Enhanced A3 adenosine receptor selectivity of multivalent nucleoside-dendrimer conjugates

Molecular Recognition Section, Laboratory of Bioorganic Chemistry, National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, Maryland 20892, USA. .
Journal of Nanobiotechnology (Impact Factor: 4.12). 10/2008; 6(1):12. DOI: 10.1186/1477-3155-6-12
Source: PubMed

ABSTRACT An approach to use multivalent dendrimer carriers for delivery of nucleoside signaling molecules to their cell surface G protein-coupled receptors (GPCRs) was recently introduced.
A known adenosine receptor (AR) agonist was conjugated to polyamidoamine (PAMAM) dendrimer carriers for delivery of the intact covalent conjugate to on the cell surface. Depending on the linking moiety, multivalent conjugates of the N6-chain elongated functionalized congener ADAC (N6-[4-[[[4-[[[(2-aminoethyl)amino]carbonyl]methyl]anilino]carbonyl]methyl]phenyl]-adenosine) achieved unanticipated high selectivity in binding to the cytoprotective human A3 AR, a class A GPCR. The key to this selectivity of > 100-fold in both radioreceptor binding (Ki app = 2.4 nM) and functional assays (EC50 = 1.6 nM in inhibition of adenylate cyclase) was maintaining a free amino group (secondary) in an amide-linked chain. Attachment of neutral amide-linked chains or thiourea-containing chains preserved the moderate affinity and efficacy at the A1 AR subtype, but there was no selectivity for the A3 AR. Since residual amino groups on dendrimers are associated with cytotoxicity, the unreacted terminal positions of this A3 AR-selective G2.5 dendrimer were present as carboxylate groups, which had the further benefit of increasing water-solubility. The A3 AR selective G2.5 dendrimer was also visualized binding the membrane of cells expressing the A3 receptor but did not bind cells that did not express the receptor.
This is the first example showing that it is feasible to modulate and even enhance the pharmacological profile of a ligand of a GPCR based on conjugation to a nanocarrier and the precise structure of the linking group, which was designed to interact with distal extracellular regions of the 7 transmembrane-spanning receptor. This ligand tool can now be used in pharmacological models of tissue rescue from ischemia and to probe the existence of A3 AR dimers.

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Available from: Kenneth A. Jacobson, Sep 26, 2015
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    • "The affinity of the QD conjugates was examined in a standard radioligand binding assay using [3H]1a in membranes of human embryonic kidney (HEK-293) cells expressing the human A2AAR (Table 1) [11]. The thiotic-acid anchored derivatives nucleoside derivatives 4-7 and the amide-anchored derivative 8 and 9 were inactive or only weakly inhibited binding at the human A2AAR at the highest concentration used (1 μM). "
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    ABSTRACT: Quantum dots (QDs) are crystalline nanoparticles that are compatible with biological systems to provide a chemically and photochemically stable fluorescent label. New ligand probes with fluorescent reporter groups are needed for detection and characterization of G protein-coupled receptors (GPCRs). Synthetic strategies for coupling the A2A adenosine receptor (AR) agonist CGS21680 (2-[4-(2-carboxyethyl)phenylethylamino]-5'-N-ethylcarboxamidoadenosine) to functionalized QDs were explored. Conjugates tethered through amide-linked chains and poly(ethyleneglycol) (PEG) displayed low solubility and lacked receptor affinity. The anchor to the dendron was either through two thiol groups of (R)-thioctic acid or through amide formation to a commercial carboxy-derivatized QD. The most effective approach was to use polyamidoamine (PAMAM) D5 dendrons as multivalent spacer groups, grafted on the QD surface through a thioctic acid moiety. In radioligand binding assays, dendron nucleoside conjugate 11 displayed a moderate affinity at the human A2AAR (Kiapp 1.02 +/- 0.15 muM). The QD conjugate of increased water solubility 13, resulting from the anchoring of this dendron derivative, interacted with the receptor with Kiapp of 118 +/- 54 nM. The fluorescence emission of 13 occurred at 565 nm, and the presence of the pendant nucleoside did not appreciably quench the fluorescence. This is a feasibility study to demonstrate a means of conjugating to a QD a small molecular pharmacophore of a GPCR that is relatively hydrophobic. Further enhancement of affinity by altering the pharmacophore or the linking structures will be needed to make useful affinity probes.
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