# Receptor-Mediated Shuttling of a D‑Amino Acid Peptide Achieves High Nanomolar Cytosolic Concentrations

## TL;DR

This study shows that receptor-mediated delivery can achieve high cytosolic concentrations of therapeutic peptides without needing endosomal escape strategies.

## Contribution

The study demonstrates that receptor-mediated shuttling can reach biologically meaningful cytosolic concentrations of peptides.

## Key findings

- Cytosolic concentrations of PMIγ over 100 nM were achieved via receptor-mediated shuttling.
- The EEP hsLMWP improved delivery but caused toxicity and was limited by rapid degradation.
- BRET confirmed PMIγ's binding to MDM2 and inhibition of the p53/MDM2 interaction.

## Abstract

Delivery of therapeutic peptides and proteins to the
cytosol is
of great interest due to their ability to inhibit intracellular protein–protein
interactions, which are mostly deemed undruggable by small molecules.
Internalization into the endosomal pathway is possible by receptor-targeted
approaches; however, endosomal escape is inefficient, and its quantification
is challenging. To improve our current understanding of cytosolic
delivery, we performed comprehensive studies on a receptor-mediated
shuttle system based on the chemokine-like receptor 1 (CMKLR1). As
a model cargo, PMIγ was used, a known D-amino acid peptide antagonist
of the MDM2/p53 interaction, which survives the harsh conditions in
the endocytic pathway. Fluorescence correlation spectroscopy (FCS)
was used to demonstrate that biologically meaningful cytosolic concentrations
(>100 nM) can be reached by receptor-mediated shuttling, even when
no endosomal escape enhancing strategies are used. Attachment of the
pH-responsive endosomal escape peptide (EEP) hsLMWP further improved
cytosolic delivery but also induced cellular toxicity at higher concentrations.
Additionally, the EEP activity was likely limited by its fast degradation
after internalization. Intracellular biological activity was confirmed
using bioluminescence resonance energy transfer (BRET) studies, which
demonstrate binding to MDM2 and inhibition of the p53/MDM2 interaction.
This study highlights the potential of receptor-mediated shuttling
for cytosolic delivery of therapeutic peptides and provides new insights
into achievable intracellular concentrations, advancing the field
of peptide therapeutics and drug delivery.