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Manuscript and Figures v9 ChemRxiv.pdf (4.73 MB)
Cellular Uptake and Cytosolic Delivery of a Cyclic Cystine Knot Scaffold
Preprints are manuscripts made publicly available before they have been submitted for formal peer review and publication. They might contain new research findings or data. Preprints can be a draft or final version of an author's research but must not have been accepted for publication at the time of submission.
submitted on 16.02.2020 and posted on 19.02.2020by Huawu Yin, Yen-Hua Huang, Kirsten Deprey, Nicholas Condon, Joshua Kritzer, David Craik, Conan Wang
Cyclotides are macrocyclic peptides that have exceptionally stable structures and been reported to penetrate cells, making them promising scaffolds for the delivery of peptide inhibitory sequences to target intracellular proteins. However, their cellular uptake and cytosolic localization have been poorly understood until now, which has limited their therapeutic potential. In this study, the recently developed chloroalkane penetration assay was combined with established assays to characterize the cellular uptake and cytosolic delivery of the prototypic cyclotide, kalata B1. We show that kalata B1 enters the cytosol at low efficiency, but introducing various epitopes, including a single hydrophobic amino acid, into its loop 6 significantly improved its cytosolic delivery. Our results provide a foundation for the further development of a structurally unique class of scaffolds for the delivery of therapeutic cargoes into cells.