ABSTRACT
Isoprenylcysteine carboxyl methyltransferases (Icmts) are a class of integral membrane protein methyltransferases localized to the endoplasmic reticulum (ER) membrane in eukaryotes. The Icmts from human (hIcmt) and Saccharomyces cerevisiae (Ste14p) catalyze the α-carboxyl methyl esterification step in the post-translational processing of CaaX proteins, including the yeast a-factor mating pheromones and both human and yeast Ras proteins. Herein, we evaluated synthetic analogs of two well-characterized Icmt substrates, N-acetyl-S-farnesyl-L-cysteine (AFC) and the yeast a-factor peptide mating pheromone, that contain photoactive benzophenone moieties in either the lipid or peptide portion of the molecule. The AFC based-compounds were substrates for both hIcmt and Ste14p, whereas the a-factor analogs were only substrates for Ste14p. However, the a-factor analogs were found to be micromolar inhibitors of hIcmt. Together, these data suggest that the Icmt substrate binding site is dependent upon features in both the isoprenyl moiety and upstream amino acid composition. Furthermore, these data suggest that hIcmt and Ste14p have overlapping, yet distinct, substrate specificities. Photocrosslinking and neutravidin-agarose capture experiments with these analogs revealed that both hIcmt and Ste14p were specifically photolabeled to varying degrees with all of the compounds tested. Our data suggest that these analogs will be useful for the future identification of the Icmt substrate binding sites.
Subject(s)
Acetylcysteine/analogs & derivatives , Peptides/chemistry , Protein Methyltransferases/antagonists & inhibitors , Protein Methyltransferases/chemistry , Saccharomyces cerevisiae/enzymology , Acetylcysteine/chemistry , Benzophenones/chemistry , Binding Sites , Biotinylation , Enzyme Inhibitors/chemistry , Humans , Mating Factor , Photoaffinity Labels/chemistry , Substrate SpecificityABSTRACT
Human protein isoprenylcysteine carboxyl methyltransferase (hIcmt) is the enzyme responsible for the α-carboxyl methylation of the C-terminal isoprenylated cysteine of CaaX proteins, including Ras proteins. This specific posttranslational methylation event has been shown to be important for cellular transformation by oncogenic Ras isoforms. This finding led to interest in hIcmt inhibitors as potential anti-cancer agents. Previous analog studies based on N-acetyl-S-farnesylcysteine identified two prenylcysteine-based low micromolar inhibitors (1a and 1b) of hIcmt, each bearing a phenoxyphenyl amide modification. In this study, a focused library of analogs of 1a and 1b was synthesized and screened versus hIcmt, delineating structural features important for inhibition. Kinetic characterization of the most potent analogs 1a and 1b established that both inhibitors exhibited mixed-mode inhibition and that the competitive component predominated. Using the Cheng-Prusoff method, the K(i) values were determined from the IC(50) values. Analog 1a has a K(IC) of 1.4±0.2µM and a K(IU) of 4.8±0.5µM while 1b has a K(IC) of 0.5±0.07µM and a K(IU) of 1.9±0.2µM. Cellular evaluation of 1b revealed that it alters the subcellular localization of GFP-KRas, and also inhibits both Ras activation and Erk phosphorylation in Jurkat cells.
Subject(s)
Amides/chemistry , Cysteine/analogs & derivatives , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Protein Methyltransferases/antagonists & inhibitors , Amides/chemical synthesis , Amides/pharmacology , Cysteine/chemistry , Enzyme Activation/drug effects , Enzyme Inhibitors/chemical synthesis , Humans , Jurkat Cells , Kinetics , Phosphorylation , Protein Methyltransferases/metabolism , Structure-Activity Relationship , ras Proteins/metabolismABSTRACT
Prenylcysteine derivatives are of interest for a variety of different biological reasons, including probing the CaaX protein processing pathway. A solid-phase synthesis protocol for the preparation of prenylcysteines using 2-chlorotrityl chloride resin as a solid support has been developed. A series of novel amide-modified farnesylcysteine analogs were synthesized in both high purity and yield under mild conditions. The farnesylcysteine analogs were evaluated using human isoprenylcysteine carboxyl methyltransferase as a biological target, and several new inhibitors, one with significantly enhanced potency, were identified.
Subject(s)
Cysteine/analogs & derivatives , Protein Methyltransferases/chemistry , Trityl Compounds/chemistry , Amino Acid Sequence , Chromatography, High Pressure Liquid , Cysteine/chemical synthesis , Cysteine/chemistry , Humans , Molecular Structure , Protein Methyltransferases/metabolism , Solid Phase ExtractionABSTRACT
N-Acetyl-S-farnesyl-L-cysteine (AFC) is the minimal substrate for the enzyme isoprenylcysteine carboxyl methyltransferase (Icmt). A series of amide-modified farnesylcysteine analogs were synthesized and screened against human Icmt. From a 23-membered library of compounds, six inhibitors were identified and evaluated further. The adamantyl derivative 7c was the most potent inhibitor with an IC(50) of 12.4 microM.
