title: Design, synthesis, pharmacological characterization of a fluorescent agonist of the P2Y₁₄ receptor.
abstract: The P2Y14R is a G(i/o)-coupled receptor of the P2Y family of purinergic receptors that is activated by extracellular UDP and UDP-glucose (UDPG). In an earlier report we described a P2Y14R fluorescent probe, MRS4174, based on the potent and selective antagonist PPTN, a naphthoic acid derivative. Here, we report the design, preparation, and activity of an agonist-based fluorescent probe MRS4183 (11) and a shorter P2Y14R agonist congener, which contain a UDP-glucuronic acid pharmacophore and BODIPY fluorophores conjugated through diaminoalkyl linkers. The design relied on both docking in a P2Y14R homology model and established structure activity relationship (SAR) of nucleotide analogs. 11 retained P2Y14R potency with EC50 value of 0.96 nM (inhibition of adenylyl cyclase), compared to parent UDPG (EC50 47 nM) and served as a tracer for microscopy and flow cytometry, displaying minimal nonspecific binding. Binding saturation analysis gave an apparent binding constant for 11 in whole cells of 21.4±1.1 nM, with a t1/2 of association at 50 nM 11 of 23.9 min. Known P2Y14R agonists and PPTN inhibited cell binding of 11 with the expected rank order of potency. The success in the identification of a new P2Y14R fluorescent agonist with low nonspecific binding illustrates the advantages of rational design based on recently determined GPCR X-ray structures. Such conjugates will be useful tools in expanding the SAR of this receptor, which still lacks chemical diversity in its collective ligands.
authors: Molecular Recognition Section, Laboratory of Bioorganic Chemistry, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892, USA.
authors: Evgeny
authors: E
authors: Kiselev
authors: Molecular Recognition Section, Laboratory of Bioorganic Chemistry, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892, USA.
authors: Ramachandran
authors: R
authors: Balasubramanian
authors: Molecular Recognition Section, Laboratory of Bioorganic Chemistry, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892, USA.
authors: Elisa
authors: E
authors: Uliassi
authors: Department of Pharmacology, University of North Carolina, School of Medicine, Chapel Hill, NC 27599, USA.
authors: Kyle A
authors: KA
authors: Brown
authors: Molecular Recognition Section, Laboratory of Bioorganic Chemistry, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892, USA.
authors: Kevin
authors: K
authors: Trujillo
authors: The Bridge Institute, Department of Biological Sciences, Dornsife College of Letters, Arts and Sciences, University of Southern California, Los Angeles, CA 90089, USA.
authors: Vsevolod
authors: V
authors: Katritch
authors: Molecular Recognition Section, Laboratory of Bioorganic Chemistry, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892, USA.
authors: Eva
authors: E
authors: Hammes
authors: The Bridge Institute, Department of Biological Sciences, Dornsife College of Letters, Arts and Sciences, University of Southern California, Los Angeles, CA 90089, USA; The Bridge Institute, Department of Chemistry, Dornsife College of Letters, Arts and Sciences, University of Southern California, Los Angeles, CA 90089, USA.
authors: Raymond C
authors: RC
authors: Stevens
authors: Department of Pharmacology, University of North Carolina, School of Medicine, Chapel Hill, NC 27599, USA.
authors: T Kendall
authors: TK
authors: Harden
authors: Molecular Recognition Section, Laboratory of Bioorganic Chemistry, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892, USA. Electronic address: kajacobs@helix.nih.gov.
authors: Kenneth A
authors: KA
authors: Jacobson
mesh_terms: D004305:Dose-Response Relationship, Drug; D015195:Drug Design; D005456:Fluorescent Dyes / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology*; D006575:Heterocyclic Compounds, 3-Ring / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology*; D006801:Humans; D008958:Models, Molecular; D015394:Molecular Structure; D011714:Pyrans / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology*; D018048:Receptors, Purinergic P2 / Q000378:metabolism*; D013329:Structure-Activity Relationship
chemical_list: D005456:Fluorescent Dyes; D006575:Heterocyclic Compounds, 3-Ring; C000608286:MRS4183; C518528:P2Y14 receptor, human; D011714:Pyrans; D018048:Receptors, Purinergic P2
keywords: Docking; G protein-coupled receptor; Homology modeling; Nucleotides; P2Y receptor; Pyrimidines