Celltransmissions The Newsletter for Cell Signaling and Neuroscience Research Vol 20, No 2 • June 2004 New Products pp. 12-14 In this Issue... Monoclonal Anti-γ Parvin: Focal adhesion protein p. 9 S(–)-Blebbistatin: Non-muscle An Update on Ligands for Prostanoid Receptors myosin II inhibitor p. 9 Robert Jones APHA Compound 8: Histone deacetylase inhibitor p. 9 17-AAG: Potent Hsp90 he natural prostanoids arise from the activity female reproductive tracts [2]. PGE2 and PGF2α inhibitor p. 10 of intracellular cyclo-oxygenase (COX) on were shown to be potent stimulants of uterine T Ro 48-8071: 2,3-Oxido- polyunsaturated fatty acids released from mem- smooth muscle and this triggered further squalene:lanosterol cyclase brane phospholipids. In the case of the most research into their roles in normal parturition. In inhibitor p. 10 important substrate, arachidonic acid (20:4ω6), addition, analogs were synthesized for the safe Anti-FKHR: Transcription the first product is prostaglandin G2 (PGG2), induction of parturition. PGF2α was also found factor marker p. 10 which is then reduced to PGH2 by an associated to induce luteolysis in many laboratory and farm 15-hydroperoxidase [1]. Depending on the animals, and in some species has been shown to OMPT: LPA3 lysophosphatidic tissue, PGH2 may be further metabolized by iso- function as a uterine luteolytic hormone [3]. acid receptor agonist p. 11 merases to PGD , PGE , PGI (prostacyclin and Thus, following release from the uterus, PGF2α 2 2 2 SB-239063: p38 MAP kinase TXA2 and by a reductase to PGF2α. induces regression of the corpus luteum, a fall in progesterone level and termination of the estrus inhibitor p. 11 cycle. The therapeutic potential of this mecha- Anti-Tuberin (IA-22): Protein The natural prostanoids perform a variety of nism for post-coital contraception and the induc- product of the tumor physiological and pathological roles. The discov- tion of abortion was soon recognized. However, suppressor gene TSC2 p. 11 ery of several E and F prostaglandins at particu- the role of research interest waned with the Bohemine: cdk inhibitor p. 11 larly high concentrations in human semen stimu- realization that luteal regression in the human lated interest in their actions on both male and female is somewhat different to that in lower RHC 80267: Diacylglycerol lipase inhibitor p. 15 continued on page 3 ACE inhibitors p. 15 Application Note: Monoclonal Anti-EPCR: Protein C anticoagulation pathway marker p. 15 Improved Ex Vivo Expansion of Functional MRS 2395: P2Y12 purino- CD34+ Cells Using Stemline™ II Hematopoietic ceptor antagonist p. 16 SB-258585: 5-HT6 serotonin Stem Cell Expansion Medium receptor antagonist p. 16 Anti-Transportin 1: Nuclear Daniel W. Allison, Stacy L. Leugers, Barry J. Pronold, Gary Van Zant, import receptor marker p. 16 and Laurel M. Donahue Chrysamine G and BTA-1: β-amyloid aggregate probes p. 17 Introduction VDM11: Anandamide mem- ematopoietic stem cells (HSC) have the ability to repopulate the brane transport inhibitor p. 17 Hhematopoietic system by differentiating into all of the necessary URB597: Fatty acid amide erythroid, lymphoid, and myeloid lineages. Due to this rare ability, HSCs hydrolase (FAAH) inhibitor p. 17 are used as therapeutic agents in the treatment of malignant and benign New Phosphorylation Assay diseases of the blood forming and immune systems. There have been Kits pp. 18-19 many advances in the area of clinical HSC research, but the availability of suitable cells for transplantation still remains a major limiting factor [1,2]. continued on page 20 sigma-aldrich.com/cellsignaling 3 An Update on Ligands for Prostanoid Receptors Robert Jones (continued from cover) order animals. PGD2 has had a chequered career, being cyclase via Gs to produce inhibitory events, while EP1, FP implicated in the control of sleep [4] and as a mediator in and TP receptors couple to phospholipase C via Gq to allergen-induced disease [5]. The latter profile has been the produce excitatory events. EP3 receptors have the most subject of major new developments (see later). complex molecular biology. They exist in several isoforms through RNA splicing and usually couple to Gi, but individ- ual isoforms may also couple to Gq, Gs and G12,13 [14]. The demonstration that TXA2, a highly labile product of arachidonate metabolism in human platelets [6], could also activate this tissue and adjacent vascular smooth muscle Synthetic agonists for each prostanoid receptor have often cells stimulated enormous interest in its role in cardiovas- been designed initially to resist metabolism, and in the cular disease. Further impetus was added by the discovery case of PGI2 and TXA2 to be chemically stable as well. of prostacyclin, which could be formed from PGH2 in blood They often bear a close structural resemblance to the natu- vessels and was also unstable in physiological milieu [7,8]. ral