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.

Malaria remains a global health burden partly due to parasite resistance to first-line therapeutics. The molecular chaperone heat shock protein 90 (Hsp90) has emerged as an essential protein for blood-stage parasites, but details about its function during malaria's elusive liver stage are unclear. We used target-based screens to identify compounds that bind to and human Hsp90, which revealed insights into chemotypes with species-selective binding. Using cell-based malaria assays, we demonstrate that all identified Hsp90-binding compounds are liver- and blood-stage inhibitors. Additionally, the Hsp90 inhibitor SNX-0723 in combination with the phosphatidylinositol 3-kinase inhibitor PIK-75 synergistically reduces the liver-stage parasite load. Time course inhibition studies with the Hsp90 inhibitors and expression analysis support a role for Hsp90 in late-liver-stage parasite development. Our results suggest that Hsp90 is essential to liver- and blood-stage parasite infections and highlight an attractive route for development of species-selective Hsp90 inhibitors that may act synergistically in combination therapies to prevent and treat malaria.

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 through many mechanisms, often leading to endosomal entrapment. Herein, 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 that a single anionic glutamate residue is responsible for maintaining the disordered bioactive state of the peptide, 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.