Peptides Acting as Efficient Shuttles for Active Substances
- Fig. 1: Potential cellular uptake pathways of CPP-cargo constructs. (1) A first step might be the interaction with negatively charged glycosaminoglycans or phospholipids at the outer plasma membrane surface leading to the initiation of different entry processes. CPP-cargo conjugates or CPP-cargo complexes enter cells either by endocytosis (2) or direct transduction (3). However, direct entry is mainly observed when small cargos are delivered. (CPP-cv: covalent CPP-cargo conjugate; CPP-nc: non-covalent CPP-cargo complex)
- Fig. 2: Peptide conjugates are generated by direct attachment of the reporter group or drug to the peptide still immobilized on the solid support. Cleavage by trifluoroacetic acid releases the peptide conjugate.
- Fig. 3: Different cell assays are used to determine cell toxicity (left) or to investigate cellular uptake (middle). (right) For 64Cu-labeled NIA-sC18 conjugates significant uptake in the tumor regions of tumor-bearing mice was demonstrated by using small animal positron emission tomography (PET) imaging or ex vivo autoradiography.
Peptides are an integral part of many biological processes and perform a wide range of essential functions in the human body. For example they play important roles as hormones, as well as antibiotics. In this work, we describe how peptides are used in a different way, namely as efficient transporters for biological active molecules.
The cellular membrane constitutes an effective barrier that protects all living cells from the surrounding environment. It consists of a lipid bilayer that is semi-permeable only for ions and small molecules but not for large, polar and charged molecules. In recent years, pharmaceutical research has provided new perspectives by the identification of novel therapeutic substances. However, a major obstacle of many biologically active molecules is their incapacity to cross cellular membranes due to their charge, size or hydrophilicity. One additional factor limiting their bioavailability is based on low solubility and/or stability in aqueous solutions. Thus, much attention has been paid on circumventing all these drawbacks and to increase particularly the effective intracellular delivery, one of the keystones in therapeutic development.
For more than two decades now, so-called cell-penetrating peptides (CPPs) have been extensively studied as a promising tool for the intracellular delivery of various kinds of (therapeutic) molecules. CPPs exist in length of 5 to 40 amino acids. The structural features among this family of peptides are highly diverse including amphipathic α-helices, β-strands or random coil secondary structures. Importantly, they all share the ability of an autonomous cell entry in a wide variety of different cell types, including mammalian cells, protists, bacteria, fungi, and also plant cells. Thereby they are able to transport covalently or non-covalently attached cargos inside the cell interior, and thus the use of CPPs as drug transporters is also known as the “Trojan horse approach”. The spectrum of attached cargos encompasses a wide range of different compounds like small organic molecules, peptides and proteins, nucleic acids, and nanoparticles.
The cellular mechanisms behind CPP translocation are still not fully understood and a matter of extensive debate. Based on the positively charged nature of most of these sequences, the interaction with negatively charged membrane constituents is most probably the first step. The enrichment and simultaneous aggregation of CPPs at the outer membrane surface then triggers different processes of cell entry, e.g. direct translocation or endocytosis. Indeed, endocytotic processes are observed in most cases when cargos are attached to the CPPs. Examples of widely used CPPs are Tat or penetratin, that both derive from natural occurring protein transduction domains; transportan, that is a chimera composed out of two different sources; or the polyarginine sequences (Arg5-9) that are of total artificial design.
sC18 – a Promising New CPP
In our group we recently developed the CPP named sC18 as a potent drug transporter. sC18 consists of 16 amino acids and is derived from the C-terminal domain of the cationic antimicrobial peptide CAP18, that belongs to the family of cathelicidines. Based on its secondary structure it can be classified as a secondary amphipathic CPP. For this class of peptides accumulation and binding near to anionic regions of the cell membrane is assumed, inducing then the processes of peptide internalization. However, when sC18 is attached to cargos, endocytosis is observed as main cellular entry pathway. During the last years, we continuously optimized sC18 with regard to uptake efficiency and proteolytic stability. Furthermore, we investigated the synthesis and biological activity of CPP-drug conjugates consisting of sC18 and metal complex-containing drugs, or other small molecule drugs, as well as radioactive probes.
Synthesis of CPP-cargo Conjugates
Synthesis of peptides in the laboratory is usually done by solid phase peptide synthesis (SPPS) with the help of an automated peptide synthesizer. Making use of a solid support (called resin), the amino acids (aa) are coupled in a stepwise manner from the C- to the N-terminus. This technique is based on the use of adequate protecting groups to circumvent side reactions, and activating reagents for coupling the carboxylic acid to the N-terminus of the other aa. In our lab we frequently run the Fmoc (fluorenylmethoxycarbonyl)/tBu (tert-butyl)-strategy, where N-terminally Fmoc-protected amino acids are activated by the addition of oxyma pure. After each coupling step the Fmoc-group is cleaved by addition of piperidine. When the final length of the peptide is reached, it is removed from the resin by concentrated trifluoroacetic acid. Simultaneously, all side chain protecting groups of the trifunctional amino acids are cleaved, too, releasing the fully unprotected peptide.
The covalent attachment of cargo molecules to the CPPs can be achieved either when the peptide is still attached to the solid support, or the cargo can be coupled to the peptide in solution, using in most cases chemoselective coupling strategies.
