6 - Experimental Challenges to Targeting Poorly Characterized GPCRs: Uncovering the Therapeutic Potential for Free Fatty Acid Receptors

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Abstract

The G protein-coupled receptors (GPCRs) are extremely successful drug targets, with recent estimates suggesting that approximately 30% of all currently available therapeutics act at these receptors. Despite this success, only a small number of the over 400 known nonodorant GPCRs are currently targeted, suggesting there is still untapped therapeutic potential. However, as most GPCRs were identified based on their sequence homology to other members of the superfamily, many still remain “orphan” receptors without known ligands. Indeed, even once a GPCR has been deorphanized, the receptor typically is still poorly characterized in terms of its pharmacology and biological functions, presenting a unique set of experimental challenges in order to define its therapeutic potential. We discuss some of these challenges and how they have been addressed in order to uncover the therapeutic potential of five recently deorphanized receptors that are activated by short- and long-chain free fatty acids.

Introduction

G protein-coupled receptors (GPCRs) represent the largest superfamily of integral membrane proteins. The human genome encodes approximately 400 nonodorant GPCRs (Fredriksson and Schioth, 2005, Takeda et al., 2002, Vassilatis et al., 2003), which are activated by extremely diverse stimuli, including hormones, neurotransmitters, Ca2+ ions, lipids, photons, and peptides (Lagerstrom and Schioth, 2008, Perez, 2003, Pierce et al., 2002). The ability of GPCRs to act as signal transducers, binding an extracellular ligand in order to produce an intracellular biological response, has made these receptors the most historically successful drug targets (Hopkins & Groom, 2002). Indeed, recent estimates suggest that approximately 30% of all currently used pharmaceutical agents target GPCRs (Fredriksson and Schioth, 2005, Jacoby et al., 2006, Summers, 2010). However, despite their success as drug targets, only approximately 30 of the more than 400 known nonodorant GPCRs are currently targeted by therapeutics (Tyndall & Sandilya, 2005), suggesting that there are still many additional GPCR drug targets yet to be exploited.

Historically, pharmacological investigations have been carried out by first identifying a biological response to a ligand, and then subsequently using that ligand to identify the receptor(s) responsible for the response (Wilson et al., 1998). However, this approach needed revision with the discovery of the GPCR superfamily, which began following the observation that the β2-adrenoceptor contained both sequence homology and a similar seven-transmembrane topography to rhodopsin (Dixon et al., 1986). The identification of the GPCR superfamily, in turn, allowed DNA hybridization and PCR-derived techniques to rapidly identify more GPCRs (Bunzow et al., 1988, Libert et al., 1989, Wilson et al., 1998). However, the discovery of GPCRs in this manner meant that the identified receptors were “orphans,” in that the nature of their ligands was unknown.

The study of orphan GPCRs necessitated a reversal of the classic approach to pharmacology, whereby now instead of using a ligand to identify its receptor, the receptor needed to be used to identify potential ligands. Initially, orphan GPCR research was commonly carried out by informed approaches, considering sequence homology with GPCRs that had known ligands, and the tissue distribution of the orphan receptor, to identify likely ligands (Civelli et al., 2006, Wise et al., 2004). However, as the number of orphan GPCRs increased, such approaches became less useful, and researchers turned to higher throughput approaches, based on screening many known, or potential, GPCR ligands against the orphan receptor (Milligan, 2002, Szekeres, 2002). This approach to GPCR research, sometimes referred to as “reverse pharmacology,” has provided a steady supply of novel potential therapeutic targets, although it has been noted that the rate of GPCR deorphanization has slowed in recent years. Moreover, due to the nature of orphan receptor research, it has also provided a new set of experimental challenges to validate these receptors as targets for potentially useful therapeutics.

