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Drug Discovery Today: Technologies Vol. 1, No. 1 2004

Editors-in-Chief

Kelvin Lam – Pfizer, Inc., USA

Henk Timmerman – Vrije Universiteit, The Netherlands

Target identification

Identifying orphan G protein coupledreceptors in drug discoveryJohn Dunlop1, Richard M Eglen2,*1Neuroscience Discovery Research, Wyeth Research, CN-8000, Princeton, NJ 08543, USA2DiscoveRx Corporation, 42501 Albrae St. Fremont, CA 94538, USA

G-protein coupled receptors (GPCRs) represent the

most tractable family of drug targets. Those GPCRs

identified by sequence only, but lacking an endogenous

ligand, are defined as orphan GPCRs (oGPCRs) and

might represent the next generation of targets for

GPCR drug discovery. Drug discovery at oGPCRs is

a resource intensive approach and frequently taken

‘at-risk’ without a clear understanding of the role in

a disease. Identification of oGPCRs is, therefore, a

prerequisite for the initiation of a drug discovery pro-

gram.

Corresponding author: (R.M. Eglen) reglen@discoverx.com

740-6749/$ � 2004 Elsevier Ltd. All rights reserved. DOI: 10.1016/j.ddtec.2004.06.004

Section Editors:Wolfgang Fischer, Rob Hooft, Michael Walker

Orphan G protein coupled receptors have been widely publicized asrepresenting the next generation of drug targets and source of future

drug candidates, yet the challenges for identification are still as great andthe impact remains to be completely realized. Many drug discovery

companies are adopting a highly integrated approach to the process ofde-orphaning these receptors.

Introduction

G-protein coupled receptors (GPCRs) are the most tractable

family of drug targets and over 40% of marketed drugs target

GPCRs. Endogenous ligands (see Glossary) have been identi-

fied for nearly 200 GPCRs, although the human genome

contains over 1000 GPCR genes, suggesting that the majority

of receptors are orphan in nature [1,2]. Although all might

not signal via G proteins (see Glossary), or even be expressed

at the cell plasma membrane, several hundred GPCRs do so

[1,2]. The complex process of drug discovery at oGPCRs is a

multidisciplinary approach using many techniques for tar-

get identification including bioinformatics, chemoinfor-

matics (see Glossary), cloning and expression, and ligand

identification strategies using cell-based assays, and high-

throughput pharmacological screening (Fig. 1). In many

drug discovery companies, therefore, de-orphaning GPCRs

is a significant activity that involves a highly integrated

approach (Outstanding issues) [3].

Comparison of technologies in oGPCR identification

The completion of the human genome project has suggested

that there are approximately 1000 GPCR genes, of which 300

have potential as drug targets. As of 2003, an additional 100

unique ligands remain to be identified for the GPCR family

[4]. However, it is implicit when pairing ligands with an

oGPCR, that naturally occurring compounds (purified either

from crude tissue extracts or expressed proteins and peptides)

act as agonists (see Glossary). This might not always be the

case, as some GPCRs do not require a ligand or even partici-

pate in signal transduction. Moreover, several oGPCRs act as

non-specific binding proteins, as well as exhibiting differing

pharmacologies that vary according to the extent and nature

of receptor dimerization. Collectively, these phenomena raise

issues regarding the traditional approach of identifying

oGPCR sequences via the endogenous ligand [5].

Consequently, a strategy widely favored in current oGPCR

identification strategies is to use a reverse pharmacology

approach (see Glossary) [6]. This involves identification of

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Drug Discovery Today: Technologies | Target identification Vol. 1, No. 1 2004

Figure 1. Classical cell-based approaches to the identification of

endogenous and surrogate agonists and inverse agonists for orphan

GPCRs. (a) Knowledge of oGPCR distribution allows intuitive selection

of tissue sources for preparation of fractionated tissue extracts to

screen for activators of the receptor, putatively the endogenous ligand.

