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Modulation of G protein-coupled adenosine receptors by strategically functionalized agonists and antagonists immobilized on gold nanoparticles

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Abstract

Gold nanoparticles (AuNPs) allow the tuning of pharmacokinetic and pharmacodynamic properties by active or passive targeting of drugs for cancer and other diseases. We have functionalized gold nanoparticles by tethering specific ligands, agonists and antagonists, of adenosine receptors (ARs) to the gold surface as models for cell surface interactions with G protein-coupled receptors (GPCRs). The AuNP conjugates with chain-extended AR ligands alone (PEGylated nucleosides and nonnucleosides, anchored to the Au via thioctic acid) were found to be insoluble in water due to hydrophobic entities in the ligand. Therefore, we added a second, biologically inactive pendant moiety to increase the water solubility, consisting of a PEGylated chain terminating in a carboxylic or phosphate group. The purity and stability of the immobilized biologically active ligand were examined by ultrafiltration and HPLC. Pharmacological receptor binding studies on these GPCR ligand-derivatized AuNPs (2–5 nm in diameter), performed using membranes of mammalian cells stably expressing human A1, A2A, and A3ARs, showed that the desired selectivity was retained with K i values (nanomolar) of A3AR agonist 21b and A2AAR antagonists 24 and 26a of 14 (A3), 34 (A2A), and 69 (A2A), respectively. The corresponding monomers displayed K i values of 37, 61, and 1,420 nM, respectively. In conclusion, we have synthesized stable, water-soluble AuNP derivatives of tethered A3 and A2AAR ligands that retain the biological properties of their monomeric ligands and are intended for therapeutic and imaging applications. This is the first prototypical application to gold carriers of small molecule (nonpeptide) GPCR ligands, which are under investigation for treatment of cancer and inflammatory diseases.

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Abbreviations

AR:

Adenosine receptor

DLS:

Dynamic light scattering

DMSO:

Dimethylsulfoxide

EDC:

N-ethyl-N′-dimethylamino-propylcarbodiimide

cAMP:

Adenosine 3′,5′-cyclic phosphate

CHO:

Chinese hamster ovary

Cl-IB-MECA:

2-Chloro-N 6-(3-iodobenzyl)-5′-N-methylcarboxamido-adenosine

DCC:

N,N′-dicyclohexylcarbodiimide

DMAP:

4-Dimethylaminopyridine

DCM:

Dichloromethane

DIEA:

Diisopropylethylamine

DMF:

Dimethylformamide

DMEM:

Dulbecco’s modified Eagle’s medium

AuNP:

Gold nanoparticle

GPCR:

G protein-coupled receptor

HEK:

Human embryonic kidney

CGS21680:

2-[p-(2-carboxyethyl)phenyl-ethylamino]-5′-N-ethylcarboxamido-adenosine

MS:

Mass spectrometry

NECA:

5′-N-ethylcarboxamidoadenosine

PTP:

Pyrazolo[4,3-e][1, 2, 4]triazolo[1,5-c]pyrimidine-5-amine

PyBOP:

Benzotriazol-1-yloxy-tripyrrolidino-phosphonium hexafluorophosphate

SPB:

Surface plasmon absorption band

TEM:

Transmission electron microscopy

THF:

Tetrahydrofuran

I-AB-MECA:

N 6-(4-amino-3-iodobenzyl)-adenosine-5′-N-methyluronamide

PEG:

Polyethylene glycol

PIA:

N 6-(2-phenylisopropyl)adenosine

Tris:

2-amino-2-hydroxymethyl-propane-1,3-diol

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Acknowledgments

We thank Dr. Noel Whittaker (NIDDK) for MS measurements. This research was supported by the Intramural Research Programs of NIDDK and NCI, National Institutes of Health.

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Authors and Affiliations

  1. Molecular Recognition Section, Laboratory of Bioorganic Chemistry, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bldg. 8A, Rm. B1A-19, Bethesda, MD, 20892-0810, USA

    P. Suresh Jayasekara, Khai Phan, Dilip K. Tosh, T. Santhosh Kumar, Steven M. Moss, Zhan-Guo Gao & Kenneth A. Jacobson

  2. Biomedical Engineering and Physical Science Shared Resource, NIBIB, National Institutes of Health, Bethesda, MD, 20892, USA

    Guofeng Zhang

  3. Chemical Biology Laboratory, Center for Cancer Research, Frederick National Laboratory for Cancer Research, Frederick, MD, 21702-1201, USA

    Joseph J. Barchi Jr.

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  1. P. Suresh Jayasekara
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Corresponding author

Correspondence to Kenneth A. Jacobson.

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Supporting information

Supporting information with details of the AuNP characterization and biological and stability assays is available online at http://. (DOCX 11420 kb)

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Jayasekara, P.S., Phan, K., Tosh, D.K. et al. Modulation of G protein-coupled adenosine receptors by strategically functionalized agonists and antagonists immobilized on gold nanoparticles. Purinergic Signalling 9, 183–198 (2013). https://doi.org/10.1007/s11302-012-9338-z

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