Hooked on a Hunt

In the late 1990's, Olivier Civelli's lab at the University of California, Irvine, bustled with nearly a dozen researchers, all devoted to hunting for missing links in one type of vital molecular signaling pathway. Half of the elements of this pathway were already in place, as several hundred G protein-coupled receptors (GPCRs) had been identified from DNA screens. One component, however, was still missing: the signal-triggering molecule - or ligand - that bound to the receptor.

Civelli's team wasn't alone. An onslaught of research groups around the globe raced to identify the missing ligands to these "orphan receptors," which are GPCRs whose ligands have yet to be discovered. Between 1986 and 2006, about 300 orphan receptors were paired with their natural ligands, and dozens of these pairings titillated academics and pharmaceutical researchers with their therapeutic potential.

Today, 30% of the 800 known GPCRs are drug targets, and nearly half of the thousands of drugs sold target a GPCR. The approximately 800 G protein-coupled receptor genes are the largest family of genes in the human genome. They encode a superfamily of membrane-bound receptors, which detect a wide range of signaling molecules outside the cell and, in response, turn on signaling cascades inside the cell. GPCRs control countless processes in the body, including appetite, vision, smell, and heartbeat, endowing the receptor class with enormous therapeutic potential. About half of the 800 receptors are chemosensory-related receptors, which are unlikely sites for drugs; the other half are already, or might one day be, viable drug targets.

Despite these advances, in 2004, with about 100 unpaired GPCR orphans remaining, the field slowed dramatically. Civelli's team dwindled. Of the seven researchers in his lab today, only two work part-time to deorphanize GPCR receptors, and they haven't had any publishable results for more than three years. "When you talk to people in the field, they say it was a little bit of a disappointment," says Graeme Milligan, a professor at the University of Glasgow in Scotland.

Drug Development of Deorphanized GPCR Ligands
Receptor	Year Deorphanized	Ligand	Ligand Source	Therapeutic Indication	Drug/Brand Name	Manufacturer	Phase
D2 (RGB-2)	1988	D2 antagonist	Brain	Schizophrenia	Clozapine	Novartis, Azur Pharma	In treatment
CCR5	1995	Rantes, MIP-1ax, MIP-1B	Immune System	HIV	Maraviroc	Pfizer	In treatment
ORL-1	1995	Nociceptin/Orphanin FQ	CNS	Blood pressure, pain	ZP120	Zealand Pharma	II
Orexin1, 2	1998	Orexin A and B	Brain	Food intake, insomnia	Almorexant	Actelion	III
H3	1999	H3 antagonist	Brain	Narcolepsy, ADHD, schizophrenia	JNJ-17216498 MK0249	Johnson & Johnson Merk	II II
GHSR	1999	Ghrelin	Stomach	Anorexia nervosa, cahexia	TZP-101 SUN-11031	Tanzyme Asubio Pharma	II II
SLC-1	1999	MCH	Brain, Islets of Langerhans	Energy homeostasis, diabetes	NGD-4715	Neurogen	I complete
GPR38	2001	Motilin	GI tract	Gastroparesia and IBS	GM-611	Chugai Pharma	II complete
LGR7	2002	H2 Relaxin	Heart	Heart failure	Relaxin	Corthera	II/II
S1P	2005	FTY720	Lymphocytes	Multiple sclerosis	Fingolimod	Novartis	III
BLT1, 2	2005	LTB4	Leukocytes	Pancreatic cancer	LY293111	Eli Lilly and Co.	II complete

 

Then slowly, drugs that target once-orphaned GPCRs started emerging: Last year, Pfizer released its new anti-HIV drug, Maraviroc, which targets CCR5, a chemokine receptor that HIV uses to bind with human T cells; in 2006 Janssen Pharmaceuticals introduced paliperidone, a new schizophrenia drug that targets five once-orphaned neuro- GPCRs; and Corthera Pharmaceuticals is about halfway through a Phase III trial of relaxin, a drug that targets the once-orphaned LGR7 receptor and mediates heart failure. It appears that we are finally beginning to see the fruits of 20 years of labor.