This approach is complemented by the use of panels of known ligands to

screen for either natural or surrogate agonists. (b) Over-expressed or

constitutively active receptors allow screening campaigns aimed at

identifying compounds with the property of reducing basal receptor

activity, inverse agonists, or the identification of surrogate agonists. This

approach represents the initial step in a drug discovery effort toward

novel pharmacological agents, potential tool compounds and, ultimately,

future drug candidates, if successful. HTS, high-throughput screening;

RBI-LOPAC, Research Biochemicals Inc. (Sigma) library of pharmaco-

logically active compounds.

Figure 2. Strategy of orphan GPCR identification using a variety of

techniques. A highly integrated approach is necessary in the oGPCR drug

discovery paradigm incorporating strategies to identify oGPCR

sequences and express receptors, thus facilitating cell-based screening

efforts. In parallel with these efforts, strategies aimed at defining the

potential physiological function and disease relevance of the receptor

are crucial to the decision to pursue the target in an extensive drug

discovery effort.

ligands (frequently antagonists; see Glossary) that act to

attenuate receptor function by reducing constitutive activity

(see Glossary). This is generally defined as initiation of signal

transduction via the receptor in the absence of ligand bind-

ing. Increased emphasis is, therefore, being placed upon

functional screening of oGPCRs in cell-based assays, as

described below. Integral to the reverse pharmacological

approach is, therefore, robust oGPCR expression and devel-

opment of high-throughput cell-based screening (Fig. 2).

Bioinformatics and chemoinformatics

Bioinformatics and chemoinformatics techniques are a col-

lection of ‘virtual’ approaches to oGPCR identification. They

are attractive as an initial approach because of their low cost

62 www.drugdiscoverytoday.com

and the widespread availability of sophisticated relational

databases. Moreover, the lack of substantial three-dimen-

sional structural data on oGPCRs, and consequent difficulties

of defining algorithms to accurately describe ligand docking,

have placed an increased reliance upon oGPCR sequence

analysis and homology comparisons [7]. Several algorithms

are available to model GPCR structures, including those by

Predix (http://www.predixpharm.com), which uses the PRE-

DICT software to suggest GPCR structure based on the amino

acid sequence only [8].

Bioinformatic ‘mining’ of sequence data are frequently

used to identify potential proteins as oGPCRs. Widely used

is homology screening, based upon degenerate primers

derived from known GPCR sequences, to identify novel,

closely related, sequences. The most extensively used

approach is the National Center for Biotechnology Basic Local

Vol. 1, No. 1 2004 Drug Discovery Today: Technologies | Target identification

Glossary

Aequorin: a jellyfish photoprotein, commercially available from Euro-

screen, used to detect transient calcium signals.

Agonist: a ligand that both binds and activates the receptor to induce a

cellular response. Almost all endogenous ligands for oGPCRs are agonists.

Antagonist: a ligand that binds to the receptor but does not cause

activation. Almost all antagonists are synthetic molecules.

b-arrestin: cytosolic proteins involved in the desensitization process of

GPCRs and induction of internalization. Labeling of b-arrestin by GFP

provides an imaging assay to measure receptor translocation.

Bioinformatics: organization of large amounts of biological informa-

tion, such as DNA and protein sequence databases. Exploration or

‘mining’ of these data bases is frequently used to classify genes and

proteins and identify sequence relationships.

Chemoinformatics: an emerging in silico technology that allows one

to predict chemical candidates from a protein structure.

Chemical tractability: desirable characteristics of chemical com-

pounds that can be used in a medicinal chemistry program to produce

clinical candidates. Various chemical criteria, generically termed drug-like

properties, are often applied to leads derived from an HTS screen to

assess their suitability for further chemical optimization.

Constitutive activity: augmented activity of GPCRs occurring in the

absence of ligand. This is induced either be selective mutations in the

sequence or by expression at high levels in the cell.

Endogenous ligand: the natural ligand for a particular GPCR. Identi-

fication of this substance is a frequently a key step in deorphanizing a

receptor.

FLIPR: fluorometric imaging plate reader. A device from Molecular

Devices extensively used in oGPCR screening, in which transient changes

in intracellular calcium levels are detected in response to agonist

activation, in 96, 384 and 1536 wells.

G proteins: a series of related proteins that are involved in coupling the

activated oGPCR to a second messenger. They are formed of alpha beta

and gamma subunits, the dissociation of which is induced by coupling to an

agonist occupied oGPCR.

