Matthew O. Barrett

Quantification of Gi-Mediated Inhibition of Adenylyl Cyclase Activity Reveals That UDP Is a Potent Agonist of the Human P2Y14 Receptor

The P2Y14 receptor was initially identified as a G protein-coupled receptor activated by UDP-glucose and other nucleotide sugars. We have developed several cell lines that stably express the human P2Y14 receptor, allowing facile examination of its coupling to native Gi family G proteins and their associated downstream signaling pathways (J Pharmacol Exp Ther 330:162–168, 2009). In the current study, we examined P2Y14 receptor-dependent inhibition of cyclic AMP accumulation in human embryonic kidney (HEK) 293, C6 glioma, and Chinese hamster ovary (CHO) cells stably expressing this receptor. Not only was the human P2Y14 receptor activated by UDP-glucose, but it also was activated by UDP. The apparent efficacies of UDP and UDP-glucose were similar, and the EC50 values (74, 33, and 29 nM) for UDP-dependent activation of the P2Y14 receptor in HEK293, CHO, and C6 glioma cells, respectively, were similar to the EC50 values (323, 132, and 72 nM) observed for UDP-glucose. UDP and UDP-glucose also stimulated extracellular signal-regulated kinase (ERK) 1/2 phosphorylation in P2Y14 receptor-expressing HEK293 cells but not in wild-type HEK293 cells. A series of analogs of UDP were potent P2Y14 receptor agonists, but the naturally occurring nucleoside diphosphates, CDP, GDP, and ADP exhibited agonist potencies over 100-fold less than that observed with UDP. Two UDP analogs were identified that selectively activate the P2Y14 receptor over the UDP-activated P2Y6 receptor, and these molecules stimulated phosphorylation of ERK1/2 in differentiated human HL-60 promyeloleukemia cells, which natively express the P2Y14 receptor but had no effect in wild-type HL-60 cells, which do not express the receptor. We conclude that UDP is an important cognate agonist of the human P2Y14 receptor.

A selective high-affinity antagonist of the P2Y14 receptor inhibits UDP-glucose-stimulated chemotaxis of human neutrophils.

The nucleotide-sugar–activated P2Y14 receptor (P2Y14-R) is highly expressed in hematopoietic cells. Although the physiologic functions of this receptor remain undefined, it 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. A series of molecules recently were identified by Gauthier et al. (Gauthier et al., 2011) that exhibited antagonist activity at the P2Y14-R. We synthesized one of these molecules, a 4,7-disubstituted 2-naphthoic acid derivative (PPTN), and studied its pharmacological properties in detail. The concentration-effect curve of UDP-glucose for promoting inhibition of adenylyl cyclase in C6 glioma cells stably expressing the P2Y14-R was shifted to the right in a concentration-dependent manner by PPTN. Schild analyses revealed that PPTN-mediated inhibition followed competitive kinetics, with a KB of 434 pM observed. In contrast, 1 μM PPTN exhibited no agonist or antagonist effect at the P2Y1, P2Y2, P2Y4, P2Y6, P2Y11, P2Y12, or P2Y13 receptors. UDP-glucose–promoted chemotaxis of differentiated HL-60 human promyelocytic leukemia cells was blocked by PPTN with a concentration dependence consistent with the KB determined with recombinant P2Y14-R. In contrast, the chemotactic response evoked by the chemoattractant peptide fMetLeuPhe was unaffected by PPTN. UDP-glucose–promoted chemotaxis of freshly isolated human neutrophils also was blocked by PPTN. In summary, this work establishes PPTN as a highly selective high-affinity antagonist of the P2Y14-R that is useful for interrogating the action of this receptor in physiologic systems.

Exploring a 2-naphthoic acid template for the structure-based design of P2Y14 receptor antagonist molecular probes.

