Enhanced Potency of Nucleotide−Dendrimer Conjugates as Agonists of the P2Y₁₄ Receptor: Multivalent Effect in G Protein-Coupled Receptor Recognition

The P2Y₁₄ receptor is a G protein-coupled receptor activated by uridine-5′-diphosphoglucose and other nucleotide sugars that modulates immune function. Covalent conjugation of P2Y₁₄ receptor agonists to PAMAM (polyamidoamine) dendrimers enhanced pharmacological activity. Uridine-5′-diphosphoglucuronic acid (UDPGA) and its ethylenediamine adduct were suitable functionalized congeners for coupling to several generations (G2.5−6) of dendrimers (both terminal carboxy and amino). Prosthetic groups, including biotin for avidin complexation, a chelating group for metal complexation (and eventual magnetic resonance imaging), and a fluorescent moiety, also were attached with the eventual goals of molecular detection and characterization of the P2Y₁₄ receptor. The activities of conjugates were assayed in HEK293 cells stably expressing the human P2Y₁₄ receptor. A G3 PAMAM conjugate containing 20 bound nucleotide moieties (UDPGA) was 100-fold more potent (EC₅₀ 2.4 nM) than the native agonist uridine-5′-diphosphoglucose. A molecular model of this conjugate docked in the human P2Y₁₄ receptor showed that the nucleotide-substituted branches could extend far beyond the dimensions of the receptor and be available for multivalent docking to receptor aggregates. Larger dendrimer carriers and greater loading favored higher potency. A similar conjugate of G6 with 147 out of 256 amino groups substituted with UDPGA displayed an EC₅₀ value of 0.8 nM. Thus, biological activity was either retained or dramatically enhanced in the multivalent dendrimer conjugates in comparison with monomeric P2Y₁₄ receptor agonists, depending on size, degree of substitution, terminal functionality, and attached prosthetic groups.