G protein coupled receptors: a large class of cell surface proteins that

generally function by coupling to G proteins to mobilize second

messenger pathways. They are divided into several large classes and

depending upon the sequences that bind a wide variety of small

molecules, peptides, proteins and ions. They are also termed 7 trans-

membrane proteins as a result of their presumed tertiary structure.

Green fluorescent protein: a widely used fluorescent protein derived

from marine animals that can be cloned onto various cell proteins, such as

b-arrestin. Movement of proteins can be tracked by measuring the

movement of the fluorescent signal under a confocal microscope of a

high-throughput imaging device.

High content screening: the rapid acquisition and analysis of diverse

data in a screening campaign. The term is frequently used in the context of

cell-based assays and their application to various automated imaging

devices.

High-throughput screening: the rapid acquisition and analysis of data

from large numbers of compounds screened against a defined target. The

approach frequently uses robotic fluid dispensing and analysis systems in

which very large compound libraries (often 500,000 to 1 million

compounds) are tested.

Homology screening: the use of nucleic acid or amino acid sequence

information to screen databases for similar sequences.

Internalization: the process undergone by most GPCRs in the face of

sustained agonist activation. It follows desensitization and binding of

b-arrestin and leads to movement of the GPCR: ligand complex to the

cell interior.

Inverse agonist: a compound that reduces the basal activity of an

oGPCR. Compounds of this nature are frequently used against con-

stitutive active GPCRs and provide a means to identify compounds that

antagonize the receptor even when the endogenous agonist is unknown

Orphan GPCR: a GPCR for which the endogenous ligand has yet to be

identified

Reverse pharmacology: the use of the novel receptor to identify the

endogenous ligand. This is the opposite of classical pharmacological

approaches where the receptor was characterized on the basis of the

ligand pharmacology.

Second messenger: cytosolic molecules such as cAMP, Ins P3 or

calcium mobilized upon activation of a cell surface oGPCR. Measurement

of changes in the intracellular concentration of these molecules provide

robust biomarkers of the oGPCR activation, there by enabling functional

screening.

Alignment Search Tool (BLAST) that allows query of a putative

oGPCR sequence against all public databases (http://

www.ncbi.nlm.nih.gov/BLAST). Because several GPCRs lack

introns, homology-screening approaches are streamlined to

identify only the long open reading frames. The sequences can

be ‘filtered’ by focusing only on those that cluster with a

particular disease or those that are expressed in a tissue with

a defined physiology.

Bioinformatics is also applied to identify oGPCR sequences

that cluster with their liganded counterparts. Several novel

ligands can be predicted using this approach, by assuming

that members in a cluster are activated by similar ligands.

Although many oGPCRs cluster in this manner, they can

have very dissimilar ligands, thereby limiting the approach.

In practice, bioinformatic inaccuracies in oGPCR identifica-

tion usually result from the paradigms used to cluster the

receptors – for example, some in silico methods employ exclu-

sively the seven transmembrane spanning domains, and

ignore crucial amino terminal residues required by many

oGPCRs for ligand binding [5].

A chemoinformatic (homology screening, see Glossary)

approach to oGPCR identification assumes that ligand iden-

tification allows identification of the receptor, for example, as

used in the Quasi II algorithm developed at De Novo Phar-

maceuticals (http://www.denovopharma.com) [9]. Many

compound screening libraries used in oGPCR high-through-

put screening (HTS) contain several privileged structures

known from repeated screening campaigns that interact with

GPCRs at a higher hit rate than would be predicted from the

random rate, allowing rapid optimization of a pharmaco-

phore. These compound structures provide insights into the

nature and validation of the oGPCR under investigation [10].