The P2Y14 receptor (P2Y14R), one of eight P2Y G protein-coupled receptors (GPCR), is involved in inflammatory, endocrine, and hypoxic processes and is an attractive pharmaceutical target. The goal of this research is to develop high-affinity P2Y14R fluorescent probes based on the potent and highly selective antagonist 4-(4-(piperidin-4-yl)-phenyl)-7-(4-(trifluoromethyl)-phenyl)-2-naphthoic acid (6, PPTN). A model of hP2Y14R based on recent hP2Y12R X-ray structures together with simulated antagonist docking suggested that the piperidine ring is suitable for fluorophore conjugation while preserving affinity. Chain-elongated alkynyl or amino derivatives of 6 for click or amide coupling were synthesized, and their antagonist activities were measured in hP2Y14R-expressing CHO cells. Moreover, a new Alexa Fluor 488 (AF488) containing derivative 30 (MRS4174, Ki = 80 pM) exhibited exceptionally high affinity, as compared to 13 nM for the alkyne precursor 22. A flow cytometry assay employing 30 as a fluorescent probe was used to quantify specific binding to P2Y14R. Known P2Y receptor ligands inhibited binding of 30 with properties consistent with their previously established receptor selectivities and affinities. These results illustrate that potency in this series of 2-naphthoic acid derivatives can be preserved by chain functionalization, leading to highly potent fluorescent molecular probes for P2Y14R. Such conjugates will be useful tools in expanding the SAR of this receptor, which still lacks chemical diversity in its collective ligands. This approach demonstrates the predictive power of GPCR homology modeling and the relevance of newly determined X-ray structures to GPCR medicinal chemistry.

4-Alkyloxyimino derivatives of uridine-5'-triphosphate: distal modification of potent agonists as a strategy for molecular probes of P2Y2, P2Y4, and P2Y6 receptors.

Extended N4-(3-arylpropyl)oxy derivatives of uridine-5′-triphosphate were synthesized and potently stimulated phospholipase C stimulation in astrocytoma cells expressing G protein-coupled human (h) P2Y receptors (P2YRs) activated by UTP (P2Y2/4R) or UDP (P2Y6R). The potent P2Y4R-selective N4-(3-phenylpropyl)oxy agonist was phenyl ring-substituted or replaced with terminal heterocyclic or naphthyl rings with retention of P2YR potency. This broad tolerance for steric bulk in a distal region was not observed for dinucleoside tetraphosphate agonists with both nucleobases substituted. The potent N4-(3-(4-methoxyphenyl)-propyl)oxy analogue 19 (EC50: P2Y2R, 47 nM; P2Y4R, 23 nM) was functionalized for chain extension using click tethering of fluorophores as prosthetic groups. The BODIPY 630/650 conjugate 28 (MRS4162) exhibited EC50 values of 70, 66, and 23 nM at the hP2Y2/4/6Rs, respectively, and specifically labeled cells expressing the P2Y6R. Thus, an extended N4-(3-arylpropyl)oxy group accessed a structurally permissive region on three Gq-coupled P2YRs, and potency and selectivity were modulated by distal structural changes. This freedom of substitution was utilized to design of a pan-agonist fluorescent probe of a subset of uracil nucleotide-activated hP2YRs.

Small Molecule Inhibitors of Phospholipase C from a Novel High-throughput Screen

Background: Phospholipase C (PLC) isozymes are increasingly attractive therapeutic targets; however, pharmacological modulators are lacking. Results: A facile fluorescent high-throughput screen was developed and used to identify small molecule inhibitors of PLC activity. Conclusion: The new assay is robust and suitable for the rapid discovery of novel PLC modulators. Significance: This new methodology eliminates the major roadblock hampering the discovery of small molecule PLC inhibitors. Phospholipase C (PLC) isozymes are important signaling molecules, but few small molecule modulators are available to pharmacologically regulate their function. With the goal of developing a general approach for identification of novel PLC inhibitors, we developed a high-throughput assay based on the fluorogenic substrate reporter WH-15. The assay is highly sensitive and reproducible: screening a chemical library of 6280 compounds identified three novel PLC inhibitors that exhibited potent activities in two separate assay formats with purified PLC isozymes in vitro. Two of the three inhibitors also inhibited G protein-coupled receptor-stimulated PLC activity in intact cell systems. These results demonstrate the power of the high-throughput assay for screening large collections of small molecules to identify novel PLC modulators. Potent and selective modulators of PLCs will ultimately be useful for dissecting the roles of PLCs in cellular processes, as well as provide lead compounds for the development of drugs to treat diseases arising from aberrant phospholipase activity.

