Article Structures of the Human PGD2 Receptor CRTH2 Reveal Novel Mechanisms for Ligand Recognition Graphical Abstract Authors Lei Wang, Dandan Yao, R.N.V. Krishna Deepak, ..., Weimin Gong, Zhiyi Wei, Cheng Zhang Correspondence [email protected] (Z.W.), [email protected] (C.Z.) In Brief Wang et al. reported crystal structures of antagonist-bound human CRTH2 as a new asthma drug target. Chemically diverse antagonists occupy a similar semi-occluded pocket with distinct binding modes. Structural analysis suggests a potential ligand entry port and an opposite charge attraction-facilitated binding process for the endogenous CRTH2 ligand prostaglandin D2. Highlights d Crystal structures of antagonist-bound human CRTH2 are solved d A well-structured N terminus covers ligand binding pocket d Conserved and divergent binding features of CRTH2 antagonists are revealed d A multiple-step binding process of prostaglandin D2 is proposed Wang et al., 2018, Molecular Cell 72, 48–59 October 4, 2018 ª 2018 Elsevier Inc. https://doi.org/10.1016/j.molcel.2018.08.009 Molecular Cell Article Structures of the Human PGD2 Receptor CRTH2 Reveal Novel Mechanisms for Ligand Recognition Lei Wang,1,7 Dandan Yao,2,3,7 R.N.V. Krishna Deepak,4 Heng Liu,1 Qingpin Xiao,1,5 Hao Fan,4 Weimin Gong,2,6 Zhiyi Wei,5,* and Cheng Zhang1,8,* 1Department of Pharmacology and Chemical Biology, School of Medicine, University of Pittsburgh, Pittsburgh, PA 15261, USA 2Key Laboratory of RNA Biology, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China 3University of Chinese Academy of Sciences, Chinese Academy of Sciences, Beijing 100049, China 4Bioinformatics Institute (BII), Agency for Science, Technology and Research (A*STAR), Singapore 138671, Singapore 5Department of Biology, Southern University of Science and Technology, Shenzhen, Guangdong 518055, China 6Hefei National Research Center for Physical Sciences at the Microscale, School of Life Sciences, University of Science and Technology of China, Hefei, Anhui 230027, China 7These authors contributed equally 8Lead Contact *Correspondence: [email protected] (Z.W.), [email protected] (C.Z.) https://doi.org/10.1016/j.molcel.2018.08.009 SUMMARY chemokine chemoattractant GPCRs, which also includes the receptors for anaphylatoxin C3a and C5a, formylpeptides, leu- The signaling of prostaglandin D2 (PGD2) through kotrienes, and some other eicosanoids (Fredriksson et al., G-protein-coupled receptor (GPCR) CRTH2 is a ma- 2003; Nagata and Hirai, 2003; Serhan, 2014)(Figure S1A). These jor pathway in type 2 inflammation. Compelling evi- non-chemokine chemoattractant receptors share a relatively dence suggests the therapeutic benefits of blocking high sequence similarity and the same preference for Gi protein, CRTH2 signaling in many inflammatory disorders. but they recognize diverse ligands, including lipids, peptides, Currently, a number of CRTH2 antagonists are under and large proteins. Despite much evidence linking this group of receptors to a number of inflammatory diseases, no drugs that clinical investigation, and one compound, fevipi- specifically target this group of GPCRs are currently commer- prant, has advanced to phase 3 clinical trials for cially available. asthma. Here, we present the crystal structures of CRTH2 is highly expressed in type 2 helper T cells (Th2), innate human CRTH2 with two antagonists, fevipiprant lymphoid cells (ILCs), eosinophils, and basophils (Cosmi et al., and CAY10471. The structures, together with dock- 2000; Hirai et al., 2001; Mjo¨ sberg et al., 2011; Nagata et al., ing and ligand-binding data, reveal a semi-occluded 1999). PGD2-CRTH2 signaling is a major pathway in type 2 pocket covered by a well-structured amino terminus inflammation, leading to the activation of immune cells and the and different binding modes of chemically diverse production of type 2 cytokines (Monneret et al., 2001; Xue CRTH2 antagonists. Structural analysis suggests et al., 2005). Thus, CRTH2 has emerged as a promising new a ligand entry port and a binding process that is target in treating type 2 inflammation-driven diseases, such facilitated by opposite charge attraction for PGD , as asthma and allergic rhinitis, which has spurred intensive 2 research efforts in developing CRTH2 antagonists for clinical which differs significantly from the binding pose investigation (Kupczyk and Kuna, 2017; Pettipher et al., 2007; and binding environment of lysophospholipids and Pettipher and Whittaker, 2012; Schuligoi et al., 2010). The first endocannabinoids, revealing a new mechanism for nonlipid CRTH2 antagonist, ramatroban, was discovered by lipid recognition by GPCRs. serendipity (Hirai et al., 2002; Sugimoto et al., 2003). Ramatro- ban was initially developed as a thromboxane receptor antago- nist drug used in