Targeted Delivery of a Ligand-Drug Conjugate via Formyl Peptide Receptor 1 through Cholesterol-Dependent Endocytosis

Article abstract of DOI:10.1021/acs.molpharmaceut.9b00188

G protein-coupled receptors (GPCRs) undergo ligand-induced internalization that carries the cognate ligands into intracellular compartments. The present study explores this property for the use of formyl peptide receptor 1 (FPR1), a class A GPCR that binds formylated peptides, as a potential target for drug delivery. A pH-sensitive peptide-drug conjugate consisting of doxorubicin (DOX), N-?-maleimidocaproic acid hydrazide (EMCH), and the formyl peptide fMet-Leu-Phe-Cys (abbreviated as DEF) was prepared. DEF retained pharmacological activities of formyl peptides in binding to FPR1 and mobilization of Ca²⁺ from intracellular stores. However, the conjugated DOX was no longer cell membrane-permeable and relied on FPR1 for cellular entry. DOX was released from DEF into acidic compartments labeled with fluorescent trackers for endosomes. Treatment of cells with pharmacological inhibitors that block clathrin- or caveolae-mediated endocytosis did not abrogate FPR1-dependent DEF internalization, nor did inhibition of macropinocytosis and phagocytosis. In contrast, cholesterol depletion abrogated DEF internalization through FPR1, suggesting characteristics of cholesterol-dependent uptake mediated by a cell surface receptor. These results demonstrate the possibility of using FPR1 for targeted drug delivery.

Full text of DOI:10.1021/acs.molpharmaceut.9b00188

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Article
Targeted delivery of a ligand-drug conjugate via formyl
peptide receptor 1 through cholesterol-dependent endocytosis
Junlin Wang, Meiwan Chen, Shaoping Li, and Richard D Ye
Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/
acs.molpharmaceut.9b00188 • Publication Date (Web): 08 May 2019
Downloaded from http://pubs.acs.org on May 9, 2019
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Molecular Pharmaceutics
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Targeted delivery of a ligand-drug conjugate via formyl peptide receptor 1
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through cholesterol-dependent endocytosis
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Junlin Wang1, Meiwan Chen1, Shaoping Li1, Richard D. Ye1*
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1 Institute of Chinese Medical Sciences and State Key Laboratory of Quality Research in
Chinese Medicine, University of Macau, Macau Special Administrative Region, China
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*Send correspondence to: Richard D. Ye, University of Macau, Tel: +853 88224690, Fax:
+853 28841358, E-mail: richardye@um.edu.mo
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Molecular Pharmaceutics
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Abstract:
G protein-coupled receptors (GPCRs) undergo ligand-induced internalization that carries
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the cognate ligands into intracellular compartments. The present study explores this property
for use of formyl peptide receptor 1 (FPR1), a class A GPCR that binds formylated peptides,
as a potential target for drug delivery. A pH-sensitive peptide-drug conjugate consisting of
doxorubicin (DOX), N-ε-maleimidocaproic acid hydrazide (EMCH) and the formyl peptide
fMet-Leu-Phe-Cys (abbreviated as DEF) was prepared. DEF retained pharmacological
activities of formyl peptides in binding to FPR1 and mobilization of Ca2+ from intracellular
stores. However, the conjugated DOX was no longer cell membrane-permeable and relied
on FPR1 for cellular entry. DOX was released from DEF into acidic compartments labeled
with fluorescent trackers for endosomes. Treatment of cells with pharmacological inhibitors
that block clathrin- or caveolae-mediated endocytosis did not abrogate FPR1-dependent
DEF internalization, nor did inhibition of macropinocytosis and phagocytosis. In contrast,
cholesterol depletion abrogated DEF internalization through FPR1, suggesting
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characteristics of cholesterol-dependent uptake mediated by a cell surface receptor. These
results demonstrate the possibility of using FPR1 for targeted drug delivery.
Key words: G protein-coupled receptors, receptor internalization, targeted drug delivery,
pH-sensitive peptides conjugates, formyl peptides.
Abbreviations
GPCRs, G protein-coupled receptors; FPR1, formyl peptide receptor 1; DOX, doxorubicin;
MDC, monodansylcadaverine; CytoD, cytochalasin D; CytoB, cytochalasin B; MβCD,
methyl-β-cyclodextrin; CHC, clathrin heavy chain; CAV1, caveolin-1; Pak1, p21-activated
kinase 1.
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Molecular Pharmaceutics
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1. Introduction
Small molecules enter into cells mainly through simple diffusion, passive diffusion
(including channel-mediated diffusion and carrier-mediated diffusion) and active transport 1.
With simple diffusion, the substance passes through plasma membrane without the aid of
energy and integral membrane proteins. The substance diffuses in a concentration gradient-
dependent manner, i.e., from a high concentration compartment to a low concentration
compartment. Similarly, passive diffusion does not need energy and the moving direction of
the substance is also from high concentration to low concentration. In contrast, active
transport moves molecules against a concentration gradient. A carrier, usually an integral
membrane protein, is required for active transport of molecules.
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In comparison, macromolecules entry into cells is mainly mediated by cell surface
receptors through different endocytic mechanisms, including clathrin-mediated endocytosis,
clathrin-independent endocytosis (such as caveolae-mediated endocytosis),
macropinocytosis and phagocytosis 2-5. A well-documented example is the transferrin
receptor (TfR), a dimeric carrier protein for transport of transferrin through clathrin-mediated
endocytosis 6 . Another example is the low-density lipoprotein receptor (LDLR) that binds
cholesterol apolipoproteins and mediates the transport of LDL into cells through clathrin-
dependent endocytosis 7 . The insulin receptor (IR) binds insulin and carries it into cells
through caveolae-mediated endocytosis 8. In addition to these receptors, the folate receptor,
CD44 receptor and sigma receptor are also involved in the transport of extracellular
substances 9. Clathrin-mediated endocytosis is characterized by the formation of clathrin-
coated vesicles that consist of the clathin proteins (termed triskelion and contains three
heavy chains and three light chains), adaptor protein-2 (AP2), dynamin and other associated
proteins 2. This pathway is most extensively studied among all known endocytic pathways. It
has been reported that sucrose and monodansylcadaverine (MDC) can block this
endocytosis pathway 10. The best studied clathrin-independent endocytosis is the caveolae-
mediated endocytosis. It is characterized by the formation of 60-80 nm flask-shaped
caveolae vesicles. Caveolae has 3 isoforms, caveolin-1 (CAV1), caveolin-2 (CAV2) and
caveolin-3 (CAV3) 11 that are sensitive to the inhibitor filipin12 . Macropinocytosis is a non-
selective endocytic process driven by the actin cytoskeleton. It is often used for engulfing
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