Click to enter: activation of oligo-arginine cell-penetrating peptides by bioorthogonal tetrazine ligations.

Cell-penetrating peptides are able to transport a wide variety of cargo across cell membranes. Although promising, they are not often considered for therapeutic purposes as they lack controllable activity and cell selectivity. We have developed an activation strategy based on a split octa-arginine cell-penetrating peptide (CPP) that can be activated by means of bioorthogonal ligation. To this end we prepared two non-penetrating tetra-arginine halves, functionalized either with a tetrazine or with a complementary bicyclo[6.1.0]nonyne (BCN) group. We demonstrate that an active octa-arginine can be reconstituted in situ upon mixing the complementary split peptides. The resulting activated peptide is taken up as efficiently as the well-established cell-penetrating peptide octa-arginine. The activation of the oligo-arginines can also be achieved using trans-cyclooctene (TCO) as a ligation partner, while norbornene appears too kinetically slow for use in situ. We further show that this strategy can be applied successfully to transport a large protein into living cells. Our results validate a promising first step in achieving control over cell penetration and to use CPPs for therapeutic approaches.

Coiled-Coil-Mediated Activation of Oligoarginine Cell-Penetrating Peptides.

A supramolecular approach was undertaken to create functionally activatable cell-penetrating peptides. Two tetra-arginines were assembled into an active cell-penetrating peptide by heterodimerizing leucine zippers. Three different leucine-zipper pairs were evaluated: activation was found to depend on the association constant of the coiled-coil peptides. The weaker-binding peptides required an additional disulfide linkage to induce cell-penetrating capability, whereas for the most-stable coiled-coil no additional stabilization was needed. The latter zipper pair was used to show that the induced formation of the coiled coils allows control over the uptake of an oligoarginine CPP-conjugated cargo protein.

Enzyme-Activatable Cell-Penetrating Peptides through a Minimal Side Chain Modification.

Activatable cell-penetrating peptides are of great interest in drug delivery because of their enhanced selectivity which can be controlled by the external stimuli that trigger their activation. The use of a specific enzymatic reaction to trigger uptake of an inert peptide offers a relevant targeting strategy because the activation process takes place in a short time and only in areas where the specific cell surface enzyme is present. To this aim, the lysine side chain of Tat peptides was modified with an enzyme-cleavable domain of minimal size. This yielded blocked Tat-peptides which were inactive but that could be activated by coincubation with the selected enzymes.

Activation of cell-penetrating peptides by disulfide bridge formation of truncated precursors.

A small library of oligoarginine peptides equipped with terminal cysteines was studied with respect to their cell-penetrating properties. The peptides themselves were inactive but gained the ability to enter cells upon extension of their sequence through disulfide bridge formation.

Self-assembling mini cell-penetrating peptides enter by both direct translocation and glycosaminoglycan-dependent endocytosis.

A small library of cell-penetrating peptides (CPPs) containing a minimized cationic domain and a lipophilic domain of different size was studied. CPPs that could self-assemble were found to enter cells more efficiently, triggering a glycosaminoglycan-dependent pathway.

Reactions inside nanoscale protein cages.

Chemical reactions are traditionally carried out in bulk solution, but in nature confined spaces, like cell organelles, are used to obtain control in time and space of conversion. One way of studying these reactions in confinement is the development and use of small reaction vessels dispersed in solution, such as vesicles and micelles. The utilization of protein cages as reaction vessels is a relatively new field and very promising as these capsules are inherently monodisperse, in that way providing uniform reaction conditions, and are readily accessible to both chemical and genetic modifications. In this review, we aim to give an overview of the different kinds of nanoscale protein cages that have been employed as confined reaction spaces.