Chapter 11 - Cysmethynil, a Specific Small-Molecule Inhibitor of Isoprenylcysteine Carboxylmethyl Transferase (Icmt)
Section snippets
Abstract
Since the discovery that Ras oncoproteins undergo processing through the CAAX prenylation pathway and that the modification is important for their function, this process had been under the spotlight in the hope to develop effective inhibitors for cancer therapy. It is increasingly recognized that C-terminal methylation, the last step of this modification process catalyzed by isoprenylcysteine carboxylmethyl transferase (Icmt), impacts the functions of both farnesylated as well as
Prenylation Process
The posttranslational modification termed prenylation is a three-step process. S-isoprenylation of the cysteine residue of the C-terminal CAAX consensus sequence, and the subsequent proteolysis of the –AAX residues, constitute the first and second step. The last step, the α-carboxylmethylation of the prenylated cysteine, occurs on the exposed carboxyl group after proteolysis. These modifications of CAAX proteins are well conserved in eukaryotic systems, from Saccharomyces cerevisiae to human,
Rationale for Targeting Icmt
Over 30% of human cancers exhibit elevated Ras activity which is important for tumor cell proliferation and survival. Ras has been an attractive target in the quest for cancer therapy, but direct inhibition of Ras activity has proven to be difficult [8]. Disrupting the protein prenylation process, particularly farnesylation, had become a focus in cancer research since the Ras oncoproteins were identified as recipients of this processing. Unprenylated Ras protein was mislocalized from plasma
Icmt and Its Functional Importance in Biology
Early information on C-terminal methylation of CAAX proteins came from the studies of mating factor of budding yeast. Anderegg and colleagues found that the hydrolysis of the carboxyl methyl ester caused a complete loss of a-factor activity in yeast [20]. It was further found the yeast carboxylmethyltansferase (Ste14) is required for the activity of a-factor—the mating pheromone. Loss of function of the Ste14 gene product resulted in reduced release of inactive a-factor, evidence for the
Development of Icmt Inhibitors
Because of the therapeutic potential for manipulating Icmt function, there has been much effort in identifying inhibitors of this enzyme. Various isoprenyl cysteine analogs were analyzed for their abilities to serve as Icmt inhibitors; the afore-mentioned AFC and N-acetyl-S-geranylcysteine (AGC) were found to inhibit well. AFC was employed to study carboxyl methylation of CAAX proteins in the early studies [24]. Using AFC as a substrate, Icmt activity was found to be abundantly present in the
Cysmethynil, a Nonstructure Analog Small-Molecule Inhibitor of Icmt
Since the initial identification of cysmethynil, various lines of evidence support its Icmt-dependent action. First, cysmethynil did not disrupt the normal localization of myristol/palmitoyl modified membrane proteins, demonstrating that this relatively hydrophobic small molecule does not disrupt global membrane protein trafficking [32]. Second, Icmt null MEFs are relatively resistant to cysmethynil-induced toxicity 32, 44. Third, even when used at 250 times the concentration of the IC50 for
Inhibition of Icmt by Cysmethynil Induces Autophagy and Cell Death, Potential for Cancer Therapy
Cysmethynil treatment of a variety of cancer cell lines has been found to elicit growth inhibition and cell death 44, 45. During the early stages of treatment, cysmethynil arrests cell growth at the G1 phase; with prolonged treatment, cell death ensues. Interestingly, cysmethynil treatment also results in increased presence of macroautophagy, evidenced by elevated level of lipidated microtubule-associated protein 1 light chain 3 (LC3-II) and the appearance of characteristic LC3-positive
Cysmethynil Inhibition of Icmt Results in Reduction of Rho-Mediated Cell Migration, Suggesting a Potential Role in Cancer Metastasis
The ability to break away from the primary site of growth, enter circulation, extravasate, and recolonize at a distal site is the hallmark of malignancy. This complicated process involves loss of cell polarity, cell detachment and cell migration, among others; several CAAX proteins are implicated in different stages of metastasis. While the Ras family of small GTPases is known to be intimately involved in the proliferation and survival of cancer cells, Rho family members are recognized to be
Current and Future Work to Identify Better Icmt Inhibitors Through Medicinal Chemistry
In the original small-molecule screen for Icmt inhibitors, cysmethynil was identified from one of the 70 series of different scaffolds; it was among 72 compounds in the library that possessed a core indole structure (Figure 11.1) [32]. These cysmethynil analogs differed primarily in the substitution at the phenyl ring and the indole nitrogen; they had IC50 values vary from 2 to 50 μM. Taking advantage of the IC50 values obtained from the initial screening, quantitative structure–activity
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