Assessing delivery of lipopeptides into the cytoplasm of intact cells by a functional assay based on PKC inhibition. I. The Jurkat model.

We have developed a functional assay to verify the delivery into the cytoplasm of a 14-amino acid hydrophilic peptide, modified by the incorporation of a palmitoyl-lysine residue into the N- or C-terminal end. This assay is based on the use of a pseudo-substrate sequence for the protein kinase C (PKC) isoenzymes of alpha and beta for the quantification of PKC-mediated tumor necrosis factor (TNF) secretion after T-cell activation by phorbol ester and anti-CD3 MAb. This cellular assay is simple, and it allows for a rapid and comparative study of several peptides. The lipidic analogues of the pseudo-substrate peptide were able to inhibit TNF secretion in intact-activated Jurkat cells, with an EC50 in the 40-60 microM range, whereas the unmodified peptide was not active. Two control lipopeptides were also inactive, demonstrating that the palmitoyl-lysine group had no effect by itself. This study confirms that the modification of a relatively long peptide by the insertion of a palmitoyl-lysine into the N- or C-terminal end is sufficient to allow entry into intact cells.