Delivery to, and Reactivation of, the p53 Pathway in Cancer Cells Using a Grafted Cyclotide Conjugated with a Cell-Penetrating Peptide

Peptides are promising drug modalities that can modulate
protein–protein
interactions, but their application is hampered by their limited ability
to reach intracellular targets. Here, we improved the cytosolic delivery
of a peptide blocking p53:MDM2/X interactions using a cyclotide as
a stabilizing scaffold. We applied several design strategies to improve
intracellular delivery and found that the conjugation of the lead
cyclotide to the cyclic cell-penetrating peptide cR10 was the most
effective. Conjugation allowed cell internalization at micromolar
concentration and led to elevated intracellular p53 levels in A549,
MCF7, and MCF10A cells, as well as inducing apoptosis in A549 cells
without causing membrane disruption. The lead peptide had >35-fold
improvement in inhibitory activity and increased cellular uptake compared
to a previously reported cyclotide p53 activator. In summary, we demonstrated
the delivery of a large polar cyclic peptide in the cytosol and confirmed
its ability to modulate intracellular protein–protein interactions
involved in cancer.