The Import Mechanism of Cationic Cell-Penetrating Peptides and Its Implications for the Delivery of Peptide Inhibitors of Signal Transduction

Inhibitors of Signal Transduction .............................................................. 170

9.3.1 Sequestration and Breakdown in the

Endosomal Compartment .............................................................. 170

9.3.2 Proteolytic Breakdown in the Cytosol .......................................... 170

9.3.3 Strategies to Induce Efficient Endosomal Release ........................ 171

9.3.4 Interference with Cellular Signal Transduction ............................ 172

9.4 Experimental Design ................................................................................ 174

9.4.1 The Nature of the CPP .................................................................. 174

9.4.2 Experimental Approaches to Analyze the Endocytic

Uptake of CPPs .............................................................................. 174

9.4.3 Monitoring Import and Distribution of CPPs .............................. 176

9.4.4 Cellular Factors .............................................................................. 176

9.5 Conclusions ................................................................................................ 176

Acknowledgments .............................................................................................. 177

References .......................................................................................................... 177

The import of peptide-based inhibitors of intracellular signal transduction pathways

has been one the earliest applications of cell-penetrating peptides (CPPs). Especially

where no low-molecular weight inhibitors are available, peptides that correspond to

pseudo-substrates or interaction motifs provide a short-cut to inhibitors by rational

design. Applications of such peptides range from tissue culture experiments to

therapeutic interventions in whole animals.1-3

The efficient cellular uptake of CPP-peptide conjugates by an uptake

mechanism that assumed direct permeation of the plasma membrane was a driving

force for the widespread application of these tools. The documented success of the

CPP-based delivery of inhibitors may serve as a justification for a rather uncritical

application of these molecules that little considered the cell biology of CPPs. Still

one must be surprised that even though detailed studies about the structure-activity

relationship of CPPs had been performed, demonstrating that import efficiency

strongly depended on the sequence of the CPP, until 2002 the impact of different

peptide cargoes on import efficiency had not been addressed quantitatively.4,5 It was

shown that import efficiency of fluorescein-labeled conjugates of peptides with the

CPP penetratin, as measured by intracellular fluorescence, was strongly affected by

even slight modifications of the cargo.6 Therefore, a quantitative control of import

efficiency is an absolute requirement for conducting analyses of structure-activity

relationships for different peptide conjugates inside the cell.7