you had to make a wild guess about the target of a certain drug, your best odds
are with G-protein coupled receptor. Drugs targeting members of this
integral membrane protein superfamily, which transmit chemical signals into a
wide array of different cell types, represent the core of modern medicine. They
account for the majority of best-selling drugs and about 40% of all prescription
pharmaceuticals on the market. Notable examples include Eli Lillys Zyprexa,
Schering-Ploughs Clarinex, GlaxoSmithKlines Zantac, and Novartiss
And there is broad consensus that GPCRs will remain at the hub of drug development
activities for the foreseeable future. According to a recent report by market
analysis firm Navigant Consulting, GPCRs are among the most heavily investigated
drug targets in the pharmaceutical industry.
These proteins are active in just about every organ system and present a wide
range of opportunities as therapeutic targets in areas including cancer, cardiac
dysfunction, diabetes, central nervous system disorders, obesity, inflammation,
and pain. Consequently, GPCRs are prominent components of pipelines in small and
large drug companies alike, and many drug discovery firms focus exclusively on
But the path to novel GPCR-targeted medicines is not routine. Most GPCR-modulating
drugs on the market werent initially targeted to a specific protein but
were developed on the basis of functional activity observed in an assay. That
they activated or inhibited a GPCR specifically was only later discovered. Post-Human
Genome Project, however, targets are the starting points for most drug discovery
endeavors. And there is still much to be learned about how GPCRs work and how
they can be selectively modulated. Fortuitously, technologies designed specifically
to tackle the GPCR challenge are blossoming.
Cell-based screening assays
The bread-and-butter of GPCR high-throughput screening is cell-based assays.
Tools such as fluorescent imaging plate readers (commonly referred to as FLIPRs)
allow multiwell plate analysis of GPCR activation events, which give good hints
of small-molecule drug leads.
GPCRs exist at the interface of a cells external and internal environments.
When the matching natural ligandwhich for the range of GPCRs could be an
amine, ion, nucleoside, lipid, peptide, protein, or, for optical receptors, lightcomes
along, it binds to a receptors active site and causes a conformational change
in the protein to form its active state. This signals the G protein coupled to
the receptor inside the cell to release components that set some predefined cellular
mechanism in motion.
The trick for high-throughput cellular screening is to find a robust marker
to monitor in cells overexpressing the GPCR of interest.
Calcium ions are one popular choice. Ca2+ is naturally produced
in cells upon activation of GPCRs coupled to Gq proteinsone of
the three main families of G proteins.
The Brussels-based company Euroscreen, for instance, has developed the AequoScreen
assay to fuel its own GPCR-based drug discovery programsas well as those
of companies that purchase its technology. AequoScreen is based on a jellyfish-derived
photoprotein called aequorin, which displays photoactivity proportional to Ca2+
concentration. Screening a library against an array of GPCR-overexpressing cells
mixed with aequorin provides a quantitative means of assessing a compounds
ability to activate a GPCR (or its ability to antagonize activation).
Even though intracellular Ca2+ levels rise directly only from Gq-protein
receptor activation, genetic expression methods have been developed that allow
Ca2+ production to proceed upon activation of GPCRs coupled to other
G protein types (i.e., Gi/Go or Gs). Thus,
fluorescent Ca2+ screening has become somewhat of a universal approach
to screening small-molecule libraries against GPCRs.
Cyclic adenosine monophosphate (cAMP), which controls myriad cellular metabolic
pathways, is one of the most important second messenger compounds
of the GPCR activation process. It also makes a good high-throughput screening
marker. Numerous commercially available and individually made cell-based GPCR
assays use luminescent tags that bind to cAMP.
Arena Pharmaceuticals entire drug screening program, for example, is
based around the cAMP approach, although the companys system doesnt
require the use of tagging compounds. With its Melanophore technology, it expresses
GPCR targets in frog skin cells containing a pigment that is highly sensitive
to changing levels of cAMP.
If you stimulate these cells such that they increase intracellular levels
of cAMP inside the cell, the pigment disperses throughout the cell and appears
black, explains Dominic Behan, co-founder and chief scientific officer of
Arena Pharmaceuticals. And if you stimulate these cells to decrease the
level of cAMP, the pigment aggregates to the center and the cell appears clear.
Because Gs- and Gq-coupled receptor activation stimulates
cAMP production, whereas Gi/Go-coupled receptors inhibit
cAMP, this is a broadly applicable screening assay.
