Introduction Eicosanoid lipid prostaglandin D
Introduction Eicosanoid lipid prostaglandin D2 (PGD2) is the major prostaglandin produced by activated mast Retigabine dihydrochloride (Lewis and Austen, 1981). The physiological function of PGD2 is mainly mediated by two G protein-coupled receptors (GPCRs), PGD2 receptor 1 and 2 (DP1 and DP2), which share modest sequence similarity and couple to different G proteins (Monneret et al., 2001, Nagata et al., 1999). DP2 is more commonly called the chemoattractant receptor-homologous molecule expressed on Th2 cells (CRTH2). While DP1 is closely related to other prostaglandin receptors, CRTH2 is more akin to a group of leukocyte non-chemokine chemoattractant GPCRs, which also includes the receptors for anaphylatoxin C3a and C5a, formylpeptides, leukotrienes, and some other eicosanoids (Fredriksson et al., 2003, Nagata and Hirai, 2003, Serhan, 2014) (Figure S1A). These non-chemokine chemoattractant receptors share a relatively high sequence similarity and the same preference for Gi protein, but they recognize diverse ligands, including lipids, peptides, and large proteins. Despite much evidence linking this group of receptors to a number of inflammatory diseases, no drugs that specifically target this group of GPCRs are currently commercially available. CRTH2 is highly expressed in type 2 helper T cells (Th2), innate lymphoid cells (ILCs), eosinophils, and basophils (Cosmi et al., 2000, Hirai et al., 2001, Mjösberg et al., 2011, Nagata et al., 1999). PGD2-CRTH2 signaling is a major pathway in type 2 inflammation, leading to the activation of immune cells and the production of type 2 cytokines (Monneret et al., 2001, Xue et al., 2005). Thus, CRTH2 has emerged as a promising new target in treating type 2 inflammation-driven diseases, such as asthma and allergic rhinitis, which has spurred intensive research efforts in developing CRTH2 antagonists for clinical investigation (Kupczyk and Kuna, 2017, Pettipher et al., 2007, Pettipher and Whittaker, 2012, Schuligoi et al., 2010). The first nonlipid CRTH2 antagonist, ramatroban, was discovered by serendipity (Hirai et al., 2002, Sugimoto et al., 2003). Ramatroban was initially developed as a thromboxane receptor antagonist drug used in Japan for treating allergic diseases; it was then proven to also be a CRTH2 antagonist. Modification of ramatroban led to the discovery of the first potent and selective CRTH2 antagonist, CAY10471 (also named TM30089), which exhibits insurmountable action, in contrast to the reversible action of ramatroban in some assays (Mathiesen et al., 2006, Ulven and Kostenis, 2005). Such early studies have inspired a number of companies to develop numerous CRTH2 antagonists with diverse chemical scaffolds and pharmacological properties in the past decade (Kupczyk and Kuna, 2017, Pettipher and Whittaker, 2012, Santus and Radovanovic, 2016). Several of these antagonists have been tested in asthma patients, but the results were mixed (Barnes et al., 2012, Busse et al., 2013, Erpenbeck et al., 2016, Kuna et al., 2016, Miller et al., 2017, Pettipher et al., 2014). It has been suggested that a subpopulation of asthmatic patients whose airway inflammation is largely driven by Th2-type inflammation would benefit most from CRTH2 antagonists (Kupczyk and Kuna, 2017). Recently, a potent CRTH2 antagonist, fevipiprant, showed promising clinical efficacy in patients with uncontrolled asthma in a few clinical trials (White et al., 2018). Thus, CRTH2 antagonists hold the promise of being a new class of asthma drugs, and the development of new CRTH2 antagonists remains highly competitive, as evidenced by the continuing clinical investigation initiated by many companies with their own compounds (Kupczyk and Kuna, 2017, Pettipher and Whittaker, 2012). Similar to PGD2, nearly all of the CRTH2 antagonists are carboxylic acid derivatives with a carboxylate moiety, which is believed to be a critical pharmacophore that interacts with the receptor (Pettipher and Whittaker, 2012) (Figure 1A). To understand the molecular mechanisms for the action of CRTH2 ligands, we solved the crystal structures of human CRTH2 bound to two antagonists, fevipiprant and CAY10471. The structures, together with the results from computational docking studies and ligand binding assays, reveal conserved and divergent structural features for the binding of diverse CRTH2 antagonists, which occupy a semi-occluded ligand-binding pocket covered by a well-structured N-terminal region with a novel conformation. Interesting characteristics of the ligand binding pocket, including a widely open end as the potential ligand entry port and a gradually increased positive charge distribution, allow us to propose a novel mechanism for the binding of PGD2. Structural comparison analysis suggests a distinct binding pose of PGD2 compared to the lysophospholipids and endocannabinoids.