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Polyamidoamine (PAMAM) dendrimer conjugates of "clickable" agonists of the A3 adenosine receptor and coactivation of the P2Y14 receptor by a tethered nucleotide.

ABSTRACT: We previously synthesized a series of potent and selective A(3) adenosine receptor (AR) agonists (North-methanocarba nucleoside 5'-uronamides) containing dialkyne groups on extended adenine C2 substituents. We coupled the distal alkyne of a 2-octadiynyl nucleoside by Cu(I)-catalyzed "click" chemistry to azide-derivatized G4 (fourth-generation) PAMAM dendrimers to form triazoles. A(3)AR activation was preserved in these multivalent conjugates, which bound with apparent K(i) of 0.1-0.3 nM. They were substituted with nucleoside moieties, solely or in combination with water-solubilizing carboxylic acid groups derived from hexynoic acid. A comparison with various amide-linked dendrimers showed that triazole-linked conjugates displayed selectivity and enhanced A(3)AR affinity. We prepared a PAMAM dendrimer containing equiproportioned peripheral azido and amino groups for conjugation of multiple ligands. A bifunctional conjugate activated both A(3) and P2Y(14) receptors (via amide-linked uridine-5'-diphosphoglucuronic acid), with selectivity in comparison to other ARs and P2Y receptors. This is the first example of targeting two different GPCRs with the same dendrimer conjugate, which is intended for activation of heteromeric GPCR aggregates. Synergistic effects of activating multiple GPCRs with a single dendrimer conjugate might be useful in disease treatment.

SUBMITTER: Tosh DK

PROVIDER: S-EPMC2845915 | biostudies-literature | 2010 Feb

REPOSITORIES: biostudies-literature

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Polyamidoamine (PAMAM) dendrimer conjugates of "clickable" agonists of the A3 adenosine receptor and coactivation of the P2Y14 receptor by a tethered nucleotide.

Tosh Dilip K DK Yoo Lena S LS Chinn Moshe M Hong Kunlun K Kilbey S Michael SM Barrett Matthew O MO Fricks Ingrid P IP Harden T Kendall TK Gao Zhan-Guo ZG Jacobson Kenneth A KA

Bioconjugate chemistry 20100201 2

We previously synthesized a series of potent and selective A(3) adenosine receptor (AR) agonists (North-methanocarba nucleoside 5'-uronamides) containing dialkyne groups on extended adenine C2 substituents. We coupled the distal alkyne of a 2-octadiynyl nucleoside by Cu(I)-catalyzed "click" chemistry to azide-derivatized G4 (fourth-generation) PAMAM dendrimers to form triazoles. A(3)AR activation was preserved in these multivalent conjugates, which bound with apparent K(i) of 0.1-0.3 nM. They we ...[more]

PMID: 20121074

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Project description:BACKGROUND: 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. RESULTS: 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. CONCLUSION: 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.

Project description:The P2Y(1) receptor is a prothrombotic G protein-coupled receptor (GPCR) activated by ADP. Preference for the North (N) ring conformation of the ribose moiety of adenine nucleotide 3',5'-bisphosphate antagonists of the P2Y(1) receptor was established by using a ring-constrained methanocarba (a bicyclo[3.1.0]hexane) ring as a ribose substitute. A series of covalently linkable N(6)-methyl-(N)-methanocarba-2'-deoxyadenosine-3',5'-bisphosphates containing extended 2-alkynyl chains was designed, and binding affinity at the human (h) P2Y(1) receptor determined. The chain of these functionalized congeners contained hydrophilic moieties, a reactive substituent, or biotin, linked via an amide. Variation of the chain length and position of an intermediate amide group revealed high affinity of carboxylic congener 8 (K(i) 23 nM) and extended amine congener 15 (K(i) 132 nM), both having a 2-(1-pentynoyl) group. A biotin conjugate 18 containing an extended epsilon-aminocaproyl spacer chain exhibited higher affinity than a shorter biotinylated analogue. Alternatively, click coupling of terminal alkynes of homologous 2-dialkynyl nucleotide derivatives to alkyl azido groups produced triazole derivatives that bound to the P2Y(1) receptor following deprotection of the bisphosphate groups. The preservation of receptor affinity of the functionalized congeners was consistent with new P2Y(1) receptor modeling and ligand docking. Attempted P2Y(1) antagonist conjugation to PAMAM dendrimer carriers by amide formation or palladium-catalyzed reaction between an alkyne on the dendrimer and a 2-iodopurine-derivatized nucleotide was unsuccessful. A dialkynyl intermediate containing the chain length favored in receptor binding was conjugated to an azide-derivatized dendrimer, and the conjugate inhibited ADP-promoted human platelet aggregation. This is the first example of attaching a strategically functionalized P2Y receptor antagonist to a PAMAM dendrimer to produce a multivalent conjugate exhibiting a desired biological effect, i.e., antithrombotic action.

Dataset Information

Polyamidoamine (PAMAM) dendrimer conjugates of "clickable" agonists of the A3 adenosine receptor and coactivation of the P2Y14 receptor by a tethered nucleotide.

Publications

Polyamidoamine (PAMAM) dendrimer conjugates of "clickable" agonists of the A3 adenosine receptor and coactivation of the P2Y14 receptor by a tethered nucleotide.

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