1521-0081/69/3/316–353$25.00 https://doi.org/10.1124/pr.116.013243 PHARMACOLOGICAL REVIEWS Pharmacol Rev 69:316–353, July 2017 Copyright © 2017 by The American Society for Pharmacology and Experimental Therapeutics ASSOCIATE EDITOR: STEPHEN P. H. ALEXANDER Chemical Tools for Studying Lipid-Binding Class A G Protein–Coupled Receptors Anna Cooper, Sameek Singh, Sarah Hook, Joel D. A. Tyndall, and Andrea J. Vernall School of Pharmacy, University of Otago, Dunedin, New Zealand Abstract. ....................................................................................317 I. Introduction. ..............................................................................317 A. Cannabinoid Receptor . ..................................................................317 1. Ligand Classes. ......................................................................318 B. Free Fatty Acid Receptor ................................................................320 1. Ligand Classes. ......................................................................320 C. Lysophospholipid Receptors. ............................................................321 1. Ligand Classes. ......................................................................321 D. Prostanoid Receptor . ..................................................................321 Downloaded from 1. Ligand Classes. ......................................................................322 E. Leukotriene Receptor . ..................................................................322 1. Ligand Classes. ......................................................................322 F. Bile Acid Receptor.......................................................................322 1. Ligand Classes. ......................................................................322 G. Platelet-Activating Factor Receptor ......................................................322 by guest on September 27, 2021 1. Ligand Classes. ......................................................................323 H. Orphan G Protein–Coupled Receptors....................................................323 I. Rational Design of Tools and Considerations for Use .....................................323 II. Radioligands . ..............................................................................324 A. Characteristics and Design Rationale ....................................................324 B. Applications of Radioligands . ............................................................324 1. Radioligands for In Vitro Experiments. ...............................................324 2. Radioligands for In Vivo Experiments. ................................................327 III. Small-Molecule Fluorescent Tools............................................................330 A. Characteristics and Design Rationale ....................................................330 B. Applications of Small-Molecule Fluorescent Tools . .....................................331 1. Fluorescent Tools for In Vitro Experiments.. ..........................................331 2. Near-Infrared Wavelength Fluorescent Tools for In Vivo Imaging. ...................334 IV. Covalent Tools ..............................................................................335 A. Characteristics and Design Rationale ....................................................335 B. Applications of Covalent Tools ...........................................................336 1. Photoactivatable Covalent Tools. .....................................................336 2. Electrophilic Covalent Ligands. ......................................................340 3. Bifunctional Covalent Ligands. ......................................................342 V. Antibodies ..................................................................................342 A. Characteristics of Antibodies. ............................................................342 B. Antibody Limitations . ..................................................................343 C. Applications of Antibodies . ............................................................343 D. Beyond Antibodies.......................................................................344 A.C. and S.S. contributed equally to this work. Address correspondence to: Dr. Andrea J. Vernall, School of Pharmacy, University of Otago, 18 Frederick Street, PO Box 56, Dunedin 9054, New Zealand. E-mail: [email protected] https://doi.org/10.1124/pr.116.013243. 