Endocytic Delivery of Vancomycin Mediated by a Synthetic Cell Surface Receptor:  Rescue of Bacterially Infected Mammalian Cells and Tissue Targeting In Vivo

Abstract

Extracellular domains of internalizing cell surface receptors are often targeted to enable drug delivery through the mechanism of receptor-mediated endocytosis. To circumvent natural receptors required for endocytic drug delivery, we constructed a small artificial cell surface receptor comprising the membrane anchor N-alkyl-3β-cholesterylamine linked to a D-Phe-D-Ala motif that binds the glycopeptide antibiotic vancomycin. By mimicking membrane association and trafficking properties of cholesterol, this cholesterol-derived synthetic receptor functions as a prosthetic molecule, inserting into plasma membranes of mammalian cells, and rapidly cycling between the cell surface and intracellular endosomes. Human HeLa cells treated with this receptor gained the ability to internalize vancomycin by endocytosis, enabling this cell-impermeable antibiotic to eradicate the lethal intracellular pathogen Listeria monocytogenes, and preserving the viability of the host cells in vitro. Moreover, when combined with the synthetic receptor, vancomycin penetrated the blood brain barrier, accumulating in the brain and other tissues of mice in vivo. Because of their ability to define new pathways across biological membrane barriers, small synthetic cell surface receptors represent promising tools for drug delivery.Receptor-mediated drug delivery is a Trojan horse approach for the release of therapeutics into cells, tissues, or organs. By coupling drugs to ligands of cell surface receptors, ligand-drug conjugates can accumulate in specific tissues and can be internalized by cells through the mechanism of receptor-mediated endocytosis. 1 Receptors targeted in this way include the low density lipoprotein (LDL) receptor, 2 transferrin receptors, 3 and folate receptors. 4 Receptors involved in the passage of nutrients across the blood-brain barrier through the related mechanism of receptor-mediated transcytosis have been similarly targeted to deliver drugs and imaging agents into the brain. 5 This latter strategy is of significant interest because less than 2% of all drugs are capable of penetrating the blood-brain barrier. 5We report here synthetic receptor-mediated drug delivery. This approach was applied to the glycopeptide antibiotic vancomycin (1, Figure 1, Panel A), a drug of last resort against bacterial pathogens such as methicillin-resistant Staphylococcus aureus. 6 By binding to peptidoglycan precursors that terminate in the peptide sequence D-Ala-D-Ala, vancomycin (1) blocks crosslinking of the bacterial cell wall, providing an effective mode of action against gram-positive bacteria. However, the high polarity and consequent low cell permeability of glycopeptide E-mail: brpeters@chem.psu.edu. antibiotics render these drugs ineffective against bacteria capable of replicating either in the cytoplasm of mammalian host cells or in organs such as the brain that are protected by membrane barriers. NIH Public AccessWe hypothesized that a synthetic receptor capable of promoting the endocytosis of vancomycin (1) might allow this...

Lipophilic Lysine−Spermine Conjugates Are Potent Polyamine Transport Inhibitors for Use in Combination with a Polyamine Biosynthesis Inhibitor

“…A series of persulfated polyanionic molecular umbrellas based on spermine tetra-lysine conjugates have been evaluated as anti-HIV and anti-HSV agents 65. Peterson’s group has designed and characterized several series of cholesterol-linked of peptides as models for receptor-mediated endocytosis 66,67,68…”

Synthetic cell surface receptors for delivery of therapeutics and probes

“…To explore the potential utility of synthetic receptor-mediated drug delivery, termed synthetic receptor targeting, our laboratory designed[78] a cholesterylamine linked to the peptide sequence D-Phe-D-Ala. This dipeptide binds[79] the glycopeptide antibiotic vancomycin, a drug of last resort against gram positive pathogens such as methicillin-resistant Staphylococcus aureus .…”

“…Control compounds that do not bind vancomycin or that exhibit lower affinity for cellular plasma membranes did not show these beneficial effects, further supporting the hypothesis that sVancoR does not simply permeabilize cells, but rather binds vancomycin on cell surfaces and promotes endocytosis by mimicking the trafficking of natural cell surface receptors. [78]…”

Practical Synthesis of 3β-Amino-5-cholestene and Related 3β-Halides Involving i-Steroid and Retro-i-Steroid Rearrangements

“…N -Alkyl derivatives of 3β-amino-5-cholestene can insert into plasma membranes of living mammalian cells and cycle between the cell surface and early/recycling endosomes, mimicking the membrane trafficking of many cell surface receptors 10. These compounds are under investigation as tools for drug delivery,11 and when linked to endosome disruptive peptides12 can deliver molecules into the cytosol and nucleus of mammalian cells.…”