Identification of indole inhibitors of human hematopoietic prostaglandin D 2 synthase (hH-PGDS) Fredrik Edfeldt a,⇑ , Johan Evenäs b , Matti Lepistö b , Alison Ward b , Jens Petersen a , Lisa Wissler a , Mattias Rohman a , Ulf Sivars b , Karin Svensson b , Matthew Perry b , Isabella Feierberg a , Xiao-Hong Zhou b , Thomas Hansson b , Frank Narjes b a Discovery Sciences, Innovative Medicines, AstraZeneca R&D, 431 83 Molndal, Sweden b Respiratory, Inflammation and Autoimmunity, Innovative Medicines, AstraZeneca R&D, 431 83 Molndal, Sweden article info Article history: Received 10 March 2015 Revised 19 April 2015 Accepted 20 April 2015 Available online 28 April 2015 Keywords: Prostaglandin D 2 synthase PGDS inhibitors Indole Focused screening Hit validation abstract Human H-PGDS has shown promise as a potential target for anti-allergic and anti-inflammatory drugs. Here we describe the discovery of a novel class of indole inhibitors, identified through focused screening of 42,000 compounds and evaluated using a series of hit validation assays that included fluorescence polarization binding, 1D NMR, ITC and chromogenic enzymatic assays. Compounds with low nanomolar potency, favorable physico-chemical properties and inhibitory activity in human mast cells have been identified. In addition, our studies suggest that the active site of hH-PGDS can accommodate larger struc- tural diversity than previously thought, such as the introduction of polar groups in the inner part of the binding pocket. Ó 2015 Elsevier Ltd. All rights reserved. Prostaglandin D 2 (PGD 2 ) is an allergic and inflammatory medi- ator produced by mast cells and Th2 cells after cross-linking of an allergen with the specific IgE antibody on the cell membrane. 1 PGD 2 acts on two G-protein-coupled receptors, DP 1 and CRTH2 that have been associated with inflammatory conditions. 2 Production of PGD 2 in the peripheral tissues and in immune and inflammatory cells from the precursor prostaglandin H 2 (PGH 2 ) is mainly catalyzed by human hematopoietic prostaglandin D syn- thase (hH-PGDS). This has led hH-PGDS to be envisioned as a target for the treatment of asthma and inflammatory diseases. 3 hH-PGDS is a 26-kDa cytosolic homodimer of the sigma class glutathione-S- transferase (GST) family. It depends on glutathione (GSH) for cat- alytic activity, which is further increased by divalent metal ions. 4 Several inhibitors of hH-PGDS, most of them containing a signature bis-aryl amide motif, have been disclosed in recent years by a number of organizations, such as (1 and 2) Pfizer, 5 (3) Sanofi- Aventis, 6 (4) Evotec, 7 (5) AstraZeneca 8 and (6) Taiho 9 (Fig. 1). For a recent review of hH-PGDS inhibitors, see Ref. 3. Our previous efforts led to the identification of compound (5) as an hH-PGDS inhibitor. The current study reports our continued work on identifying novel inhibitors of this enzyme applying a fragment based screening approach, aiming to identify smaller and more ligand efficient hits. A fluorescence polarization assay 10 was used to screen two library subsets of 25,000 and 17,000 com- pounds at 125 lM and 250 lM, respectively. IC 50 values were determined for compounds showing a >30% displacement effect, resulting in 1040 compounds with IC 50 values <100 lM. To further validate the actives a 1D NMR binding assay was deployed. K d val- ues were determined using the competitive displacement of repor- ter ligands with known affinity. 11 The hits were clustered using structural similarity and 216 clus- ter representatives were selected for testing in the NMR assay. 187 of these hits were confirmed in the NMR assay, corresponding to a validation rate of 87%. Validated hits were then tested in a GST enzymatic assay using CDNB (1-chloro-2,4-dinitrobenzene) as chromogenic substrate 12 and IC 50 values were generated. We chose to monitor the GST enzymatic activity of hH-PGDS rather than its PGD 2 synthase activity due to technical feasibility. Both enzymatic activities reside in the same binding pocket. 4b,13 For a set of four reference compounds (1–3, and 5) activity in the NMR and GST assays, as well as isothermal titration calorimetry (ITC), 14 corre- lated overall well with the inhibition of PGD 2 production in megakaryoblast cells 15 (Table 1). We therefore felt confident that our screening set-up was fit for purpose in driving hit expansion chemistry in a high throughput manner. We observed excellent correlation between the NMR binding assay and inhibition of GST activity for our screening hits. 168 compounds had an IC 50 http://dx.doi.org/10.1016/j.bmcl.2015.04.065 0960-894X/Ó 2015 Elsevier Ltd. All rights reserved. ⇑ Corresponding author. Tel.: +46 31 776 1604; fax: +46 31 776 3792. E-mail address: Fredrik.Edfeldt@astrazeneca.com (F. Edfeldt). Bioorganic & Medicinal Chemistry Letters 25 (2015) 2496–2500 Contents lists available at ScienceDirect Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier.com/locate/bmcl