The P2Y14R is a Gi/o-coupled receptor, a member of the P2Y family of purinergic receptors, which is activated by extracellular UDP and UDP-glucose. The development of fluorescent affinity probes for P2Y14R will be described. The structure of P2Y14R was modeled based on recent P2Y12R X-ray structures (45% sequence identity). Analogues of potent and selective antagonist PPTN and nucleotide agonists were docked into the P2Y14R homology model to determine sites suitable for fluorophore conjugation. Subsequently, the antagonist PPTN (a 2-naphthoic acid derivative) and agonist UDP-glucuronic acid (UDPGA) were modified with appropriate linkers and conjugated to fluorescent moieties through alkyne-azide click reaction or amide coupling. As a result, two of the fluorescent conjugates, an AlexaFluor488 conjugate of functionalized PPTN (MRS4174, Ki 80 pM in cyclic AMP functional assay) and BODIPY conjugate of UDPGA (MRS4183) were found to bind specifically to the P2Y14R expressed in CHO cells. The cell binding of both agonist and antagonist fluorescent conjugates was inhibited by other known P2Y14R agonists and antagonist PPTN. The level of fluorescence binding inhibition seems to correlate with the relative potencies of competing ligands. The further studies of these ligands with regard to fluorescence binding and receptor pharmacology as well as their suitability for ligand affinity screening are currently underway. The success in the identification of new P2Y14R fluorescent ligands illustrates the advantages of rational design based on homology modeling and highlights the value of X-ray structures to the medicinal chemistry of GPCRs.

Development of fluorescent affinity probes for the P2Y14 G protein-coupled receptor

249th National Meeting (2015)

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