316 Radioligands, Antibodies, Fluorescent, and Covalent Tools 317 VI. Conclusions and Future Outlook . ............................................................345 Acknowledgments . ........................................................................345 References ..................................................................................345 Abstract——Cannabinoid, free fatty acid, lysophospha- receptors is balancing the often lipophilic requirements tidic acid, sphingosine 1-phosphate, prostanoid, leukotri- of the receptor-binding pharmacophore with favorable ene, bile acid, and platelet-activating factor receptor physicochemical properties to optimize highly specific families are class A G protein–coupled receptors with binding. In this study, we review the radioligands, endogenous lipid ligands. Pharmacological tools are fluorescent ligands, covalent ligands, and antibodies crucial for studying these receptors and addressing the that have been used to study these lipid-binding many unanswered questions surrounding expression receptors. For each tool type, the characteristics and of these receptors in normal and diseased tissues. An design rationale along with in vitro and in vivo inherent challenge for developing tools for these lipid applications are detailed. I. Introduction GPCR structure, the static nature somewhat limits the use in unraveling dynamic receptor processes. The Cannabinoid (CB), free fatty acid (FFA), lysophos- chemical tools for CB, FFA, LPA, S1P, prostanoid, phatidic acid (LPA), sphingosine 1-phosphate (S1P), leukotriene, GPBA, and PAF receptors discussed in this prostanoid (DP, EP, FP, IP, TP), leukotriene [leukotri- review can help elucidate receptor physiologic role and ene B(4) (LTB4), cysteinyl leukotriene (CysLT), oxoei- dynamic signaling events in live cells and in native cosanoid (OXE), formyl peptide receptor (FPR)2], bile environments, thus complementing available structural acid receptor (GPBA receptor), and platelet-activating information. factor (PAF) receptors are class A G protein–coupled receptors (GPCRs) that are activated by lipid-derived A. Cannabinoid Receptor endogenous ligands. GPCRs are the largest family of Two CB receptor subtypes (CB and CB ), endocan- transmembrane-signaling proteins in the human ge- 1 2 nabinoid (ECB) endogenous ligands, and the corre- nome and convey signals from a wide range of stimuli, sponding regulatory enzymes are part of the ECB including neurotransmitters, peptide hormones, and system (Katona and Freund, 2012). CB1 receptor (the autocrine factors (Katritch et al., 2012), thus offering then only identified CB receptor) was characterized in huge potential for disease therapy (Jacobson, 2015). 1988 through investigation of binding of the known CB Chemical tools are invaluable for interrogating GPCR receptor radioligand [3H]CP-55,940 (Fig. 1; Table 1) in structure and physiologic role, which facilitates effica- rat brain membranes (Devane et al., 1988), and it was cious drug development. GPCRs can mediate different cloned in 1990 (Matsuda et al., 1990). CB1 receptor is signaling pathways and are dynamic, flexible, and abundant in the brain, particularly on central and sensitive to many proximal stimuli (Kobilka and Deupi, peripheral neurons in the presynapse (Kano et al., 2007); therefore, studies using live cells in native 2009), postsynaptic cells, and astrocytes (Castillo environments often reveal more relevant information. et al., 2012), and is found in low levels in peripheral However, scientists face challenges with GPCR re- organs (Engeli et al., 2005). CB1 receptor can activate search, including very low receptor expression levels multiple, diverse signaling pathways; for example, it in native endogenous cells and tissues (Fredriksson and can couple with Gi,Gs, and/or Gq proteins (Bosier et al., Schiöth, 2005). In addition, GPCRs are difficult to 2010) and modulate calcium and potassium channels. crystallize due to conformational flexibility, a fluid CB2 receptor was cloned from spleen in 1993 (Munro native membrane environment, and lack of stability in et al., 1993) and is expressed predominantly in immune detergents (Salom et al., 2013). In spite of this, the cells, lymphoid tissues, such as the spleen and tonsils number of reported GPCR crystal structures has ex- (Galiegue et al., 1995), and at low levels in central ploded in recent years, opening up opportunities for nervous system (CNS) tissues such as microglial cells rational structure-based design of drugs and tools (Savonenko et al., 2015). Two crystal structures of (Cooke et al., 2015; Ghosh et al., 2015). Although the human CB1 receptor have recently been published, X-ray crystallography offers invaluable insights into one complexed with a modified rimonabant-based ABBREVIATIONS: 2-AG, 2-arachidonylglycerol; 2-AGE, 2-arachidonyl glyceryl ether; ALX, lipoxin A4; AEA, anandamide; BBB, blood brain barrier; BRET, bioluminescence resonance energy transfer; CB, cannabinoid; CNS, central nervous system; CysLT, cysteinyl leukotriene; DBT, delayed