Traditionally, researchers studying signal transduction pathways have started with a discrete physiological response in assays or tissues; further study would reveal the receptor and the ligand that binds to it. The discovery of many GPCRs took the reverse route: Researchers identified the receptors first without knowing either the receptors' functions or ligands.

In 1986, Robert Lefkowitz's group at Duke University successfully cloned the mammalian beta 2-adrenergic receptor (beta 2AR), which binds hormones such as adrenaline, and then generates a second messenger - cAMP in the case of beta 2AR - to signal a cellular response such as increased heart rate or dilated pupils.¹ The receptor showed a remarkable homology to the seven-transmembrane receptor rhodopsin, involved in nighttime light perception, and the only receptor known at the time to act through a G protein. The new beta 2AR genomic sequence suggested that a new family of receptors might exist, and they all might share a common structure.

Though it was early in his career, Civelli, then at the Vollum Institute at the Oregon Health & Science University, gambled that he could use the sequence of the beta 2AR gene as a screen for similar receptor genes. In the same year that the Duke group published, Civelli recloned beta 2AR and used it as a probe to screen a rat genomic library. At the time, many researchers told him that the approach was a long shot and that his team wouldn't touch a new receptor with a "10-foot pole." Other researchers were using DNA homology to find new genes, but since Civelli had only two other models - rhodopisn and beta 2AR - there was a high chance for false-positive results. "People were telling me this is just a fishing expedition," he says, "and that I'd come out with plenty of data but no fish," in this case, receptors.

On a spring evening in 1987, his research assistant, Jim Bunzow, while preparing to give a presentation at the weekly lab meeting the next morning, came into Civelli's office. "'I looked at my data,' Bunzow said, 'and I think that we have two transmembrane domains in one of the clones [identified by the screens],'" Civelli recalls. Civelli looked at the data, and the sequence was similar enough to the beta 2AR sequence that he knew: They'd found the gene for a new receptor.

Civelli's new receptor turned out to be the D2 receptor, one of five subtypes of the dopamine receptor family. "That [discovery] was sufficient for realizing that this approach could lead to the discovery of new receptors," says Civelli, "and that was a highlight of my research career." The receptor had seven transmembranes and no ligand, and it was one of the first two "orphan" GPCRs discovered (the other was the serotonin receptor 5-HT1A), although the term had not yet been coined.

Civelli went on to show that the receptor was preferentially expressed in the brain, particularly in the caudate, putamen, and pituitary. "This told us it could be the D2 receptor," he says, since only D2 is expressed in the pituitary gland. The team transfected cells with the putative D2 receptor gene, to see if the cells would express the receptor. Civelli then extracted the membrane of the cells and treated them with a radiolabeled D2 receptor antagonist, haloperidol, which several pharmaceutical companies had developed to treat schizophrenia. Haloperidol bound Civelli's receptor with the same affinity that the pharma companies had shown with the D-2 receptor in the rat brain, before they had known the sequence or structure of the receptor. Civelli's clone was the D2 receptor, and no longer an orphan.²

The orphan-receptor field exploded. "Initially the work was driven by the excitement of identifying a receptor you didn't know anything about," says Jeffrey Benovic, now professor and chair at the Thomas Jefferson University Kimmel Cancer Center, who was an author of the Duke beta 2AR cloning paper. "There was a lot of excitement in the '80s and up through the next 10 years; from then on you were left with labs where [deorphanizing] was the primary focus." The ultimate goal: identify new drug targets.

Using Civelli's homology-screening techniques, and then turning to a more rapid PCR-based approach, which also relied on sequence homology, researchers identified hundreds of receptors without their ligands. To then match the receptor to a ligand, researchers expressed the receptor in eukaryotic cells by DNA transfection. They then exposed the orphan receptor to tissue extracts that researchers hoped contained the natural ligand - guessing, in many cases, that the ligand would live in brain or gut tissue, for example, depending on the orphan receptor's homology to other receptors. Changes in second-messenger responses, which researchers measure by also making educated guesses about what to look for, indicated that the orphan receptor had found its ligand. By the mid-1990s, high-throughput tools had been developed for the detection of second-messenger responses.

