The isoprenoid substrate specificity of isoprenylcysteine carboxylmethyltransferase - Development of novel inhibitors

Isoprenylcysteine carboxylmethyltransferase (Icmt) is an integral membrane protein localized to the endoplasmic reticulum of eukaryotic cells that catalyzes the post-translational alpha-carboxylmethylesterification of CAAX motif proteins, including the oncoprotein Ras. Prior to methylation, these protein substrates all contain an isoprenylcysteine residue at the C terminus. In this study, we developed a variety of substrates and inhibitors of Icmt that vary in the isoprene moiety in order to gain information about the nature of the lipophilic substrate binding site. These isoprenoid-modified analogs of the minimal Icmt substrate N-acetyl-S-farnesyl-L-cysteine (AFC) were synthesized from newly and previously prepared farnesol analogs. Using both yeast and human Icmt enzymes, these compounds were found to vary widely in their ability to act as substrates, supporting the isoprenoid moiety as a key substrate recognition element for Icmt. Compound 3 is a competitive inhibitor of overexpressed yeast Icmt (K-I = 17.1 +/- 1.7 mu M). Compound 4 shows a mix of competitive and uncompetitive inhibition for both the yeast and the human Icmt proteins (yeast K-IC = 35.4 +/- 3.4 mu M, K-IU = 614.4 +/- 148 mu M; human K-IC = 119.3 +/- 18.1 mu M, K-IU = 377.2 +/- 42.5 mu M). These data further suggest that differences in substrate specificity exist between the human and yeast enzymes. Biological studies suggest that inhibition of Icmt results in Ras mislocalization and loss of cellular transformation ability, making Icmt an attractive and novel anticancer target. Further elaboration of the lead compounds synthesized and assayed here may lead to clinically useful higher potency inhibitors.