GPCR ligand dendrimer (GLiDe) conjugates: adenosine receptor interactions of a series of multivalent xanthine antagonists

Previously, G protein-coupled receptor (GPCR) agonists were tethered from polyamidoamine (PAMAM) dendrimers to provide high receptor affinity and selectivity. Here, we prepared GPCR ligand--dendrimer (GLiDe) conjugates from a potent adenosine receptor (AR) antagonist; such agents are of interest for treating Parkinson's disease, asthma, and other conditions. Xanthine amine congener (XAC) was appended with an alkyne group on an extended C8 substituent for coupling by Cu(I)-catalyzed click chemistry to azide-derivatized G4 (fourth-generation) PAMAM dendrimers to form triazoles. These conjugates also contained triazole-linked PEG groups (8 or 22 moieties per 64 terminal positions) for increasing water-solubility and optionally prosthetic groups for spectroscopic characterization and affinity labeling. Human AR binding affinity increased progressively with the degree of xanthine substitution to reach K(i) values in the nanomolar range. The order of affinity of each conjugate was hA(2A)AR > hA(3)AR > hA(1)AR, while the corresponding monomer was ranked hA(2A)AR > hA(1)AR ≥ hA(3)AR. The antagonist activity of the most potent conjugate 14 (34 xanthines per dendrimer) was examined at the G(i)-coupled A(1)AR. Conjugate 14 at 100 nM right-shifted the AR agonist concentration--response curve in a cyclic AMP functional assay in a parallel manner, but at 10 nM (lower than its K(i) value), it significantly suppressed the maximal agonist effect in calcium mobilization. This is the first systematic probing of a potent AR antagonist tethered on a dendrimer and its activity as a function of variable loading.