Cell Penetrating Peptides (CPPs)

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The efficient passage of drugs through the plasma membrane remains a major hurdle for drug delivery. Good cell uptake often requires the administration of high quantities of drugs in order to obtain the expected intracellular biological effect. Therefore, improving the translocation process across the plasma membrane will significantly reduce the quantity of drug to be administered, and the side effects on healthy tissues that are currently observed in most of the cases.

In the nineties, several proteins have been shown to translocate spontaneously through the plasma membrane when incubated in the extracellular medium. In particular, two of these molecules have been extensively studied in order to define the structural or sequence elements needed for the translocation:  the Tat protein from the HIV-1 virus [1, 2] and the Drosophila melanogaster Antennapedia homeodomain [3]. A study on the structure-activity relationship was carried out on these two small proteins of 101 and 60 amino acids, respectively, and the minimal domain needed for translocation was defined [4-6]. This corresponds to short sequences of 10 to 16 amino acids, thus opening the way to the chemical synthesis of different mutants and analogues that are called “Cell Penetrating Peptides” (CPPs) or “Protein Transduction Domains” (PTDs). However, since “non-natural” peptide sequences that are quite different from the conventional PTD have also been used as cell translocating units for drug delivery [7, 8], we prefer the acronym CPP for such family of peptides.

Both Tat and Antennapedia peptides contain several basic amino acids. The native Tat peptide is composed of several cationic amino acids, including 6 arginine and 2 lysine residues. Starting from this native composition, the potential of arginine homopeptides to promote cellular uptake has been rapidly realized [9, 10], as it was demonstrated that the arginine-rich peptide is more efficient than the other cationic (i.e. poly-lysine, poly-histidine or poly-ornithine) homopolymers [11]. The strong impact of arginine residues has been described by Futaki’s group [10, 12] and further investigated by Rothbard and Wender, and others, who performed a systematic replacement of arginine residues with alanine residues [11, 13]. Such substitutions induced a strong reduction of peptide uptake that was directly correlated with the number of substituted arginines. Therefore, Rothbard et al. proposed that a bidentate hydrogen-bonding interaction between the guanidinium group of arginine residues and phosphate groups in the membrane [14] is implicated in the mechanism of translocation.

Surprisingly, the role of cationic amino acids in the Antennapedia peptide has not been so extensively studied, whereas the influence of the tryptophane residues has been comprehensively investigated [4]. In line with this, a peptide made of arginine and tryptophane residues only and showing an efficient translocating potency has been recently designed [15].

Altogether, the studies on Tat and Antennapedia peptides represent more than 75% of the published work on CPPs (for reviews, see [16] and [17]), and in the last two years this percentage even increased with several publications being reported weekly in the literature. The Antennapedia peptide has been also marketed as “Penetratin”. Under this commercial version, an activated group sensitive to nucleophilic attack by a sulfhydryl function conveniently allows the spontaneous formation of a disulfide bridge between any cargo molecule and “Penetratin”. At the time of writing this review however, only 172 results appeared in Medline for the keyword “Penetratin”. Most of them are fundamental studies mainly about the entry mechanism of CPPs or the biological evaluation of a coupled drug, whereas only a little number concerns clinical applications. It is noteworthy to consider that a stable covalent linkage has to be formed between CPP and cargo to allow translocation, at least for Tat, Antennapedia, or poly-Arg peptides, although a couple of publications also reported a surprising efficacy upon simple mixing with the cargo entities [18, 19]. Similarly, another CPP, Pep-1, which has been marketed as “Chariot” [20], can induce internalization of a cargo molecule just by being mixed with it [21]. A very little number of references on “Chariot” can be however found in the literature despite its apparent ease of use. As for other CPPs, the debate about the mechanism of entry of “Chariot” is still ongoing. Although being described initially as energy-independent, further works have proposed different mechanisms, such as the association of helices [22] or the formation of discrete nano-particles [23]. Controversies about the formation of pores through the membrane have also been reported [24, 25].