Cellular pharmacology of cerulenin analogs that inhibit protein palmitoylation

S-palmitoylation is a dynamic post-translational modification of certain proteins, which helps determine membrane association and may function to enhance the interactions of signaling molecules with their activated receptors and effector systems. Unlike enzymes that catalyze other protein lipidation reactions, e.g. farnesylation and N-myristoylation, protein palmitoyltransferase is virtually uncharacterized biochemically. We have described previously the synthesis of cerulenin analogs including cis-2,3-epoxy-4-oxononadecanamide (16C) and cis-2,3-epoxy-4-oxododecanamide (9C) that inhibit protein palmitoylation (Lawrence et al., J Med Chem 1999;42:4932-41), most likely through covalent alkylation of protein palmitoyltransferase. [3H]9C and [3H]16C were prepared by catalytic incorporation of 3H2 into unsaturated precursors for further study of their cellular pharmacology. After 4 hr, T24 bladder carcinoma cells in the absence of serum accumulated a 4-fold higher intracellular level of [3H]16C than of [3H]9C. Uptake of [3H]9C and [3H]16C was reduced by the presence of serum in the medium, suggesting their binding to serum proteins. [3H]9C and [3H]16C alkylated unique patterns of proteins in T24 cells, with proteins of approximately 80 and 31 kDa being labeled by each compound. A panel of human tumor cell lines demonstrated half-maximal proliferation inhibition at concentrations of 7-30, 4-16, and 8-36 microM, for cerulenin, 9C, and 16C, respectively, indicating that the cell lines have approximately equal sensitivity to these compounds. Different cell lines have similar patterns of protein alkylation by [3H]9C or [3H]16C, with labeling intensity related to cytotoxicity of the compounds. Since both 9C and 16C inhibit palmitoylation, the commonly labeled proteins are candidates for human protein palmitoyltransferase.