Structure-activity relationship of truncated and substituted analogues of the intracellular delivery vector Penetratin

Peptides derived from the third alpha-helix of the homeodomain (residues 43 -58; Penetratin) of Antennapedia, a Drosophila homeoprotein, were prepared by simultaneous multiple synthesis. Sets of N- and C-terminally truncated p eptides, as well as a series of alanine substitution analogues, were studie d. Cell penetration assays using human cell cultures with these peptides re vealed that the C-terminal segment (52)Arg-Arg-Met-Lys-Trp-Lys-Lys(58) Of t he parent sequence was necessary and sufficient for efficient cell membrane translocation. Individual Ala substitutions of the peptide's basic residue s led to markedly decreased cell internalization ability, whereas replaceme nt of hydrophobic residues was tolerated surprisingly well. Subcellular loc alization was seen to be affected by substitutions, with analogues being ad dressed preferentially to the cytosol or to the nucleus. Conformational con striction of the Penetratin sequence through placement and oxidation of fla nking cysteine residues afforded a cyclic disulfide peptide which had lost most of its membrane translocation capacity.