Multivalent dendrimeric conjugates of GPCR ligands may have increased potency or selectivity in comparison to monomeric ligands, a phenomenon that was tested in a model of cytoprotection in mouse HL-1 cardiomyocytes. Quantitative RT-PCR indicated high expression levels of endogenous A1 and A2A adenosine receptors (ARs), but not of A2B and A3ARs. Activation of the heterologously expressed human A3AR in HL-1 cells by AR agonists significantly attenuated cell damage following 4h exposure to H2O2 (750μM) but not in untransfected cells. The A3 agonist IB-MECA (EC50 3.8μM) and the non-selective agonist NECA (EC50 3.9μM) protected A3 AR-transfected cells against H2O2 in a concentration-dependent manner, as determined by lactate dehydrogenase release. A generation 5.5 PAMAM (polyamidoamine) dendrimeric conjugate of a N6-chain-functionalized adenosine agonist was synthesized and its mass indicated an average of 60 amide-linked nucleoside moieties out of 256 theoretical attachment sites. It non-selectively activated the A3AR to inhibit forskolin-stimulated cAMP formation (IC50 66nM) and, similarly, protected A3-transfected HL-1 cells from apoptosis-inducing H2O2 with greater potency (IC50 35nM) than monomeric nucleosides. Thus, a PAMAM conjugate retained AR binding affinity and displayed greatly enhanced cardioprotective potency.
Bibliographical noteFunding Information:
This research was supported by the Intramural Research Program of the NIH, National Institute of Diabetes and Digestive and Kidney Diseases . We thank Lena Yoo (NIDDK) for proofreading this manuscript.
- G protein-coupled receptor
- HL-1 cells
- Polymeric drugs