Here we describe an entirely new class of cell-penetrating peptide (CPP) represented by the short peptide Xentry (LCLRPVG) derived from an N-terminal region of the X-protein of the hepatitis B virus. Xentry permeates adherent cells using syndecan-4 as a portal for entry, and is uniquely restricted from entering syndecan-deficient, non-adherent cells, such as resting blood cells. Intravenous injection of Xentry alone or conjugated to β-galactosidase led to its delivery to most tissues in mice, except circulating blood cells. There was a predilection for uptake by epithelia. Anti-B-raf antibodies and siRNAs linked to Xentry were capable of killing B-raf-dependent melanoma cells. Xentry represents a new class of CPP with properties that are potentially advantageous for life science and therapeutic applications.
Here we describe a structure-function analysis of the cell-penetrating peptide Xentry derived from the X-protein of the hepatitis B virus. Remarkably, the tetrapeptide core LCLR retains the cell-penetrating ability of the parental peptide LCLRPVG, as either an L- or D-enantiomer. Substitution of the cysteine with leucine revealed that the cysteine is essential for activity. In contrast, the C-terminal arginine could be substituted in the L-isomer with lysine, histidine, glutamic acid, glutamine, and asparagine, though the resulting peptides displayed distinct cell-type-specific uptake. Substitution of the leucines in the D-isomer with other hydrophobic residues revealed that leucines are optimal for activity. Surprisingly, linear di- and tetra-peptide forms of Xentry are not cell-permeable. Protease-activatable forms of Xentry were created by fusing Xentry to itself via a protease-cleavable peptide, or by attaching a heparin mimic peptide to the N-terminus. These novel activatable forms of Xentry were only taken up by MCF-7 cells after cleavage by matrix metalloproteinase 9, and could be used to deliver drugs specifically to tumours.
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