S-Palmitoylation is a dynamic post-translational modification of certain pr
oteins, which helps determine membrane association and may function to enha
nce 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 palmitoyltrans
ferase is virtually uncharacterized biochemically. We have described previo
usly the synthesis of cerulenin analogs including cis-2,3-epoxy-4-oxononade
canamide (16C) and cis-2,3-epoxy-4-oxododecanamide (9C) that inhibit protei
n palmitoylation (Lawrence et al., J Med Chem 1999;42:4932-41), most likely
through covalent alkylation of protein palmitoyltransferase. [H-3]9C and [
H-3]16C were prepared by catalytic incorporation of H-3(2) into unsaturated
precursors for further study of their cellular pharmacology. After 4 hr, T
24 bladder carcinoma cells in the absence of serum accumulated a 4-fold hig
her intracellular level of [H-3]16C than of [H-3]9C. Uptake of [H-3]9C and
[H-3]16C was reduced by the presence of serum in the medium, suggesting the
ir binding to serum proteins. [H-3]9C and [H-3]16C alkylated unique pattern
s of proteins in T24 cells, with proteins of approximately 80 and 31 kDa be
ing labeled by each compound. A panel of human tumor cell lines demonstrate
d half-maximal proliferation inhibition at concentrations of 7-30, 4-16, an
d 8-36 muM, for cerulenin, 9C, and 16C, respectively, indicating that the c
ell lines have approximately equal sensitivity to these compounds. Differen
t cell lines have similar patterns of protein alkylation by [H-3]9C or [H-3
]16C, with labeling intensity related to cytotoxicity of the compounds. Sin
ce both 9C and 16C inhibit palmitoylation, the commonly labeled proteins ar
e candidates for human protein palmitoyltransferase. (C) 2001 Elsevier Scie
nce Inc. All rights reserved.