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

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.