Cellular pharmacology of cerulenin analogs that inhibit
protein palmitoylation.
De Vos ML, Lawrence DS, Smith CD.
Department of Pharmacology, H078, Pennsylvania State University, College of
Medicine, 500 University Drive, Hershey, PA 17033-2390, USA.
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.