A more straightforward approach to a universal GPCR assay is to monitor a mechanism
common and consistent for all receptorG-protein couplings. GPCR desensitization
following ligand activation is, perhaps, the only process identified that fits
this requirement. The need to continually increase dosages of morphine, which
targets the GPCR µ-opiate receptor, to maintain a constant clinical effect
is a familiar example of this desensitization process.
|Signal send-off. Ligand binding to
a GPCRs extracellular region triggers changes in the proteins transmembrane
region. This causes the release of guanosine diphosphate (GDP) and the uptake
of guanosine triphosphate (GTP) from the G protein, spurring activation of predefined
A receptor has to have the ability to signal independent of other GPCRs,
says Carson Loomis, senior vice president for research at Norak Biosciences, a
company focused exclusively on GPCR drug discovery and technology licensing. That
is why the G-protein signaling pathway is so diverse. But desensitizing has one
common mechanism for virtually all [GPCRs].
The desensitization pathway begins with binding of the cytoplasmic protein
to the activated receptor, which turns off the GPCR. The receptorarrestin
complex then enters the cell, where the ligand is removed and the receptor is
recycled back to the membrane. Noraks Transfluor assay is designed to monitor
the highly characteristic movements of -arrestininto
successive spatial arrangements called pits and vesiclesduring
this recycling process. By genetically engineering cell lines to express green
fluorescent protein-tagged arrestin and to overexpress the GPCR of interest, various
commercial imaging instruments can be used to measure and quantify GPCR activation.
Several other companies including 7TM, another GPCR-focused drug company, and
Perkin-Elmer have developed GPCR screening technologies that take advantage of
the arrestinreceptor binding. Each of these companies applies a technique
called bioluminescence resonance energy transfer, which measures changes in light
emission based on the interaction between an electron donor attached to b-arrestin
and an acceptor expressed with the GPCR target.
Besides the many GPCRs that function as basic receptors for sensory functions
like seeing and smelling, which are not prime therapeutic targets, there are more
than 300 other GPCRs up for grabs for drug discovery initiatives. About 200 of
thesea portion of which account for currently marketed GPCR drug targetshave
known natural ligands. And ligands are not known for about 150 nonsensory receptors
identified as GPCRs from the Human Genome Project. These so-called orphan GPCRs
have become a primary focus of many investigators and companies, because of the
largely uncharted path of discovery they offer.
Typically, an initial goal is to deorphanize these GPCRs using
high-throughput screening. Determining the endogenous ligand provides a first
hint of function, structural cues for lead design, and a particular receptor-activating
entity to antagonize.
For example, in 1999, researchers at SmithKline Beecham (now GlaxoSmithKline)
identified the orphan GPCR GPR14s natural ligand as urotensin II (UII),
a cyclic peptide associated with cardiovascular homeostasis and pathology (1).
More recently, researchers at Aventis found a potent nonpeptide GPR14/UII antagonist
by screening UII analogues against GPR14-transfected cells in a Ca2+
FLIPR assay and designing small molecules based on the important pharmacophores
for activating the receptor (2).
Despite this type of success, many scientists focused on discovering new drugs
appear to be bypassing the conventional deorphanizing step.
It is really hard to develop peptide libraries to look for the ligand,
Loomis says. Instead, he notes, drug researchers more often perform initial high-throughput
assays to find synthetic small-molecule agonists, which then can be used to go
back to the cell and work out the physiology of the receptor, he adds.
Arena Pharmaceuticals, on the other hand, does substantial reconnaissance work
on potential GPCR targetsspecifically using genomic analysis for determining
expression in specific organs and cells of interest and the intracellular signaling
mechanismprior to having any knowledge of natural or nonnatural ligands.
The company subsequently uses its constitutively activated receptor technology
(CART), a generic technique to trick GPCRs into activation, Behan
says, by mutating key sequence regions. CART aflows researchers to screen libraries
against an orphans active state without the need for a ligand.
By forcing GPCR signaling, scientists can readily validate agonist response
and directly determine antagonists and inverse agonists (i.e., ligands that expressly
If you wait too long to identify the natural ligand, you will miss out
on the opportunity of finding the actual drugs, Behan explains.
Assays like those designed by Norak, 7TM, and Arena are helping to populate
pipelines. But there is some consensus that more structural data is needed to
truly exploit the value of GPCRs.
It is absolutely imperative to have more structural information,
Loomis says. These receptors have so many subtypes, he explains. [For
example,] I think there are 16 serotonin receptors. The importance of being able
to target one and not the others is critical. You cant get that kind of
specificity in compounds that are already binding at 109 M without
some structural work.