agonist, although non-prostanoid agonists exist for EP3 Prostacyclin had opposing actions to TXA2 and the concept and IP receptors. Antagonists have emerged from the emerged of an imbalance between these agents contribut- chemical modification of a partial agonist, and more ing to various pathological states. Around the same time, recently from high throughput screening assays using the unique therapeutic profile of aspirin (acetyl salicyclic cloned prostanoid receptors. Useful ligands for each of the acid, Prod. No. A 5376) became apparent: at low doses it prostanoid receptors will be briefly discussed. The reader is irreversibly inhibits TXA2 biosynthesis in platelets without also referred to the ‘Prostanoid Receptors’ chart in The compromising the protective value of PGI2 on the blood Sigma-RBI Handbook - see http://www.sigma-aldrich.com/ vessel, while at higher doses it suppresses the pro-inflam- sigma/rbi-handbook/sg_ls_cs_rbibook_prostanoid.pdf ostanoid Receptors r matory effects of PGE2 similar to other COX inhibitors [9]. P Update on Ligands for Figure 1. DP Receptor Ligands Classification of Prostanoid Receptors Agonists COOH It thus became clear that the five primary prostanoids COOH 5 1 exhibit distinctive pharmacological profiles. In the 1970s HO HO this was the starting point for defining five types of 9 N prostanoid receptor, referred to as DP, EP, FP, IP and TP 15 N 11 CH3 [10], based on three experimental approaches: O O OH OH BW 245C • The ranking of agonist potencies on isolated tissue PGD2 (Prod. No. P 5172) (Prod. No. B 9305) preparations (e.g. PGD > PGF > PGE = PGI = TXA sigma-aldrich.com/cellsignaling 2 2α 2 2 2 COOH signifies a DP receptor) COOH 3 • The use of radiolabeled prostanoids (e.g. [ H]-PGE2) to identify saturable binding sites on cell membranes with O appropriate affinity rankings for competing ligands OH • The use of competitive receptor antagonists, although in OH SQ 27986 some cases the specificity of the antagonist was relatively OH COOH RS 93520 low. Antagonist echnical Service: 1-800-325-5832 O T N Further studies divided the EP receptor into four subtypes, N N each denoted by a subscript numeral (i.e. EP , EP , EP and H 1 2 3 O OH EP4). BW A868C (Prod. No. B 9180) der: 1-800-325-3010 In the late 1990s, verification of this classification system r came through the isolation and structural determination of the eight prostanoid receptors, and their expression in con- The DP receptor mediates inhibition of platelet aggrega- venient cell lines [11]. In addition, the development of tion [15], relaxation of vascular smooth muscle [16] and antagonists with higher affinity and specificity continued, plasma exudation [17]. Similar to other prostanoid recep- although it is surprising that useful antagonists for the FP tors, an S-configuration for the C15-secondary alcohol in receptor and some EP receptors are only now emerging ol 20, No. 2, 2004 O the natural prostanoid (PGD2) is critically important to ago- V some 40 years after the discovery of PGE2 and PGF2α. nist activity. The hydantoin BW 245C (Prod. No. B 9305) has been widely used as a selective DP agonist [18-20]; it ® Prostanoid receptors belong to the rhodopsin super family may exist as 9-oxo,11-oxo and 9,11-dioxo tautomers. of G protein-coupled receptors, and their evolution from a Inversion of all chiral centers in the ring systems of prosta- ® common PGE ancestor has been postulated [12,13]. In cyclin analogs (e.g. RS-93520) [21,22] and PGH analogs general, DP, EP , EP and IP receptors couple to adenylyl (e.g. SQ 27986) [23] also leads to selective DP agonists. 2 4 Celltransmissions 4 Ligands for Prostanoid Receptors...(continued) The DP antagonist BW A868C, a relative of BW 245C, has CRTH2 receptor [32]; its routine use to suppress prostanoid proved useful in identifying DP receptors [20,24,25]. It biosynthesis in isolated tissue systems may therefore behaves competitively (pA2 7.8 - 9.5) and shows good require reconsideration. There is much interest in develop- specificity. However, its affinity for the EP4 receptor (pA2 ing antagonists for this receptor as therapeutic agents for 5.1) may result in a right-shift of the concentration- immunological diseases. response curve for PGE2 in blood vessel preparations with highly sensitive EP4 systems [26]. EP1 receptors have a limited tissue distribution, and their activation causes contraction of smooth muscle in gut and It has been known for some time that PGD2 induces trachea. 16,16-Dimethyl PGE2 is a highly potent but non- ω effects that cannot be attributed to either the classical DP selective EP1 agonist [33], while 17-phenyl- -trinor PGE2 is receptor or FP and TP receptors; two examples are arterial more selective, and in combination with sulprostone constriction [27,28] and suppression of short-circuit current (Prod. No. S 8692) (EP3 > EP1) and SC-46275 (EP3 >> EP1) in colonic mucosa [29].