Japan for treating allergic diseases; it was INTRODUCTION then proven to also be a CRTH2 antagonist. Modification of ram- atroban led to the discovery of the first potent and selective Eicosanoid lipid prostaglandin D2 (PGD2) is the major prosta- CRTH2 antagonist, CAY10471 (also named TM30089), which glandin produced by activated mast cells (Lewis and Austen, exhibits insurmountable action, in contrast to the reversible ac- 1981). The physiological function of PGD2 is mainly mediated tion of ramatroban in some assays (Mathiesen et al., 2006; Ulven by two G protein-coupled receptors (GPCRs), PGD2 receptor 1 and Kostenis, 2005). Such early studies have inspired a number and 2 (DP1 and DP2), which share modest sequence similarity of companies to develop numerous CRTH2 antagonists with and couple to different G proteins (Monneret et al., 2001; Nagata diverse chemical scaffolds and pharmacological properties in et al., 1999). DP2 is more commonly called the chemoattrac- the past decade (Kupczyk and Kuna, 2017; Pettipher and Whit- tant receptor-homologous molecule expressed on Th2 cells taker, 2012; Santus and Radovanovic, 2016). Several of these (CRTH2). While DP1 is closely related to other prostaglandin antagonists have been tested in asthma patients, but the results receptors, CRTH2 is more akin to a group of leukocyte non- were mixed (Barnes et al., 2012; Busse et al., 2013; Erpenbeck 48 Molecular Cell 72, 48–59, October 4, 2018 ª 2018 Elsevier Inc. Figure 1. CRTH2 Ligands and Overall Structures (A) Chemical structures of PGD2, fevipiprant, CAY10471 and ramatroban and the conserved carboxylate group. 3 (B) Competition radioactive ligand binding assays with HEK293T cell membranes expressing wtCRTH2 and CRTH2-mT4L. For each experiment, 2 nM [ H] PGD2 was used and various concentrations of PGD2 (top), CAY10471 (middle), and fevipirant (bottom) were added as competing ligands. Data points are presented as the mean values ± SEM, n = 3. (C) Overall structures of fevipiprant-bound and CAY10471-bound CRTH2 are colored in blue and slate, respectively. Fevipiprant and CAY10471 are shown as orange and yellow spheres. et al., 2016; Kuna et al., 2016; Miller et al., 2017; Pettipher et al., Interesting characteristics of the ligand binding pocket, including 2014). It has been suggested that a subpopulation of asthmatic a widely open end as the potential ligand entry port and a patients whose airway inflammation is largely driven by Th2- gradually increased positive charge distribution, allow us to pro- type inflammation would benefit most from CRTH2 antagonists pose a novel mechanism for the binding of PGD2. Structural (Kupczyk and Kuna, 2017). Recently, a potent CRTH2 antago- comparison analysis suggests a distinct binding pose of PGD2 nist, fevipiprant, showed promising clinical efficacy in patients compared to the lysophospholipids and endocannabinoids. with uncontrolled asthma in a few clinical trials (White et al., 2018). Thus, CRTH2 antagonists hold the promise of being RESULTS a new class of asthma drugs, and the development of new CRTH2 antagonists remains highly competitive, as evidenced Crystallization of CRTH2 and Overall Structures by the continuing clinical investigation initiated by many com- To crystallize human CRTH2, a construct of human CRTH2 was panies with their own compounds (Kupczyk and Kuna, 2017; generated by inserting an engineered T4 lysozyme (mT4L) Pettipher and Whittaker, 2012). (Thorsen et al., 2014) with an additional N-terminal 8-amino Similar to PGD2, nearly all of the CRTH2 antagonists are car- acid linker into the intracellular loop 3 (ICL3) for crystallization boxylic acid derivatives with a carboxylate moiety, which is (Figure S1B). The 8-amino acid linker greatly improved crystal believed to be a critical pharmacophore that interacts with the quality, which was achieved unintentionally. To further facilitate receptor (Pettipher and Whittaker, 2012)(Figure 1A). To under- crystallogenesis, the flexible C-terminal region from R340 to stand the molecular mechanisms for the action of CRTH2 li- S395 was removed before crystallization, and the potential gands, we solved the crystal structures of human CRTH2 bound glycosylation site N25 was mutated to alanine. No other muta- to two antagonists, fevipiprant and CAY10471. The structures, tions were introduced. Ligand competition binding assays together with the results from computational docking studies showed that the sequence modifications did not significantly and ligand binding assays, reveal conserved and divergent affect the ligand-binding properties of CRTH2 (Figure 1B). Using structural features for the binding of diverse CRTH2 antagonists, this construct, we solved the crystal structures of human CRTH2 which occupy a semi-occluded ligand-binding