But right now, he adds, it is trial and error.
GPCRs, like other membrane proteins, are notoriously difficult to crystallize.
All GPCRs are known to have a common motif of seven transmembrane helical structures,
but the only GPCR crystal structure published at atomic resolution is of the inactive
conformation of rhodopsin, the optical receptor protein solved in 2000 by University
of Washington chemistry and ophthalmology professor Krzysztof Palczewski (3).
Although GPCR crystallography is the focus of several companies business
plans, little else has been accomplished since this report.
|Visual clue. The visual photoreceptor
rhodopsin, shown above in its inactive state, is the only GPCR with a solved crystal
structure. (Adapted with permission from Ref. 3.)
The key bottleneck of GPCR crystallography is sample, says Juan
Ballesteros, CSO of Novasite, a GPCR-focused drug discovery company that retains
Palczewski as a crystallography consultant. Obtaining high-quantity and high-purity
GPCR proteins is very challenging, because membrane proteins are typically produced
in a heterogeneous manner by cells with substantial variability in glycosylation,
But Novasite has developed a new approach using retinas from transgenic frogs
and mice as GPCR bioreactors. It takes advantage of the efficient mechanism by
which the eye produces rhodopsin to express other GPCRs. Every second, you
are expressing 80,000 new rhodopsin molecules that are 98% chemically identical,
Novasite will soon publish a structure of rhodopsin in its active state, which,
he says, will itself be a significant accomplishment and will also validate the
new expression system. The company then plans to use this expression system to
generate receptor samples for 20 GPCR therapeutic targets and start crystallization
trials by the end of 2004.
This will have tremendous value to the drug discovery effort, Ballesteros
insists. Nevertheless, with more than 300 potential GPCR targets of interest,
crystallization efforts clearly have a long way to go.
Receptor structures are sometimes able to inform drug discovery efforts through
extrapolation from rhodopsins structure. Researchers from the University
of Michigan and the University of Kansas, for example, recently used computational
homology modeling techniques to determine a three-dimensional structure of dopamine
3 (D3R, a potential target for drug addiction, Parkinsons disease,
and schizophrenia (4). They found potential ligands
via computational pharmacophore and structure-based screening, several of which
displayed substantial inhibition in a D3 binding assay.
Ballesteros notes that the rhodopsin structure can serve as a useful guide
for family 1 GPCRs, which are homologous to the optical protein, but it provides
less practical information for the other two GPCR families. The farther
away [structurally] from rhodopsin, the more valuable is a target GPCR structure,
Predix Pharmaceuticals, however, has developed an in silico GPCR structure-based
method that does not rely on rhodopsin homology. Its PREDICT algorithm combines
protein sequence information with membrane environment property factors to determine
the most stable three-dimensional structure of a receptors transmembrane
domain. The company recently published five examples of successful early-stage
discovery projects that led to very promising lead compounds validated
via in vitro and in vivo assays (5). Each was
initiated by screening libraries virtually against PREDICT-generated structures,
including of two different serotonin receptors.
However, according to Ballesteros, the new hot thing is allosteric
GPCR modulators. These are compounds that bind away from a proteins active
site and modulate its activity independent of the natural ligand. Big pharmaceutical
companies are now very interested in this, he says. Amgens Sensipar,
approved by the FDA in March and indicated for secondary hyperparathyroidism and
elevated calcium levels, became the first allosteric GPCR modulator. There are
others in several different company pipelines.
Computational tools are not often very effective at modeling allosteric binding
sites, he says. This is where you really need the structure of a particular
receptor to guide discovery, he stresses.
Another tool for structure-based development, whether in the active site or
in an allosteric region, is high-throughput mutagenesis screening, in which different
mutations at a GPCR binding site are analyzed against multiple ligands. This helps
uncover the key ligandreceptor contacts responsible for drug recognition
by the receptor. By this means, the information unearthed in functional assays
is connected to structural data determined by X-ray crystallography or computation.
Place your bets
Norak, Arena, 7TM, Novasite, and Predix are prime examples of firms completely
focused on the GPCR drug discovery effort. And the extensive partnerships and
licensing agreements each has with the likes of Aventis, Merck, Eli Lilly, AstraZeneca,
Hoffman-La Roche, and individual endeavors by other big pharmaceutical companies
point to the far-reaching investments ongoing in this area. Rather than a wild
guess, GPCRs seem to be a good bet for future drug discovery successes.