Figure 6: Antagonism by an A1AR antagonist of [35S]GTPγS binding induced by compound 17. Compound 17 was incubated with increasing concentrations of A1 antagonist DPCPX, [35S]GTPγS, and a CHO A1 membrane suspension. The amount of [35S]GTPγS bound was measured, and the results were interpreted with Prism software. The results shown are means ± S.E.M. of three independent experiments.

Mentions: Radioligand binding was completed for each of the G2.5 dendrimer conjugates. Compound 17 showed a 2.4 nM affinity for the A3 AR while compound 16 had a 14 nM affinity for this receptor. Interestingly, 16 displayed at least a 10-fold selectivity, and compound 17 displayed over a 100 fold selectivity for the A3 AR in comparison to the A1 and A2A ARs (Figure 4). Compound 17 was also 100 fold selective for the A3 AR in comparison to A1 AR in assays of adenylate cylase inhibition with an EC50 value of 1.6 nM at the A3 AR (Figure 5). However, in this assay, 16 was only 8 fold more potent at the A3 AR than at the A1 AR. In GTPγS studies, 16 was 15 fold less potent at the A1 AR than in an assay measuring the suppression of cAMP production; however, 17 had similar potency at both A1 AR functional assays, and both compounds were full agonists in both assays. In the GTPγS study, DPCPX, an A1 antagonist, was able to fully inhibit the binding of [35S]GTPγS when incubated with 17 (Figure 6), showing that the binding is due to the specific interaction of 17 with the A1 receptor. The control dendrimer 15 showed no binding or activity in either cAMP or GTPγS assays of A1 AR activation. The stably transfected CHO A1 and A3 cells had Bmax values of 530 ± 210 fmol/mg protein and 253 ± 19 fmol/mg protein, respectively, showing that there is similar receptor expression in both cell lines.

Klutz AM, Gao ZG, Lloyd J, Shainberg A, Jacobson KA - J Nanobiotechnology (2008)

Bottom Line: 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.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.

Affiliation: Molecular Recognition Section, Laboratory of Bioorganic Chemistry, National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, Maryland 20892, USA. kajacobs@helix.nih.gov.

Abstract: 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.