Synthesis and in vitro Testing of Metal-containing Peptide-Drug Conjugates
During the last years anti-proliferative organometallic complexes have come into focus of research based on exciting new activity spectra. In collaboration with Prof. Dr. Ulrich Schatzschneider (University of Würzburg) we investigated half-sandwich cyclopentadienyl tricarbonyl complexes containing manganese (cymantrene) or rhenium (cyrhetrene) as metal center. Accordingly, we synthesized metal-complex peptide conjugates made of sC18 and the respective metal complexes with the help of SPPS methods.
The biological properties were tested in several cancer cell lines using different cell assays for investigating cytotoxicity, apoptosis induction, etc. Fluorescently labeled variants of the peptide-drug conjugates were also generated, and cellular uptake was monitored by confocal laser scanning microscopy. Moreover, it is possible to quantify the intracellular uptake by flow cytometry methods. All investigated conjugates exhibited high cytotoxic activity against the tested cancer cell lines compared to the metal-complexes alone. This underscores the promising combination of CPPs with the used metal-containing drugs.
Multifunctional Peptide Platforms
One of the emerging fields of the last years is theranostics, the combination of therapeutics and diagnosis. In collaboration with Dr. Ralf Bergmann (Helmholtz-Zentrum Dresden-Rossendorf) we focused on the selective targeting of hypoxic tumor tissues by 2-nitroimidazole conjugated peptides. Hypoxia is one the characteristics of locally advanced solid tumors, where oxygen supply and consumption is typically unbalanced. Due to insufficient blood supply, hypoxic tumor cells receive lower amounts of anticancer drugs making them more drug resistant. Targeting hypoxic tumor regions is possible by 2-nitroimidazoles that are reduced intracellularly by an enzyme-mediated one-electron reaction. The resulting radical anions are not reoxidized in hypoxic cells. Thus, they undergo further reduction steps ultimately leading to linkage to macromolecules and accumulation in hypoxic tissue. By SPPS including orthogonal protecting group strategy we synthesized peptide conjugates that were composed of sC18, (2-(2-nitroimidazol-1-yl) acetic acid (NIA), and a radioactive label. The novel multifunctional conjugates were investigated in vivo in tumor bearing mice. Interestingly, increased uptake of these conjugates even in less well-perfused hypoxic regions of the tumor was demonstrated. This observation indicated a successful capture of the conjugate as a result of the attached NIA unit. With this new approach we created a targeted delivery system that is applicable to act both as an imaging agent and as a platform to deliver therapeutics through conjugation.
Peptides have come more and more into focus of pharmaceutical research because of their high selectivity, efficacy, and good tolerance. Doubtless, the application of cell-penetrating peptides is one of the future directions within the peptide field. Many approaches including CPPs in peptide drug conjugates have been described and present promising new alternative avenues to common biopharmaceuticals. Yet a certainly important aspect is the exploration of new design routes to eliminate probable loss of efficacy by low metabolic stability. Another challenge is to better understand their mechanism of action that would of course be the base for the development of more active CPPs and CPP cargo constructs.
More articles on drug discovery:
 T. Skotland, T. G. Iversen, M. L. Torgersen, K. Sandvig, Cell-penetrating peptides: possibilities and challenges for drug delivery in vitro and in vivo, Molecules 20, 13313-13323 (2015) – DOI: 10.3390/molecules200713313
 S. Reissmann, Cell penetration: scope and limitations by the application of cell-penetrating peptides, J. Pept. Sci. 760-784 (2014) – DOI: 10.1002/psc.2672
 I. Neundorf, R. Rennert, J. Hoyer, F. Schramm, K. Löbner, I. Kitanovic, S. Wölfl, Fusion of a short HA2-derived peptide sequence to cell-penetrating peptides improves cytosolic uptake, but enhances cytotoxic activity, Pharmaceuticals 2, 49-65 (2009) – DOI: 10.3390/ph2020049
 M. Horn, F. Reichart, S. Natividad-Tietz, D. Diaz, I. Neundorf, Tuning the properties of a novel short cell-penetrating peptide by intramolecular cyclization with a triazole bridge, Chem. Commun., published online 10 December 2015 – DOI: 10.1039/C5CC08938G
 J. Hoyer, U. Schatzschneider, M. Schulz-Siegmund, I. Neundorf, Dimerization of a cell-penetrating peptide leads to enhanced cellular uptake and drug delivery, Beilstein J. Org. Chem. 8, 1788-1797 (2012) – DOI: 10.3762/bjoc.8.204
 K. Splith, R. Bergmann, J. Pietzsch, I. Neundorf, Specific targeting of hypoxic tumor tissue with nitroimidazole-peptide conjugates, ChemMedChem 7, 57-61 (2012) – DOI: 10.1002/cmdc.201100401
 K. Splith K, W. Hu, U. Schatzschneider, R. Gust, I. Ott, L. A. Onambele, A. Prokop, I. Neundorf, Protease-activatable organometal-peptide bioconjugates with enhanced cytotoxicity on cancer cells, Bioconjugate Chem. 21, 1288-96 (2010) – DOI: 10.1021/bc100089z
1Institute of Biochemistry, University of Cologne, Cologne, Germany
Prof. Ines Neundorf
Institute of Biochemistry
University of Cologne