Characterizing orphan receptors using a reverse pharmacology approach

Although complex and technically challenging, the strategy that has become standard for the identification of ligands for orphan GPCRs is known as the 'reverse pharmacology' approach, which is to clearly distinguish it from the more classical approach to drug discovery (Stadel et al. 1997). Historically, the classical approach was initiated by the discovery of a biological or physiological activity that could be attributed to a specific ligand, this was then used to pharmacologically characterize the tissue to determine its therapeutic potential. Subsequently, the ligand was used to isolate its corresponding receptor for use as a drug target in high-throughput screening. The reverse approach begins with an orphan receptor of unknown function that is used as a 'hook' to fish for its ligand. The ligand is then used to characterize the function of the receptor in normal cell signalling and to establish its

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NmU/FM4

ADP/SP1999 Histamine/H4 Tyramine/TAl NPFF/NGPR KiSS-1/GPR54

LTD4-LTC4/CysLT2 MCH-2/SLT LTB4/Fishboy

LPC/G2A

SPC/GPR4

ADP/GPR86

NmU/GPR66

NPFF/85

CTACK/CCR10

NmU/FM4

ADP/SP1999 Histamine/H4 Tyramine/TAl NPFF/NGPR KiSS-1/GPR54

LTD4-LTC4/CysLT2 MCH-2/SLT LTB4/Fishboy

LPC/G2A

SPC/GPR4

ADP/GPR86

NmU/GPR66

NPFF/85

CTACK/CCR10

Urotensin/GPR14

TECK/CCR9 Motilin/GPR38

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May-Jul Aug-Oct Nov-Jan Feb-Apr May-Jul Aug-Oct Nov-Jan Fep-Apr May-Jul Aug-Oct

Urotensin/GPR14

TECK/CCR9 Motilin/GPR38

May-Jul Aug-Oct Nov-Jan Feb-Apr May-Jul Aug-Oct Nov-Jan Fep-Apr May-Jul Aug-Oct

Fig. 10.1 Demonstrates the linear rise in the number of publications describing the pairing of orphan GPCRs with their cognate ligand. A number of examples have been highlighted illustrating the important new biological mediators that have been discovered.

pathophysiological role or therapeutic potential. In parallel, high-throughput screening is initiated in order to develop selective chemical tools that will realize the therapeutic value of the receptor.

A possible strategy to characterize orphan GPCRs is described in Fig. 10.2. Using bio-informatic analyses such as basic local alignment search tool (BLAST) (Altschul et al. 1990) searching with the known members of the GPCR superfamily as a probe set, plus the use of Hidden Markov Models (Grundy et al. 1997) and motif searches, we believe we have identified almost all the 'orphan' GPCRs contained within the human genome (see chapter 9). This totals, at the time of writing, to around 140 family 1: rhodopsin like, 30 family 2: calcitonin like and 5 family 3 receptors: metabotropic like. Full-length cDNAs have been isolated using standard molecular biology techniques or are available as off-the-shelf reagents. In order to prioritize those receptors for screening, tissue expression patterns are determined. In the first instance, information from expressed sequence tag (EST) databases such as that produced by Incyte Pharmaceuticals provides a limited view of expression. This is because GPCRs are generally expressed at low levels, therefore frequently below the sensitivity of their database, so a negative result is not particularly informative, but the results can be obtained instantly and direct a more focused study. More sensitive techniques using either quantitative PCR, for example, Taqman against a panel of cDNA's from important therapeutic tissues, or immunohistochemistry studies to identify expression in important cell types of therapeutic interest (e.g. Lifespan Inc, Seattle) provides data of the highest quality. In addition, Deltagen (Redwood City) produces a database containing information on numerous mouse knockouts. This is used increasingly to highlight important orphan receptors based on observed phenotypes. Once prioritized, the receptors are expressed in mammalian cells for functional analysis. The expression system chosen is of critical importance for success; cell lines with a good history of GPCR expression, which contain a wide

Orphan GPCRs—Target Validation

Orphan GPCRs—Target Validation

Fig. 10.2 Reverse pharmacology strategy for the identification and characterization of novel receptor: ligand pairs. The identification and cloning of the GPCR gene family is now complete. The remaining challenge is to identify ligands for orphan receptors and to define their pathophysiological role.

Fig. 10.2 Reverse pharmacology strategy for the identification and characterization of novel receptor: ligand pairs. The identification and cloning of the GPCR gene family is now complete. The remaining challenge is to identify ligands for orphan receptors and to define their pathophysiological role.

variety of G proteins to allow functional downstream coupling to effector systems are key. Human embryonic kidney 293 (HEK293) and Chinese hamster ovary (CHO) are often the cell lines of choice. The expressed receptor is then screened in a variety of functional assays to identify an activating ligand and since it is very difficult to predict the coupling system of a receptor, it is essential to configure the assays so as to detect the broadest array of coupling mechanisms (Kostenis 2001). However, such systems are still unlikely to work universally. Challenges to overcome include, for example, the presence of endogenous receptors in such mammalian host cell lines, and especially clonal variations in the patterns of endogenous receptor expression in cells derived from the same parental cell line.

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