Orphan GPCR expression and constitutive

signaling activity

Expression of the oGPCR is a prerequisite of validation, and is

vital for initiation of HTS assays for lead identification. Sev-

eral expression systems are used, including yeast, baculoviral

and mammalian cells. Cloning of the oGPCR mRNA is under-

taken by reverse transcription–polymerase chain reaction

(RT–PCR) studies, whereas expression of the protein is gen-

erally assessed using Western analysis (if antibodies are avail-

able) or by epitope tagging the receptor with imunogenic

peptides including FLAG, HA, myc or His. Detection of the

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Table 1. Assessment of oGPCR validation techniques

Identification

process

Bioinformatics Chemoinformatics Cellular

expression

Cell-based

assays – second

messenger

coupling

Cell-based

assays – second

messenger

coupling

Cell-based

assays – second

messenger

coupling

Cell-based

assays – high

content

screening

Cell-based

assays – high

content

screening

Technique Cluster analysis Cluster analysis Recombinant

cloning and

stable cell

expression

Changes in

intracellular

calcium

Changes in

intracellular

cAMP

Changes in NFAT

(calcium signaling) or

CRE (cAMP signaling)

reporter gene activity

b-arrestin

redistribution

Internalization

of CypHer

dye – oGPCR

epitope tags

Advantages Rapid in silico

measurement

of oGPCR

phylogeny and

potential linking

to ligands and

or disease

In silico

identification

of preferred

GPCR ligands,

including putative

endogenous ligands

Allows use of

mammalian

cells with a null

oGPCR

background

Highly sensitive

automated screening

system to detect

agonists and

antagonists

Highly sensitive

automated screening

system to detect

agonists and

antagonists

Highly sensitive

and automatable

screening systems

Many GPCRs

induce arrestin

translocation

upon activation

including several

orphans

Allows

measurement

of ligand induced

internalization

Rapid identification

of novel

pharmacophores

Over expression

to induce

constitutive

activity

Allows use of

promiscuous

G proteins to

couple to PLC

or chimeric

oGPCRs and

G protein

constructs

Detect elevations

in basal cAMP

due to constitutive

activity

Generic screening

system for agonists

and antagonists

Can detect

constitutive

internalization

Expression of

constitutively

active mutants

Disadvantages Clustering based

on part of the

sequence that

might not

include the

ligand binding

domain

Lack of high

resolution

crystal

structure

Need to ensure

that the oGPCR

is expressed at

the cell surface

in the correct

orientation

No changes

in basal levels

in intracellular

calcium with

constitutive

activity

Only detects

Gs and

Gi coupled

oGPCRs

Downstream from

ligand activation

and requires

prolonged

incubation with

oGPCR ligands,

results in many

false positives

Cells require over

expression of both

the oGPCR and

b-arrestin

Requires the

use of epitope

tagged oGPCRs

and internalization

of antibody/receptor

complex

Ambiguous

identification

of agonist vs.

antagonist

Need to counter

screen hits against

a non transfected

cell

Difficult to

detect inverse

agonists

Modification

of the carboxy

termini might

influence ligand

pharmacology

Need to assess

agonist efficacy

and potential

changes in agonist

pharmacology

Extensive amounts

of data generated

during screening

campaigns

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Vol. 1, No. 1 2004 Drug Discovery Today: Technologies | Target identification

receptor is then done using immunostaining approaches and

subsequent confocal microscopy. Some systems clone and

express the oGPCR as a fusion protein with green fluorescent

protein (GFP, see Glossary). This allows for direct assessment

of expression and spatial distribution in living cells, although

the possibility exists that the GFP protein fusion might

influence ligand pharmacology, as well as membrane inser-

tion [11]. A crucial issue is to ensure that the oGPCRs are

expressed at the cell surface membrane in the correct orienta-

tion for ligand recognition (Table 1).

In the absence of a ligand, it is difficult to ascertain if the

oGPCR, though expressed, retains functional activity. Most

oGPCRs are expressed at high levels, so that over expression

induces constitutive activation of cell signaling. Alterna-

tively, over expression of the G protein (see Glossary) or

expression of the receptor as a G protein fusion construct

might also result in constitutive oGPCR activity (see Glos-

sary). These approaches, although allowing signal to be

detected in a bioassay might be criticized in terms of potential

alterations in the pharmacology of the ligand binding site,

and thus accurate detection of compounds in HTS. A related

approach is the development of the Constitutive Active

Receptor Technology (CART) approach by Arena Pharmaceu-

ticals (http://www.arenapharm.com) [12]. This utilizes spe-

cific mutations of the oGPCR such that it is constitutively

active. This technique is combined with expression of the

mutant oGPCR in Xenopus melanophores, so that activation

of receptor results in redistribution of the melanin-contain-

ing melanosomes and thus changes in absorbed light [13].

