base. However, further optimization of chemicals targeted to functional groups is needed to improve the characteristics of ICMT inhibitors related to their application as drugs, such as solubility, effectiveness, and safety, to facilitate clinical use.
The post-translational modification of Ras family members is a crucial step in inducing translocation of Ras proteins from the endoplasmic reticulum to the plasma membrane for management of cell proliferation triggered by growth factor signaling. Mutations in Ras family proteins (e.g., K-RAS) are also closely related to the incidence of various types of cancers in humans, such as pancreatic cancer (Downward 2003; Pylayeva-Gupta et al. 2011). Normally, a series of post-translational modifications of Ras proteins are a prerequisite for localization of Ras in the plasma membrane; however, mutations in Ras proteins can induce uncontrolled activation of Ras signaling pathways and Ras-mediated oncogenesis in the cell membrane. The post-translational modification of Ras first requires farnesylation or geranylgeranylation of the C-terminal CAAX motif, in which covalent bonding of farnesyl or geranylgeranyl isoprenoid lipids to the cysteine residue of CAAX is mediated by farnesyl transferase (FTase) or geranylgeranyl transferase (GGTase I), respectively, as summarized in Fig. 1 (Winter-Vann and Casey 2005; Clarke 1988). During this step, the CAAX endoprotease RCE1 cleaves the three C-terminal amino acids (–AAX) (Ashby 1998), and isoprenyl carboxyl methyltransferase (ICMT) methylates a prenylated cysteine (Bergo et al. 2000). Isoprenyl carboxyl methylated proteins anchor to the cell membrane (Eisenberg et al. 2013) and influence cell signaling pathways. (Bergo et al. 2004; Goodman et al. 1990). ICMT (MW 32 kDa) is essential for post-translational modification of Ras proteins and is localized in the endoplasmic reticulum (Zhang and Casey 1996). Since Ras plays an important role in cancerous signaling pathways, ICMT is also a substantial focus of studies on anti-cancer agents (Wahlstrom et al. 2008). Suppression of the carcinogenic transformation of RAS by inhibition of the enzymes involved in farnesylation or geranylgeranylation has been studied for treatment of cancer. There are several inhibitors of FTase, including lonafarnib (IC50 = 1.9 nM) and tipifarnib (IC50 = 7.9 nM) (Basso et al. 2006). Although some inhibitors of FTase have shown inhibitory activities in mouse models, they had no notable effect on the clinical score in cancer patients. Moreover, the geranylgeranylated Ras proteins maintain biological activities when FTase is blocked by inhibitors, limiting the effects of those inhibitors (Anderson et al. 2005; Doll et al. 2004; Mazieres et al. 2004; Lene 2009). Numerous reports have also demonstrated that inhibition of ICMT leads to an anti-proliferative effect in cancer (Clarke 1988; Bos 1989; Bishop et al. 2003; Gibbs et al. 1994). Pharmacologic or genetic inactivation of ICMT resulted in cell cycle arrest and apoptosis (Bergo et al. 2000, 2004). Therefore, the strategy of suppressing ICMT with chemical inhibitors could be considered an alternative therapeutic approach targeting Ras-mediated tumorigenic responses (Teh et al. 2014).Fig. 1Schematic diagram of the RAS/ICMT regulatory process in a growth factor-inducing signaling cascade. FTase farnesyltransferase, RCE1 Ras-converting CAAX endopeptidase 1, ICMT isoprenylcysteine methyltransferase, AdoMet S-adenosyl-l-methionine, AdoHcy S-adenosyl-l-homocysteine, GF growth factor
Isoprenyl carboxyl methyltransferase inhibitors are divided into three classes based on their properties. The first class of these inhibitors includes S-adenosyl-l-homocysteine (AdoHcy) and its precursors. During methylation, S-adenosyl-l-methionine (AdoMet) donates a methyl group to the substrate proteins and is converted to AdoHcy (Pylayeva-Gupta et al. 2011; Anderson et al. 2005; Kim et al. 2013; Perez-Sala et al. 1992), a by-product that acts in a negative feedback reaction and leads to reduced enzyme activity of methyltransferase until it is cleaved by AdoHcy hydrolase (Shi and Rando 1992; Kramer et al. 2003). Therefore, AdoHcy suppresses the activity of ICMT by acting as a non-selective inhibitor of other methyltransferases (Winter-Vann et al. 2003), and specific inhibitors of ICMT are still needed for treatment. N-Acetyl-S-farnesyl-l-cysteine (AFC) and N-acetyl-S-geranylgeranyl-l-cysteine (AGGC) can competitively bind to the same substrate tunnel of farnesylated proteins. These structural analogs of prenylcysteine represent the second class of ICMT inhibitors by acting as actual substrates that can compete with Ras (Henriksen et al. 2005). In fact, there are three structural mimics of AFC. The first analog of AFC, which has a sulphonamide linkage in place of an amide bond, had an IC50 