ASSESSING DELIVERY OF LIPOPEPTIDES INTO THE CYTOPLASM OF INTACT-CELLSBY A FUNCTIONAL ASSAY BASED ON PKC INHIBITION .1. THE JURKAT MODEL

We have developed a functional assay to verify the delivery into the c ytoplasm of a 14-amino acid hydrophilic peptide, modified by the incor poration of a palmitoyl-lysine residue into the N- or C-terminal end. This assay is based on the use of a pseudosubstrate sequence for the p rotein kinase C (PKC) isoenzymes alpha and beta for the quantification of PKC-mediated tumor necrosis factor (TNF) secretion after T-cell ac tivation by phorbol eater and anti-CD3 MAb. This cellular assay is sim ple, and it allows for a rapid and comparative study of several peptid es. The lipidic analogues of the pseudosubstrate peptide were able to inhibit TNF secretion in intact-activated Jurkat cells, with an EC(50) in the 40-60 mu M range, whereas the unmodified peptide was not activ e. Two control lipopeptides were also inactive, demonstrating that the palmitoyl-lysine group had no effect by itself. This study confirms t hat the modification of a relatively long peptide by the insertion of a palmitoyl-lysine into the N- or C-terminal end is sufficient to allo w entry into intact cells.