An Intrinsically Disordered Peptide Facilitates Non‐Endosomal Cell Entry

Abstract

Many cell penetrating peptides (CPPs) fold at cell surfaces, adopting α- or β-structure that enable their intracellular transport. However, the same structural folds that facilitate cellular entry can also elicit potent membrane-lytic activity limiting their use in delivery applications. Further, a distinct CPP can enter cells via many mechanisms, often leading to endosomal entrapment. Here, we describe an intrinsically disordered peptide (CLIP6) that exclusively employs non-endosomal mechanisms to cross cellular membranes, while being remarkably biocompatible and serum stable. We show the presence of a single anionic glutamate residue is responsible for maintaining the peptide’s disordered bioactive state, defines its mechanism of cellular entry and is central to its biocompatibility. CLIP6 can deliver membrane impermeable cargo directly to the cytoplasm of cells, suggesting its broad utility for delivery of drug candidates limited by poor cell permeability and endosomal degradation.

Conformationally constrained peptides for drug delivery

“…This suggests that the activity of CHAP peptides is unaffected by serum proteins and other components. A possible explanation for such an observation could be that the amino acid, Aib, in the sequence of CHAPs, protects the designed peptides from proteolytic cleavage, similar to the observations reported previously while incorporating other unnatural amino acids in CPP sequences …”

“…10,11 The rearrangement of secondary structure results in the formation of a definite secondary amphipathicity profile and spatial charge distribution, unique to each peptide model, which can be envisaged as spatial fingerprints responsible for the permeation of the cell membrane. Though disordered peptides can also penetrate live cell membranes, 12 the designability of such intrinsically disordered peptides as CPPs is limited. 13 The uncertainty in adopting a specific fold further adds to the obstacles for designing CPPs with tailored functions.…”

“…presence of serum/treatment of peptides with serum has been reported to decrease the activity of CPPs, due to their instability in biological fluids. Use of unnatural amino acids is known to increase the stability of peptides in biological systems; however, the stability in biological system may not warrant that the activity of peptides is conserved 3,12,41. Therefore, to test the biocompatible activity of the CHAP peptides in bovine serum, we treated CHAP-CF conjugates with bovine serum for 1 hour at 37 C. MDA-MB-231 cells were treated with same stock of CHAP-CF conjugates for 4 hours at 37 C and analyzed for cell associated fluorescence using flow cytometry.…”

Where in the Cell Is our Cargo? Methods Currently Used To Study Intracellular Cytosolic Localisation

“…The delivered compound (e.g., small peptide) has to be tagged with the corresponding molecular ligand (tamoxifen or 4‐hydroxycyclofen). Therefore, when the ligand‐tagged molecule reaches the cytosol, it triggers the Cre nuclear translocation and thus recombines the reporter sequence …”

“…Therefore, when the ligandtaggedm olecule reaches the cytosol, it triggerst he Cre nucleart ranslocation and thus recombines the reporter sequence. [101] As econd, different methodi sb ased on the glucocorticoid receptor( GR) and its ligand dexamethasone. Twod ifferent assays have been devised:g lucocorticoid-induced eGFP induction (GIGI) and glucocorticoid-induced eGFP translocation (GIGT,S cheme 3D).…”