Once an orphan GPCR has been successfully paired with its endogenous ligand(s), it typically is still very poorly characterized and, therefore, there are many additional steps that need to be carried out before it can be considered a viable therapeutic target. Initially, screening approaches aimed at GPCR deorphanization often utilize readouts of GPCR activation that are independent of the specific signaling pathways activated by the receptor. These include assays based on receptor internalization, β-arrestin association, and Ca2+ mobilization promoted by promiscuous or chimeric G proteins (Eglen et al., 2007, Kostenis et al., 2005, Milligan, 2002, Szekeres, 2002). As a result, one of the initial challenges is often to identify and define the G protein coupling and signaling pathways normally activated by the receptor. Additional key steps at the level of receptor pharmacology include developing an understanding of how the ligand binds to the receptor and, in turn, using this information to identify novel selective ligands (Jacoby et al., 2006, Mobarec et al., 2009). In parallel with such basic characterization of receptor pharmacology and function, the other primary requirement for developing a poorly characterized receptor toward a potential therapeutic target is prediction and confirmation of specific disease states and pathologies the receptor may be useful in treating. The first steps for this normally involve establishing the biological role of the receptor, through a variety of in vitro and in vivo approaches including knockdown or knockout of the receptor in cellular or animal models and searches for disease associations via genetic linkage or understanding of polymorphic variation. Once the biological functions of the receptor have been uncovered, this information can be used to inform likely therapeutic targets for ligands that regulate the receptor, which will then require verification by proof-of-principle studies (Chung et al., 2008, Jacoby et al., 2006).

While the specific set of challenges associated with deorphanization and transforming each poorly characterized GPCR into a useful drug target will be unique, much can be learned by considering how these issues have been addressed for other receptors. One group of poorly characterized GPCRs that have recently received substantial interest as potential therapeutic targets for a wide range of pathologies are the GPCRs activated by free fatty acids (FFAs) (Hirasawa et al., 2008a, Hirasawa et al., 2008b, Milligan et al., 2006, Rayasam et al., 2007). These include the three receptors currently classified as the FFA family, FFA1, FFA2, and FFA3, as well as two additional receptors GPR120 and GPR84 (Milligan et al., 2006, Stoddart et al., 2008a, Stoddart et al., 2008b). Herein, we describe the experimental challenges that have arisen in attempting to develop FFA receptors into therapeutic targets, how these have been, at least partially, addressed and what challenges still remain to be resolved before the full therapeutic value of these receptors can be realized.

Section snippets

Deorphanization of the FFA Family

The FFA family of GPCRs was first identified by Sawzdargo et al. (1997) who used degenerate primers to search human genomic DNA for subtypes of the galanin receptor. They amplified a noncoding section of CD22 that was found to contain two intronless putative GPCRs: GPR40 (now known as FFA1) and GPR41 (now called FFA3). Further examination of the following sequence revealed two additional intronless sequences predicted to encode GPR42 and GPR43 (also known as FFA2). Initial deorphanization

G Protein Coupling and Signal Transduction

Once a receptor has been deorphanized, one of the next key steps in developing it into a potential therapeutic target is the elucidation of the signaling pathways to which the receptor is coupled. For GPCRs, this initially involves the identification of which G proteins the receptor couples with, but given that it is now clear that GPCRs may also produce G protein-independent signaling responses (Lefkowitz, 2007, Lefkowitz and Shenoy, 2005), it is important to also consider additional signaling

Synthetic Ligands for FFA Receptors

The identification of selective synthetic ligands is critical to elucidate the pharmacology and therapeutic potential of any GPCR in vitro and in vivo. This is particularly important for the FFA receptors given the poor potencies observed for the endogenous FFAs at these receptors. In addition, the significant overlap among the endogenous ligands of the two LCFA receptors FFA1 and GPR120 (Suzuki et al., 2008), as well as the overlap in ligands for the two SCFA receptors FFA2 and FFA3 (Milligan

Therapeutic Potential for FFA Receptors

The final step in developing a GPCR into a viable drug target is the identification and subsequent validation of its therapeutic potential in the treatment of a specific pathology. Typically, the tissue expression pattern for the receptor is the first piece of information available and this is often used to infer likely biological function of the receptor. From there, a variety of in vitro and in vivo approaches must be taken in order to define the function of the receptor and ultimately

Conclusion

Uncovering the pharmacology, biological functions, and resulting therapeutic potential of the FFA receptors has presented significant experimental challenges. In particular, the low potencies observed for all endogenous FFAs at these receptors and the lack of available tools, including suitable radioligands for binding assays and antagonists for many of the receptors, have made establishing the pharmacology of the FFA receptors difficult. Overlap in the endogenous ligands and the lack of

Acknowledgments

This work was supported by The Welcome Trust (Grant 089600/Z/09/Z), the Biotechnology and Biosciences Research Council (grant BB/E019455/1) and an Australian C.J. Martin National Health and Medical Research Council and National Heart Foundation Overseas Research Fellowship (to N.J.S.).

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