Quantitative Estimation of Cyclotide-Induced Bilayer Membrane Disruption by Lipid Extraction with Mesoscopic Simulation

Cyclotide-induced membrane disruption is studied at the microsecond timescale by Dissipative Particle Dynamics (DPD) to quantitatively estimate a kinetic rate constant for membrane lipid extraction with a “sandwich” interaction model where two bilayer membranes enclose a cyclotide/water compartment. The obtained bioactivity trends for cyclotides Kalata B1, Cycloviolacin O2 and selected mutants with different membrane types are in agreement with experimental findings: For all membranes investigated, Cycloviolacin O2 shows a higher lipid extraction activity than Kalata B1. The presence of cholesterol leads to a decreased cyclotide activity compared to cholesterol-free membranes. Phosphoethanolamine-rich membranes exhibit an increased membrane disruption. A cyclotide’s “hydrophobic patch” surface area is important for its bioactivity. A replacement of or with charged amino acid residues may lead to super-mutants with above-native activity but without simple charge-activity patterns. Cyclotide mixtures show linearly additive bioactivities without significant sub- or over-additive effects.<br>

Quantitative Estimation of Cyclotide-Induced Bilayer Membrane Disruption by Lipid Extraction with Mesoscopic Simulation

Cyclotide-induced membrane disruption is studied at the microsecond timescale by Dissipative Particle Dynamics (DPD) to quantitatively estimate a kinetic rate constant for membrane lipid extraction with a “sandwich” interaction model where two bilayer membranes enclose a cyclotide/water compartment. The obtained bioactivity trends for cyclotides Kalata B1, Cycloviolacin O2 and selected mutants with different membrane types are in agreement with experimental findings: For all membranes investigated, Cycloviolacin O2 shows a higher lipid extraction activity than Kalata B1. The presence of cholesterol leads to a decreased cyclotide activity compared to cholesterol-free membranes. Phosphoethanolamine-rich membranes exhibit an increased membrane disruption. A cyclotide’s “hydrophobic patch” surface area is important for its bioactivity. A replacement of or with charged amino acid residues may lead to super-mutants with above-native activity but without simple charge-activity patterns. Cyclotide mixtures show linearly additive bioactivities without significant sub- or over-additive effects.<br>

Cellular Uptake and Cytosolic Delivery of a Cyclic Cystine Knot Scaffold

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.<br>

Cellular Uptake and Cytosolic Delivery of a Cyclic Cystine Knot Scaffold

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.<br>