One of the major unsolved problems in drug delivery is how to transport poorly permeable molecules across membrane barriers. The way that Nature solves this problem is through membrane trafficking;cell impermeable ligands such as the iron-carrying transferrin protein bind cognate receptors, in this case the transferrin receptor, that reside in dynamic membrane trafficking pathways. The pathway occupied by the transferrin receptor involves rapid cycling between the cell surface and early/recycling endosomes, delivers transferrin to every cell of vertebrate animals, and represents a remarkable natural delivery vehicle. In the last grant cycle, we demonstrated that derivatives of the synthetic membrane anchor N-alkyl-3?-cholesterylamine exhibit a unique biological activity: when added to mammalian cells, they efficiently engage the membrane trafficking pathway occupied by the transferrin receptor and rapidly cycle between the plasma membrane and early/recycling endosomes. In this way, N-alkyl-3?-cholesterylamines can function as small artificial cell surface receptors;when linked to drug-binding motifs, they can shuttle drugs into cells and tissues via endocytosis. These compounds appear to functionally mimic free cholesterol, a key component of plasma and endosomal membranes. They bind cholesterol-laden LDL and HDL particles and presumably participate in natural cholesterol uptake and trafficking mechanisms. Because delivery of transferrin mediated by the transferrin receptor is so efficient, the ability of cholesterylamines to efficiently engage the same membrane trafficking pathway offers unprecedented potential to create transformative new tools for drug delivery. This proposal is focused on elucidating mechanisms that control the unique biological activities of N-alkyl-3?-cholesterylamines. By synthesizing and evaluating the biological properties of a panel of compounds bearing structural diversity in the linker region proximal to this membrane anchor, we will obtain quantitative structure-property relationships (QSPR) designed to predict cellular uptake, trafficking, efflux, metabolism, and toxicity properties. Structurally related photocrosslinkers, RNAi methods, and protein overexpression will be used to identify receptors and ligands involved in cellular uptake and efflux. These mediators of biological activity will have a profound effect on the pharmacokinetics of N-alkyl-3?-cholesterylamines in vivo. We further propose to construct the first anticancer drug delivery systems designed to cross membrane barriers by accessing a defined membrane trafficking pathway. To gain specificity for metastatic cancer cells over normal cells, peptide substrates of tumor-specific proteases will be used to link cholesterylamines to the anticancer drug doxorubicin. This drug will be selectively released when cleaved by the MMP-2 protease expressed by metastatic cancer cells. We will optimize the delivery of doxorubicin and related molecular probes into cancer cells in vitro to validate a fundamentally new concept: compounds that engage a defined membrane trafficking pathway offer a new strategy for the selective delivery of anticancer agents.

Public Health Relevance

The research proposed here is highly relevant to the advancement of human health. In the past grant cycle we discovered that synthetic compounds termed N-alkyl-3?-cholesterylamines engage the specific membrane trafficking pathway occupied by the transferrin receptor. This biological activity enables these compounds to define new pathways across membrane barriers in mammalian cells. By elucidating biological mechanisms of these compounds, we propose to create innovative new tools for anticancer drug delivery.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA083831-12
Application #
8253791
Study Section
Synthetic and Biological Chemistry B Study Section (SBCB)
Program Officer
Yovandich, Jason L
Project Start
2000-01-01
Project End
2014-04-30
Budget Start
2012-05-01
Budget End
2014-04-30
Support Year
12
Fiscal Year
2012
Total Cost
$232,058
Indirect Cost
$74,195
Name
University of Kansas Lawrence
Department
Pharmacology
Type
Schools of Pharmacy
DUNS #
076248616
City
Lawrence
State
KS
Country
United States
Zip Code
66045
Woydziak, Zachary R; Fu, Liqiang; Peterson, Blake R (2014) Efficient and Scalable Synthesis of 4-Carboxy-Pennsylvania Green Methyl Ester: A Hydrophobic Building Block for Fluorescent Molecular Probes. Synthesis (Stuttg) 46:158-164
Hymel, David; Woydziak, Zachary R; Peterson, Blake R (2014) Detection of protein-protein interactions by proximity-driven S(N)Ar reactions of lysine-linked fluorophores. J Am Chem Soc 136:5241-4
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Bender, Aaron; Woydziak, Zachary R; Fu, Liqiang et al. (2013) Novel acid-activated fluorophores reveal a dynamic wave of protons in the intestine of Caenorhabditis elegans. ACS Chem Biol 8:636-42
Woydziak, Zachary R; Fu, Liqiang; Peterson, Blake R (2012) Synthesis of fluorinated benzophenones, xanthones, acridones, and thioxanthones by iterative nucleophilic aromatic substitution. J Org Chem 77:473-81
Lönnfors, Max; Engberg, Oskar; Peterson, Blake R et al. (2012) Interaction of 3?-amino-5-cholestene with phospholipids in binary and ternary bilayer membranes. Langmuir 28:648-55
Hymel, David; Peterson, Blake R (2012) Synthetic cell surface receptors for delivery of therapeutics and probes. Adv Drug Deliv Rev 64:797-810
Sun, Qi; Wu, Runzhi; Cai, Sutang et al. (2011) Synthesis and biological evaluation of analogues of AKT (protein kinase B) inhibitor-IV. J Med Chem 54:1126-39
Law, Mankit; Morales, J Luis; Mottram, Laurie F et al. (2011) Structural requirements for the inhibition of calcium mobilization and mast cell activation by the pyrazole derivative BTP2. Int J Biochem Cell Biol 43:1228-39
Zhang, Jun; Cai, Sutang; Peterson, Blake R et al. (2011) Development of a cell-based, high-throughput screening assay for cholesterol efflux using a fluorescent mimic of cholesterol. Assay Drug Dev Technol 9:136-46

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