Membrane-induced Allosteric Control of Phospholipase C-β Isozymes

Background: Phospholipase C-β (PLC-β) isozymes hydrolyze phosphatidylinositol 4,5-bisphosphate to propagate signals for several physiological responses. Results: Membranes are essential for the allosteric release of autoinhibition of PLC-β isozymes. Conclusion: Activators of PLC-β release autoinhibition by orientating the isozymes at the membrane. Significance: The model described provides a better understanding of PLC-β regulation and potential mechanisms to inhibit their activation. All peripheral membrane proteins must negotiate unique constraints intrinsic to the biological interface of lipid bilayers and the cytosol. Phospholipase C-β (PLC-β) isozymes hydrolyze the membrane lipid phosphatidylinositol 4,5-bisphosphate (PIP2) to propagate diverse intracellular responses that underlie the physiological action of many hormones, neurotransmitters, and growth factors. PLC-β isozymes are autoinhibited, and several proteins, including Gαq, Gβγ, and Rac1, directly engage distinct regions of these phospholipases to release autoinhibition. To understand this process, we used a novel, soluble analog of PIP2 that increases in fluorescence upon cleavage to monitor phospholipase activity in real time in the absence of membranes or detergents. High concentrations of Gαq or Gβ1γ2 did not activate purified PLC-β3 under these conditions despite their robust capacity to activate PLC-β3 at membranes. In addition, mutants of PLC-β3 with crippled autoinhibition dramatically accelerated the hydrolysis of PIP2 in membranes without an equivalent acceleration in the hydrolysis of the soluble analog. Our results illustrate that membranes are integral for the activation of PLC-β isozymes by diverse modulators, and we propose a model describing membrane-mediated allosterism within PLC-β isozymes.

4-Alkyloxyimino-cytosine nucleotides: tethering approaches to molecular probes for the P2Y6 receptor

4-Alkyloxyimino derivatives of pyrimidine nucleotides display high potency as agonists of certain G protein-coupled P2Y receptors (P2YRs). In an effort to functionalize a P2Y6R agonist for fluorescent labeling, we probed two positions (N4 and γ-phosphate of cytidine derivatives) with various functional groups, including alkynes for click chemistry. Functionalization of extended imino substituents at the 4 position of the pyrimidine nucleobase of CDP preserved P2Y6R potency generally better than γ-phosphoester formation in CTP derivatives. Fluorescent Alexa Fluor 488 conjugate 16 activated the human P2Y6R expressed in 1321N1 human astrocytoma cells with an EC50 of 9 nM, and exhibited high selectivity for this receptor over other uridine nucleotide-activated P2Y receptors. Flow cytometry detected specific labeling with 16 to P2Y6R-expressing but not to wild-type 1321N1 cells. Additionally, confocal microscopy indicated both internalized 16 (t1/2 of 18 min) and surface-bound fluorescence. Known P2Y6R ligands inhibited labeling. Theoretical docking of 16 to a homology model of the P2Y6R predicted electrostatic interactions between the fluorophore and extracellular portion of TM3. Thus, we have identified the N4-benzyloxy group as a structurally permissive site for synthesis of functionalized congeners leading to high affinity molecular probes for studying the P2Y6R.

Synthesis of extended uridine phosphonates derived from an allosteric P2Y2 receptor ligand.

In this study we report the synthesis of C5/C6-fused uridine phosphonates that are structurally related to earlier reported allosteric P2Y2 receptor ligands. A silyl-Hilbert-Johnson reaction of six quinazoline-2,4-(1H,3H)-dione-like base moieties with a suitable ribofuranosephosphonate afforded the desired analogues after full deprotection. In contrast to the parent 5-(4-fluoropheny)uridine phosphonate, the present extended-base uridine phosphonates essentially failed to modulate the P2Y2 receptor.

A membrane-associated, fluorogenic reporter for mammalian phospholipase C isozymes

A diverse group of cell-surface receptors, including many G protein-coupled receptors and receptor tyrosine kinases, activate phospholipase C (PLC) isozymes to hydrolyze phosphatidylinositol 4,5-bisphosphate into the second messengers diacylglycerol and 1,4,5-inositol trisphosphate. Consequently, PLCs control various cellular processes, and their aberrant regulation contributes to many diseases, including cancer, atherosclerosis, and rheumatoid arthritis. Despite the widespread importance of PLCs in human biology and disease, it has been impossible to directly monitor the real-time activation of these enzymes at membranes. To overcome this limitation, here we describe XY-69, a fluorogenic reporter that preferentially partitions into membranes and provides a selective tool for measuring the real-time activity of PLCs as either purified enzymes or in cellular lysates. Indeed, XY-69 faithfully reported the membrane-dependent activation of PLC-β3 by Gαq. Therefore, XY-69 can replace radioactive phosphatidylinositol 4,5-bisphosphate used in conventional PLC assays and will enable high-throughput screens to identify both orthosteric and allosteric PLC inhibitors. In the future, cell-permeable variants of XY-69 represent promising candidates for reporting the activation of PLCs in live cells with high spatiotemporal resolution.