Membrane permeation of arginine-rich cell-penetrating peptides independent of transmembrane potential as a function of lipid composition and membrane fluidity

Cell-penetrating peptides (CPPs) are prominent delivery vehicles to confer cellular entry of (bio-) macromolecules. Internalization efficiency and uptake mechanism depend, next to the type of CPP and cargo, also on cell type. Direct penetration of the plasma membrane is the preferred route of entry as this circumvents endolysosomal sequestration. However, the molecular parameters underlying this import mechanism are still poorly defined. Here, we make use of the frequently used HeLa and HEK cell lines to address the role of lipid composition and membrane potential. In HeLa cells, at low concentrations, the CPP nona-arginine (R9) enters cells by endocytosis. Direct membrane penetration occurs only at high peptide concentrations through a mechanism involving activation of sphingomyelinase which converts sphingomyelin into ceramide. In HEK cells, by comparison, R9 enters the cytoplasm through direct membrane permeation already at low concentrations. This direct permeation is strongly reduced at room temperature and upon cholesterol depletion, indicating a complex dependence on membrane fluidity and microdomain organisation. Lipidomic analyses show that in comparison to HeLa cells HEK cells have an endogenously low sphingomyelin content. Interestingly, direct permeation in HEK cells and also in HeLa cells treated with exogenous sphingomyelinase is independent of membrane potential. Membrane potential is only required for induction of sphingomyelinase-dependent uptake which is then associated with a strong hyperpolarization of membrane potential as shown by whole-cell patch clamp recordings. Next to providing new insights into the interplay of membrane composition and direct permeation, these results also refute the long-standing paradigm that transmembrane potential is a driving force for CPP uptake

A critical assessment of the synthesis and biological activity of p53/Hdm2 stapled peptide inhibitors

The covalent linkage of amino acid side chains to conformationally constrain -helices has been employed as a powerful mean to enhance the activity of peptides that interact with a target protein in a helical conformation. These staples are also supposed to change the pharmacokinetics of the molecules and promote cell entry including cytoplasmic targeting. In particular, stapled peptides inhibiting the interaction of p53 with the human double minute 2 (Hdm2) protein were of interest for this approach. Here, we scrutinized to which degree the pharmacokinetic characteristics are a function of the staple and differ from those of a standard cationic cell-penetrating peptide (CPP). Stapled peptides and their linear counterparts were synthesized to verify activity in biochemical and cellular assays. All peptides showed potent sub-nanomolar potency to Hdm2. Assessing uptake for carboxyfluorescein-labeled variants, for short incubation times, there was only little difference in uptake efficiency for the stapled peptides and their linear counterpart and both were taken up less efficiently than the prototypic CPP nonaarginine (R9). Fluorescence was restricted to vesicular structures. Only following long-term incubation, and for SJSA-1 cells expressing the Hdm2 target protein, the stapled peptides and also the linear counterparts, albeit to a lesser degree, showed an enhanced cytoplasmic and nuclear accumulation. For HeLa cells, lacking target expression no such accumulation was observed. These findings demonstrate that the cytosolic and nuclear accumulation are not an intrinsic property of the stapled peptide but result from capture by the target Hdm2 once leaking out of the endo-lysosomal compartment

Exploration of the Design Principles of a Cell-Penetrating Bicylic Peptide Scaffold

Cell-penetrating peptides (CPPs) possess the capacity to induce cell entry of themselves and attached molecular cargo, either by endocytosis or by direct translocation. Conformational constraints have been described as one means to increase the activity of CPPs, especially for direct crossing of the plasma membrane. Here, we explored the structure–activity relationship of bicyclic peptides for cell entry. These peptides may be considered minimal analogues of naturally occurring oligocyclic peptide toxins and are a promising scaffold for the design of bioactive molecules. Increasing numbers of arginine residues that are primarily contributing to cell-penetrating activity were introduced either into the cycles, or as stretches outside the cycles, at both ends or at one end only. In addition, we probed for the impact of negatively charged residues on activity for both patterns of arginine substitution. Uptake was investigated in HeLa cells by flow cytometry and confocal microscopy. Overall, uptake efficiency showed a positive correlation with the number of arginine residues. The subcellular distribution was indicative of endocytic uptake. One linear stretch of arginines coupled outside the bicycle was as effective in promoting uptake as substituting the same number of arginines inside the bicycles. However, the internally substituted analogues were more sensitive to the presence of negatively charged residues. For a given bicyclic peptide, uptake was more effective than for the linear counterpart. Introduction of histidine and tryptophans further increased uptake efficiency to comparable levels as that of nonaarginine despite the larger size of the bicyclic backbone. The results demonstrate that both arginine clustering and spatial constraints are uptake-promoting structural principles, an observation that gives freedom in the introduction of cell-penetrating capacity to structurally constrained scaffolds