In several approaches, the presence of endogenous recep-

tors, orphan or otherwise, might also contribute to second

messenger (see Glossary) generation that might influence the

detection of ligands. Consequently, it is essential that puta-

tive leads are counter screened against the untransfected host

cells, to assess the selectively of the signal.

Cell-based assays to detect oGPCR function

By definition, the orphan status of oGPCRs precludes the use

of radioligand binding assays as a strategy to search for

natural and surrogate ligands. Consequently, functional

cell-based assays are the mainstay of oGPCR assay develop-

ment and de-orphaning efforts [14]. High-throughput plat-

forms have typically utilized reporter gene approaches or

assays monitoring the mobilization of intracellular calcium.

In the case of reporter gene assays, GPCR signal transduction

is monitored using expression systems engineered with cis-

acting enhancer elements, DNA sequence motifs targeted by

binding partners promoting gene expression, upstream of a

reporter gene such as luciferase or GFP. GPCR signaling via

changes in cAMP are assayed using the CRE (cAMP response

element) enhancer of gene expression, whereas GPCRs

coupled to changes in calcium utilize the calcium-sensitive

AP1 (activator protein 1) and NFAT (nuclear factor of acti-

vated T cells) elements [15]. Vector systems are commercially

available offering the various combinations of enhancer

element and reporter gene as the Mercury (http://www.

clontech.com) and PathDetect (http://www.stratagene.com)

systems.

Functional studies using intracellular calcium measure-

ments utilize cell lines engineered to express the oGPCR of

interest in the presence either of the promiscuous calcium

coupling G-protein Ga15/16, or G-protein chimeras compris-

ing Gaq, the normal calcium coupling transducer G-protein,

with the C-terminal amino acids replaced with those from

Gai, Gao, or Gas. The C-terminal substitution permits appro-

priate receptor/G-protein recognition, whereas the Gaq allows

for measurement of the functional response via calcium mobi-

lization. Platforms such as the fluorometric imaging plate

reader (FLIPR, see Glossary; http://www.moleculardevices.

com) or functional drug screening system (FDSS, http://

www.hamamatsu.com) are used with calcium sensitive fluor-

escent dyes and are compatible with HTS applications (see

Glossary). Given that oGPCR-coupling specificity is typically

unknown, a cocktail of G-protein chimeras might be used,

although bioinformatic predictions of GPCR coupling speci-

ficity might allow selection of one specific chimera. In addi-

tion to the FLIPR and FDSS platforms using calcium-sensitive

fluorescent dyes, the bioluminescent protein aequorin (see

Glossary; http://www.euroscreen.com) can also be used, based

on its calcium ion binding property resulting in the oxidation

of an added substrate coelenterazine with concomitant pro-

duction of CO2 and light emission, captured by a lumines-

cence plate reader.

More recently, the introduction of high content screening

(HCS) platforms has extended the use of cell-based assays to

those where the imaging of the cellular redistribution of

components of the GPCR signaling pathway forms the basis

of the assay [16]. HCS instruments represent enabling tech-

nology platforms capable of fully automated imaging acqui-

sition and analysis. Several platforms are currently available

including the ArrayScan (http://www.cellomics.com), IN Cell

Analyzer (http://www.amershambiosciences.com), Acumen

Explorer (http://www.acumenbioscience.com), Discovery-1

(http://www.moleculardevices.com), Opera (http://www.

