Maximizing serendipity: strategies for identifying ligands for orphan G-protein-coupled receptors

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Abstract

G-protein-coupled receptors (GPCRs) represent the largest family of cell-surface receptors within the human genome, and historically these have been a rich source of targets for small-molecule modulation and therapeutic intervention. As a result of genome closure, numerous novel GPCRs that have unknown ligands and function were identified, and termed ‘orphans’. These are considered potential new targets for drug discovery, and many companies have been focusing on ligand identification using high-throughput functional assays in the quest to discover a tool to further probe the pathophysiolgical role of these new receptors. In the past five years, approximately 50 receptors have been ligand-paired, although putative functions have only been described for the minority. The number of new small-molecule modulators that ultimately make it to the market will measure the success of this initiative.

Introduction

G-protein-coupled receptors (GPCRs) are members of one of the largest known gene families. Approximately 250 genes encode established non-sensory GPCRs, and a further 140 genes are predicted by bioinformatic analysis of sequence databases. These novel genes encode non-sensory receptors for which the endogenously relevant, or cognate, ligand is still to be identified, and which have been termed ‘orphan’ GPCRs. Historically, GPCRs, especially those in the aminergic receptor subfamily, have proved extremely tractable to the design and synthesis of synthetic modulators of their activity, both stimulatory and inhibitory. Indeed, of the top selling prescription drugs in 2000, more than a third act through GPCRs and provide greater than $25 billion in world-wide pharmaceutical sales [1]. With this impressive source of drug targets, it is not surprising that many companies have, over the past 5–10 years, invested heavily in the analysis of orphan GPCRs, with respect to identification of new receptor sequences, expression analysis, cognate and/or surrogate ligand identification and the assignment of function to these novel receptor–ligand pairings. With the completion of the human genome and the probable identification of all human GPCR sequences, it is reasonable to assume that most scientific research in this area is now focusing on the discovery of cognate or surrogate ligands for these orphan GPCRs (commonly called the ‘reverse pharmacology approach’; Figure 1), and the subsequent role of these receptors–ligand pairs in human physiology/pathophysiology. From a drug discovery viewpoint, the single most crucial aspect is the identification of a modulator of activity (agonist or antagonist). Currently, little progress can be made in the absence of these pharmacological tools, despite any subsequent discoveries about the function of a particular orphan receptor (e.g. by antisense or gene knockout). Therefore, this review highlights recent advances in the methodologies employed to aid the selection of a diverse ligand library, and the various assays that can be used to identify cognate or surrogate ligands for orphan GPCRs, as well as commenting on possible future directions that these technologies may take.

Section snippets

Selection of a ligand library

Discovery of the natural ligand for an orphan GPCR is the preferred strategy, as it provides additional biological information derived from the ligand that might well give initial clues to the utility of the receptor in disease and address pharmacological anomalies (e.g. histamine H4 receptor) [2]. Typically, an orphan GPCR screening file includes small molecules such as the biogenic amines, peptides, chemokines, bioactive lipids and metabolic pathway intermediates. Indeed, approximately 1200

Assay design

Another major consideration in the ligand-pairing strategy is the assay design. Because the ligand for the receptor is unknown, it is not possible to carry out competition binding studies and, therefore, the use of a functional screen is necessary. Furthermore, predictions of the downstream G-protein-coupling system of a receptor from bioinformatic analysis are unreliable, so it is essential to configure the assays to detect the broadest array of coupling mechanisms. However, one caveat is that

Conclusion

There are numerous high-throughput technologies to aid the identification of cognate or surrogate ligands for orphan GPCRs. Arguably the greatest advances have been in both the miniaturization of assay formats to permit high-throughput screening capabilities, and the sensitivity of the assays to increase the signal-to-noise ratio. Indeed, it is now standard to be screening in 384-well format and, recently, 1,536-well format has been introduced for some assay designs. Several assays utilized for

References and recommended reading

Papers of particular interest, published within the annual period of review, have been highlighted as:

  • of special interest

  • ••

    of outstanding interest

Acknowledgements

The authors would like to thank Geoff Johnston and Lee Beeley for their support and helpful suggestions in reviewing this manuscript.

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