Cell-Permeant Fluorescently-labelled Peptides

Peptide-based fluorescent reporters of enzyme activity in living cells have been proposed to complement, or provide alternatives to, genetically-encoded probes, such as those based on the Green Fluorescent Protein and its colourful variants. The use of well-established chemical methodologies – especially solid phase synthesis – and the vast array of fluorescent dyes available allow the preparation and the evaluation of compounds with different specificities, making a wide range of investigations possible. However, cell uptake of these compounds is generally poor, and normally requires invasive methods such as microinjection or electroporation. In these conditions, the usefulness of these probes can be severely limited. The discovery of the so-called cell-penetrating peptides (CPPs), more than ten years ago, has actually revealed that some of these compounds can cross efficiently the cell membranes and are able to deliver hydrophilic cargoes, such as DNA, proteins or even nanoparticles inside the cell. Unfortunately, much of the initial enthusiasm dissipated when it was found that endocytosis plays a major role in the uptake process. The entrapment in endosomal vesicles may in fact impose strict limits in terms of modification and degradation of the cargo of interest. This thesis describes the synthesis of fluorescent peptides and focuses on the problem of their delivery into the cytoplasm of living cells. A negatively charged peptide corresponding to one of the autophosphorylation sequences of the epithelial grow factor receptor (EGFR), namely DADEY992L, was taken as an example to investigate the possibility of creating a cell-permeable fluorescent sensor for the EGFR kinase activity in living cells. The use of a conjugate of the cell-penetrating peptide Penetratin and the probe revealed an endosomal localization in one cell type, and surprisingly no uptake in a second cell line tested. Different fluorescent derivatives of Penetratin demonstrated variations in cell uptake under different experimental conditions. During this study, red fluorescent derivatives of the cell-penetrating TAT peptide were also assessed. Although they also showed predominantly accumulation in membrane-bound compartments, these appeared to be different from the endosomal vesicles. One TAT variant in particular, namely TAT(K-LRh), may be potentially exploited as a novel vector, provided that further studies will clarify unambiguously the compartment of its accumulation. To overcome the limits inherent in the use of Penetratin conjugates, a different strategy was implemented, which relied on the chemical modification of the probe with bioactivatable protecting groups. The rationale behind these modifications was to increase the hydrophobic character of the peptide and allow its diffusion into the cells. In particular, acetoxymethyl (AM) esters were used to temporarily mask the negative charges of the aspartate and glutamate residues present in the peptide sequence. These biodegradable esters are promptly removed by ubiquitous non-specific esterases present in cells. The modified fluorescent peptide was able to penetrate easily and distribute homogeneously into the cytoplasm and the nucleus of several cell lines, with a mechanism resembling passive diffusion. Unfortunately, FRET-based assays did not measure any interaction with the EGFR. In vitro analyses showed unmasking of the carboxylate groups, albeit with modifications to the peptide backbone, which may have resulted from intramolecular condensations and formation of aspartimide residues. In particular, mass spectrometry data support this model. A loss of substrate specificity may be expected to result from such modifications, which may explain why the fluorescent peptide did not appear to interact with its enzyme target despite intracellular delivery having been achieved. This work highlights the feasibility of a tailored modification of peptides, either by masking negatively charged groups as, for example, AM esters, or by conjugating to variant of the TAT peptide, for the intracellular delivery of peptidic cargoes. Further refinements may solve the problems of poor endosomal escape or backbone modifications, and may yet offer small molecule alternatives to genetically encoded probes