evotec-technologies.com) and Pathfinder (http://www.im-

star.fr). Direct visualization of GPCR internalization (see

Glossary), an almost ubiquitous property of GPCRs involved

in the process of receptor desensitization following activa-

tion, is facilitated by tagging the receptor with a fluorescent

biomarker, most commonly, GFP. Upon agonist activation

the GPCR–GFP construct can be visualized moving from

uniform plasma membrane localization to internalized recy-

cling compartments resulting in the formation of intensely

bright spots (Fig. 3). Automated image capture and quanti-

fication of these trafficking events permits screening for both

natural and surrogate receptor agonists. However, although a

www.drugdiscoverytoday.com 65

Drug Discovery Today: Technologies | Target identification Vol. 1, No. 1 2004

Figure 3. Imaging GPCR internalization. Two potential strategies for imaging GPCR internalization are discussed. The first uses a tagged GPCR receptor

construct to directly follow the movement of the receptor. The second uses the acid pH-sensitive dye CypHer5 conjugated to an appropriate tag to observe

the bright fluorescence of CypHer dye in the acidic endosomal compartment. (a) Expression of a C-terminal tagged 5-hydroxytryptamine–GFP (5-HT2–GFP)

construct in human embryonic kidney (HEK) cells results in expression predominantly localized to the plasma membrane. (b) Upon stimulation with 5-HT the

receptor is detected in intensely bright intracellular compartments. Imaging algorithms automatically quantitate the translocation into the endosomal

compartments. (c) Using CypHer5 monoester conjugated to an HA antibody and applied to cells expressing an N-terminal HA-tagged neuropeptide receptor,

intensely bright red fluorescence ‘spots’ are evident following receptor activation. No fluorescence was observed under basal conditions (not shown). (Data

from J. Dunlop, A. Baudy, B. Schlag, M. Pausch, and M. Fennell).

seemingly straightforward approach many obstacles are fre-

quently presented including inappropriate targeting of the

GPCR–GFP construct to the plasma membrane and/or high

levels of internalized receptor under basal conditions due to

organelle trapping or constitutive recycling. In addition, as

discussed above, the pharmacological properties of an oGPCR

might be altered by the presence of a GFP-tag.

Another approach gaining in popularity more recently has

been to monitor the cellular translocation of proteins parti-

cipating in the GPCR desensitization/internalization cycle,

specifically b arrestins (see Glossary). Commercialized as the

Transfluor technology (http://www.norakbiosciences.com)

this approach monitors the change in distribution of a b-

arrestin–GFP construct from a relatively homogeneous and

diffuse intracellular localization to aggregated pit- or vesicu-

lar-like compartments, as a consequence of ligand-dependent

internalization [17]. Theoretically, Transfluor is universally

applicable across GPCRs and potentially overcomes several

the limitations associated with directly tagging the GPCR.

Provided some degree of plasma membrane localization of

the GPCR of interest is achieved b arrestins translocation

follows only those receptors committed toward the interna-

lization pathway, offering a high signal–noise ratio.

Yet another strategy focused on the internalization path-

way exploits the acidic nature of the endosomal recycling

compartments. The pH-sensitive cyanine dye CypHer 5

(http://www.amershambiosciences.com) is non-fluorescent

at pH 7.4 and brightly fluoresces in acidic environments

[18]. This approach utilizes a small N-terminal epitope tag

(VSV-G, HA, FLAG, c-myc) on the GPCR recognized by a

CypHer 5 conjugated antibody. Binding of the antibody to

cell surface GPCRs fails to produce a fluorescence signal and

66 www.drugdiscoverytoday.com

upon ligand-dependent internalization to endosomal com-

partments intensely bright fluorescence spots of CypHer 5 are

observed (Fig. 3).

Recent de-orphaning successes

The pairing of an oGPCR with its cognate ligand is a signifi-

cant step towards the elucidation of the physiological role of

the receptor. Successes in oGPCR deorphaning have recently

been reviewed [5,19] and have revealed new ligand/receptor

pairings of potential significance in a wide variety of physio-

logical functions including regulation of feeding behavior,

cardiac function, nociception, inflammation, gastrointestinal

motility, bronchoconstriction and central nervous system

function [20]. Although offering numerous possibilities for

drug discovery programs, target validation in the form of

some definitive link between the receptor, or its malfunction,

and a disease state represents the next major challenge in the

process. Identification of the endogenous activator now

allows implementation of screening campaigns to identify

receptor antagonists or agonist mimetics, potential tools to

assist in target validation and ultimately the starting points

for design of potential drugs of the future.

