Development of P2Y Receptor Ligands

National Institute Of Diabetes And Digestive And Kidney Diseases

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Abstract

Extracellular ATP acts as a neurotransmitter and modulator of cellular function by binding to and activating two classes of cell surface receptors: P2X ion channels and P2Y G protein-coupled receptors. The signaling function of ATP, ADP, UTP, UDP, UDP-sugars and other nucleotides by activating P2Y receptors is fertile ground for drug discovery, because of the important influence of these receptors on cell proliferation and death, cytokines and immune responses, stem cell differentiation, communication at the tripartite synapse in the nervous system, and many other functions. This class of receptors is responsible for maintaining homeostasis in a variety of organ systems, and their pharmacological manipulation is especially relevant to chronic disease states such as inflammation, digestive disorders, neurodegeneration, endocrine disorders and cardiac failure. The challenge to the medicinal chemist is both to enhance selectivity of the nucleotide derivatives within the family of eight P2Y receptor subtypes and to enhance the stability and bioavailability of normally unstable native ligands. The introduction of numerous selective agonists and antagonists of P2Y receptors (P2YRs) by our laboratory, and their availability for use as pharmacological research tools, has greatly facilitated research in this area. Exploration of the role of P2YRs in large part using ligands designed and synthesized in our laboratory (given the number designation MRS, after the Molecular Recognition Section) and which are made available to many other research laboratories. This availability has led to the introduction of new therapeutic concepts, such as use of P2Y1R antagonists as antithrombotic agents, P2Y2 agonists for cardioprotection, P2Y6R agonists or P2Y13R antagonist for treatment of diabetes, P2Y6R agonists for skeletal muscle protection, and P2Y14R antagonists for asthma and inflammation. For example, we have explored in detail the signaling pathways related to the action of UDP on the P2Y6R of pancreatic beta islet cells, including preventing apoptosis and promoting insulin release, and we're currently studying P2Y6 KO mice. We reported the crystallographic P2Y12R structural determination, the first within this class of GPCRs. We achieved the structure-functional analysis of various other P2YRs, by indirect means, using mutagenesis and molecular modeling. We also designed and synthesized new agonists of the P2Y6 nucleotide receptor using classical and computational approaches. The most potent of these agonists, UDP analogue MRS2795, contains a methanocarba ring system in place of the ribose moiety, which was prepared as a pure stereoisomer using a novel synthetic route. This ring system is locked in the South (S) envelope conformation, which is greatly preferred over other conformations in agonist recognition at the P2Y6R. Thus, the conformational requirements of the ribose moiety of UDP analogues in binding to the P2Y6R are very different from those of the P2Y1R, which prefers a North (N) conformation. We also explored the structure activity relationship at the P2Y6R of pyrimidine nucleotides that contain an alpha,beta-methylene bridge for increased stability. Using mouse lines in which the P2Y6 or P2Y14 receptor was knocked out specifically in adipocytes, we established the potential utility of antagonists of those receptors for treating diabetes and obesity. KO of P2Y6R in the whole body or in an adipocyte-specific manner protected mice from diet-induced obesity to improve glucose tolerance, insulin sensitivity and to reduce systemic inflammation. This improvement was associated with reduced JNK signaling with consequent increase of peroxisome proliferator-activated receptor (PPAR)- and beiging of white fat. The P2Y14R is found in human adipocytes and is highly upregulated in subcutaneous fat in human obesity. Mice lacking this receptor in adipocytes are protected against metabolic dysregulation from a high fat diet. This attenuation is evident in reduced body weight, enhanced glucose tolerance and insulin sensitivity and reduced inflammation. P2Y14R expression in visceral white adipocytes was increased in high fat diet and decreased in fasting. The protective mechanism in adipocyte P2Y14 KO mice involves enhanced lipolysis, but only during fasting. The enzymes required for complete lipolysis were increased and activated during fasting in adipocyte-specific P2Y14 KO mice compared to wild type. The fatty acids and glycerol produced in this manner from white adipocytes can serve as an alternative energy source for other tissues during periods of caloric deprivation. Mice with adipocyte P2Y14 KO on a high fat diet had less liver steatosis. Mice lacking P2Y14R, the receptor for UDP-G, had decreased airway eosinophilia and airway hyperresponsiveness (AHR) compared with wild type mice in a protease-mediated model of asthma. P2Y14R was dispensable for allergic sensitization, and for the production of type 2 cytokines in the lung after challenge. However, UDP-G increased chemokinesis in eosinophils and enhanced their response to the eosinophil chemoattractant, CCL24. In turn, eosinophils triggered the release of UDP-G into the airway, thereby amplifying eosinophilic recruitment. This positive feedback loop was sensitive to therapeutic intervention, as a small molecule antagonist of P2Y14R inhibited airway eosinophilia. These findings thus reveal a novel pathway that can be therapeutically targeted to treat asthma exacerbations and glucocorticoid-resistant forms of this disease. We have introduced novel ligands of the P2Y14, including high affinity antagonists and fluorescent probes, using a structure-based approach. A novel chemical scaffold has proven to be a good basis for P2Y14 antagonists. P2Y14R is highly expressed in hematopoietic cells and has been strongly implicated recently in immune and inflammatory responses. Lack of availability of receptor-selective high-affinity antagonists has impeded progress in studies of this and most of the eight nucleotide-activated P2Y receptors. We carried out the first systematic SAR studies on the P2Y14R leading to UDP analogues of nanomolar affinity, such as selective agonist MRS2905. We synthesized derivatives of a 4,7-disubstituted 2-naphthoic acid (PPTN, Ki 0.4 nM) as selective P2Y14 antagonists, and studied their pharmacological properties in detail. PPTN blocked chemotaxis of differentiated human promyelocytic leukemia cells promoted by UDP-glucose. P2Y14R-promoted chemotaxis of freshly isolated human neutrophils also was blocked by PPTN. A fluorescent derivative MRS4174 was designed as a high affinity ligand probe for drug screening (using flow cytometry and other means) and receptor characterization. Thus, PPTN and its derivatives are highly selective high-affinity antagonists that are useful for interrogating the action of P2Y14R in physiologic systems. We reported that UDP-glucose released into the airways activates the P2Y14R to act as a local mediator of neutrophil inflammation. Furthermore, UDP-glucose activates inflammation in renal intercalated cells via the P2Y14 receptor. We are designing novel potent antagonists of the P2Y14 receptor for application in inflammatory and pulmonary diseases. We are performing a comprehensive study of the SAR of PPTN analogues, and we have discovered potent antagonists that have more favorable drug-like physical-chemical properties. In summary, we emphasize four aspects of studying the P2Y receptors: 1) design and synthesis of novel and selective agonists and antagonists based on SAR; 2) protein structure-function studies; 3) exploring the novel biological role of such receptors; and 4) conceptualization of future therapeutics. In this effort, there is a tight coupling of organic synthetic methodology, structural biology, and pharmacology.