The tetrapeptide core of the carrier peptide Xentry is cell-penetrating: novel activatable forms of Xentry

Abstract

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.

Intracellular oligonucleotide delivery using the cell penetrating peptide Xentry

“…Therefore, the use of CPPs to deliver Cx43AsODN could have significant therapeutic advantages. A new class of CPP, Xentry, derived from the X-protein of the hepatitis B virus has previously been used to successfully transport large molecules such as peptides, antibodies as well as smaller molecules such as siRNA into cells with high efficiency 32 , 33 . An added advantage of Xentry is that it binds to cell surface expressed Syndecan-4 to initiate clathrin-mediated endocytosis 33 .…”

Organic Nanocarriers for Delivery and Targeting of Therapeutic Agents for Cancer Treatment

“…Furthermore, the residue cysteine was the most pivotal for the bioactivity of Xentry which cannot be substituted. On the other hand, the C‐terminal arginine could be substituted by other residues; however, the modified tetrapeptides showed different cellular uptake profiles …”

“…On the other hand, the C-terminal arginine could be substituted by other residues; however, the modified tetrapeptides showed different cellular uptake profiles. [401] Similar to the concept of ACPPs, the cell-penetrating activity of TAT peptide could be manipulated to specifically target cancer cells by blocking TAT with certain enzyme-responsive peptides. Liu et al prepared liposomes through the attachment of alaninealanine-asparagine (AAN) to the fourth Lys on the TAT peptide which leads to decrease in the transmembrane capacity of TAT by 72.65%.…”

Inhibition of regulated cell death by cell-penetrating peptides

“…Negligible permeability of these drugs across the intestinal barrier and low pH conditions of the gastrointestinal tract reduce the delivery of intact agents to the target and add to the short half-life and low bioavailability of CPPs. In this regard, it is worth noting that Xentry [ 119 ], as already mentioned, represents a new class of cell-penetrating peptides that is not able to invade resting blood cells, which offers a therapeutic advantage when the drug must be administrated intravenously. Furthermore, nano-carrier-based delivery represents an appropriate choice to significantly improve uptake of CPPs and protect cargo from proteolytic degradation with the intention of increasing the retention time of these molecules in the body, leading to a reduction of treatment cost [ 120 ].…”