Two common strategies used successfully in the search

for endogenous ligands are screening of either fractionated

tissue extracts or panels of known transmitter compounds.

Knowledge of the tissue distribution of the target of interest

allows intuitive selection of a tissue source for preparation of

tissue extracts postulated to contain the natural ligand. In a

more random approach, panels of known transmitters can be

assembled or obtained commercially such as the RBI-LOPAC

(Library of Pharmacologically Active Compounds, http://

www.sigmaaldrich.com).

Vol. 1, No. 1 2004 Drug Discovery Today: Technologies | Target identification

Outstanding issues

� Realizing return on significant investment in oGPCR programs.

� Translating ligand-receptor pairing into validated drug target and

clinical development candidate.

� Decreased effectiveness of de-orphaning efforts.

� Recent obvious decline in number of new ligand-receptor pairings.

� Elucidating signaling mechanisms of non-liganded and non cell surface

expressed GPCRs.

� Realizing potential of new high-content screening approaches.

� Chemical tractability (see Glossary) of novel leads from screening

campaigns.

Successful ligand-receptor pairings have recently been

comprehensively reviewed [5] with some of the more recent

examples including GPR7/8 (activated by the novel neuro-

peptides B and W), GPR 40 and 41 (activated by fatty acids),

HM74A (activated by nicotinic acid), GPR 77 (activated by

complement fragments) and BG37/TGR5 (activated by bile

acids). Most recently, a limited number of ligand/receptor

pairings have been reported in an apparent trend suggestive

of a process that is becoming more difficult, or put another

way that the ‘easier’ oGPCRs are the ones to already have

succumbed to ligand identification. Demonstration of the

activation of GPR43, a receptor with potential physiological

roles in the immune system and the gut, by short-chain fatty

acids has led to the proposed re-designation of the receptor to

free fatty acid 2 receptor (FFA2R) [21]. The endogenous ligand

for ChemR23 was recently discovered by screening peptide

fractions derived from human hemofiltrate, with the activat-

ing peptide TIG2 isolated from the activating fraction and

identified by a combination of HPLC, mass spectrometry and

Edman sequencing [21]. Human placental tissue was the

source of peptide fractions used in the identification of

hemaphorins VV-H-7 and LVV-H-7 as low-affinity agonists

for the orphan bombesin receptor 3 [22,23]. The approach of

using fractions enriched in peptides represents one of the

standard methods employed in the search for endogenous

ligands.

These most recent ligand/receptor pairings have utilized

measurements of intracellular calcium as the screening assay.

In addition, the historical deorphaning successes have seen

cell-based assays using calcium measurements used in greater

than 50% of the studies. Clearly, this is partly a function of

the early availability of technology facilitating screening

campaigns with this approach. In the future, it is anticipated

that the impact of the newer HCS platforms and associated

technologies will be realized in oGPCR ligand identification.

Several pharmaceutical companies have invested in this tech-

nology with a view to application to the oGPCR drug dis-

covery paradigm and the continued search for future drug

targets and candidates.

Conclusions

The multidisciplinary approach, outlined above, for the iden-

tification of oGPCRs as drug discovery targets captures the

challenges of such an endeavor. Many drug discovery pro-

grams now employ a battery of techniques as part of the

identification process, resulting in a target suitably expressed

for high-throughput and or HCS. Because most of the latter

employ cell-based techniques, numerous advances have been

made in this area to accelerate the throughput and to increase

the quality of the data. Consequently, the utility of oGPCRs

as targets in drug discovery relies heavily of the identification

process employed. In reality, however, the current situation

is one of newly identified receptor–ligand pairings, whose

significance as validated drug discovery targets is largely

hypothesized. Successful screening campaigns leading to

the identification of novel pharmacological ligands, in com-

bination with a better understanding of the physiological,

and potentially pathological, role of such receptors will

determine the success of (newly identified) GPCR drug dis-

covery in the future.

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human and mouse. Proc. Nat. Acad. Sci. USA 100, 4903–4908

3 Lee, D.K. et al. (2003) Continued discovery of ligands for G protein-coupled

receptors. Life Sci. 74, 293–297

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