value of 8.8 μM in a vapor diffusion assay (Hrycyna and Clarke 1990). The other analog of AFC has a triazole moiety in place of an allylic thioether, which can be easily degraded by enzymatic and chemical processes. This analog has an IC50 value of 19.4 μM. The third structural mimic possesses an aryl alkyl moiety in the farnesyl side chain of AFC instead of two isoprenoid units and has an IC50 value of 34.6 μM (Ramanujulu et al. 2013; Buchanan et al. 2007, 2008a, b). The third class of ICMT inhibitors includes molecules that have a low molecular weight (<900 Da) and are utilized as drugs due to their rapid diffusion through the cell membrane (Macielag 2012). Cysmethynil (2-[5-(3-methylphenyl)-l-octyl-lH-indolo-3-yl]acetamide), which was identified from 10,000 compounds in the chemical library, is a representative small-molecular inhibitor that competes with isoprenylated cysteine of other CAAX-containing proteins, but not AdoMet (Lene 2009). Cysmethynil induces incorrect localization of Ras and interrupts the signaling pathway of cancer cells. Consequently, cysmethynil causes autophagic cell death through pharmacological inhibition of ICMT (Winter-Vann et al. 2003; Henriksen et al. 2005; Wang et al. 2008; Clarke and Tamanoi 2004; Baron et al. 2007). Because the desired concentration of a drug in circulation is determined by its solubility, this measure is a very important aspect of drug development (Savjani et al. 2012). Cysmethynil is insoluble in water and has high lipophilicity and structural flexibility and is, therefore, considered to be non-applicable for clinical use (Kerns and Di 2010), implying the need for improvement through further structure–activity relationship (SAR) studies. The discovery of an effective ICMT inhibitor is essential for the development of cancer drugs, because Ras is associated with numerous cancer signaling pathways according to in vitro and in vivo tumorigenic models (Lau et al. 2014). We described the current patent literature focusing on inhibitors of ICMT as an attractive biological target. In 2009, Richard A. Gibbs, Brian S. Henriksen, Christine A. Hrycyna, and Jessica L. Anderson (patent requestor: Purdue Research Foundation) filed for a patent for inhibitors of ICMT for use in treating neoplasms and cancer (Donelson et al. 2009). Gibbs’ laboratory has also published a number of papers on ICMT inhibitors (Bergman et al. 2011a, b; Majmudar et al. 2011, 2012; Donelson et al. 2006). In 2011, Patrick J. Casey, Rudi A. Baron, and Ann M. Winter-Vann at Duke University applied for a patent for ICMT inhibitors with potential anti-cancer activity (Casey et al. 2011). From 2013 to 2014, a patent of small-molecule inhibitors of ICMT was filed by the National University of Singapore (Ramanujulu et al. 2013; Leow et al. 2014). We used Worldwide Intellectual Property Service (WIPS) global, the Korea Intellectual Property Rights Information Service (KIPRIS), the Google patent search system, and the PubMed website to examine patents or papers on ICMT inhibitors. In section “Patent evaluation”, we present the types of ICMT inhibitors and their chemical structures or enzymatic therapies relating to cancer biological activities. The subsections are organized in chronological order by patent applicant. The Ras protein family has been found to play a significant role in proliferative activity in many oncogenic cell lines. Mutations in Ras family members leading to their abnormal activation are common and are found in one-third of all human tumors. Therefore, there is a high demand for development of Ras inhibitors. Since the Ras protein undergoes a number of post-translational modifications during its “switching on” process, various types of inhibitors with distinct mechanisms of action are under development. Previously, farnesylation of Ras by FTase was the prime target in the Ras-mediated proliferative signaling pathway. Such FTase inhibitors (FTIs) showed promising inhibitory effects with low toxicity in mouse models; however, results of human clinical trials were rather disappointing. It is mostly agreed that the explanation for their poor inhibitory effect is “alternate prenylation”, in which the empty farnesylation site of Ras due to use of FTIs is geranylgeranylated by GGTase I. As a result, FTIs show limited inhibitory effects, because geranylgeranylated Ras maintains biological activity similar to farnesylated Ras, ultimately leading to oncogenic proliferation. In contrast, methylation of Ras by ICMT is both crucial and nonspecific to either type of prenylation. Because the geranylgeranylated Ras will still be affected by ICMT inhibitors, such inhibitors can ensure more effective inhibition of Ras-induced abnormal growth factor signaling in cancer cells compared with the previous approaches. Source: http://doi.org/10.1007/s00726-017-2454-x