1 research outputs found
Exploration of the Design Principles of a Cell-Penetrating Bicylic Peptide Scaffold
Cell-penetrating peptides (CPPs)
possess the capacity to induce
cell entry of themselves and attached molecular cargo, either by endocytosis
or by direct translocation. Conformational constraints have been described
as one means to increase the activity of CPPs, especially for direct
crossing of the plasma membrane. Here, we explored the structureāactivity
relationship of bicyclic peptides for cell entry. These peptides may
be considered minimal analogues of naturally occurring oligocyclic
peptide toxins and are a promising scaffold for the design of bioactive
molecules. Increasing numbers of arginine residues that are primarily
contributing to cell-penetrating activity were introduced either into
the cycles, or as stretches outside the cycles, at both ends or at
one end only. In addition, we probed for the impact of negatively
charged residues on activity for both patterns of arginine substitution.
Uptake was investigated in HeLa cells by flow cytometry and confocal
microscopy. Overall, uptake efficiency showed a positive correlation
with the number of arginine residues. The subcellular distribution
was indicative of endocytic uptake. One linear stretch of arginines
coupled outside the bicycle was as effective in promoting uptake as
substituting the same number of arginines inside the bicycles. However,
the internally substituted analogues were more sensitive to the presence
of negatively charged residues. For a given bicyclic peptide, uptake
was more effective than for the linear counterpart. Introduction of
histidine and tryptophans further increased uptake efficiency to comparable
levels as that of nonaarginine despite the larger size of the bicyclic
backbone. The results demonstrate that both arginine clustering and
spatial constraints are uptake-promoting structural principles, an
observation that gives freedom in the introduction of cell-penetrating
capacity to structurally constrained scaffolds