Thanks to the development of high-throughput assays in the 1990s, on average, researchers deorphanized 10 receptors per year for nearly a 20-year stretch. Even so, in the 1980s and into the 1990s, hunting for missing ligands and deorphanizing receptors was truly uncharted water for the field of molecular biology. In searching for unknown ligands, researchers faced two unknowns: the nature of the transmitter (the type of tissue where it would likely be found), and the nature of the second messenger.

In less than two decades, industry and academia worked rapidly and deorphanized about 300 receptors. "We suddenly had a plethora of targets by 2002 or 2003," Civelli says. Then, the pace of discovery changed remarkably.

Following that rapid burst of activity and success, researchers began to identify barely one ligand every two to three years. The old techniques for deorphanization simply stopped working. Currently, approximately 100 GPCR orphans have not been united with their natural ligands.

Why? "I think that one of the possibilities is that some of these GPC receptors may not have a ligand," says Anthony Davenport, a researcher in the clinical pharmacology unit at the University of Cambridge, UK. Civelli says he suspected even in the 1990s that the receptors outnumbered the ligands; it just took more than 10 years for the pool of easily matched pairs of receptors and ligands to deplete.

Now, some researchers who once searched for ligands have shifted their focus toward determining the function of orphan receptors and explaining why their ligands remain elusive. Currently, "in academic labs, 99% of the work [on GPCRs] is focused on trying to better understand how these receptors function - anywhere from structure to biology," says Benovic. His group is concentrating on the proteins expressed downstream of the receptor itself, namely the arrestins and GPCR kinases (GRKs), which work in tandem to regulate GPCR interactions inside the cell.

A major realization has been that some receptors don't act alone as homodimers, but rather work with other receptors in heterodimers. In one instance, the orphan GPR50 receptor was found in 2006 to inhibit another receptor. This finding has given researchers pause, says Jeffrey Conn, director of the Vanderbilt University Program in Drug Discovery. By dimerizing with the MT1 melatonin receptor, which is involved in signaling for sleep, GPR50 could prevent MT1's signaling cascade.³ Therefore, researchers may never find GPR50's natural ligand, because it doesn't have one: Its primary purpose is to act on another receptor.

Timing may be another element preventing further deorphanizations. "Several ligands are missing because we don't know when to find them," says Civelli. For instance, some ligands might be expressed only at a particular time in the life of the organism or under particular conditions. The ligands are "probably presenting in low levels, or only secreting at a time when there is a particular response. If we don't know when the response happens, we can't find the ligand, and don't know when to look for it."

Some receptors, even though they act through G proteins, might not signal a second messenger at all, but rather might link to yet undefined transducing pathways. Other receptors might bind more than one ligand, and still others might require the expression of accessory proteins for their activity, thus complicating the traditional method of expressing the receptor alone. "The low-hanging fruit for receptors has been exhausted," says Conn.

Pharmaceutical companies had another reason for slowing down their search for ligands for orphan receptors. "Companies have stopped [deorphanizing] because companies realized they had too many GPCRs, and pharmacology had to catch up to the pairing with molecular biology," says Davenport. "And in most cases the new transmitter systems were blank canvases; they knew very little about the ligand or receptor."

Mounting evidence is showing that these receptors alone could have drastic effects on development and other processes, indicating that even orphaned receptors might serve vital yet undiscovered purposes: A recent study of a mutant once-orphaned GPR54 in mice and humans showed that without the functioning receptor, puberty never begins, whereas overexpression of the receptor leads to precocious puberty.⁴

In the mid- to late-1990s, one-third of the 125 employees of Lundbeck Pharmaceuticals (formerly Synaptic), in Paramus, NJ, was devoted to deorphanizing GPCRs. None of the researchers are working on this project now, says Mary Walker, principal scientist in GPCR research at Lundbeck.

"In the late '90s we put a lot of effort into the various activities that would support deorphanization," Walker says, including developing universal functional assays, cellular reporters to record signaling output, and cell-surface tagging. Now, the company has disbanded the deorphanization program and shifted its focus to studying the function of neuro-GPCRs in the larger context of disease in the body - what the company calls systems biology. The deorphanization focus was an "opportunistic approach without strong rationale scientifically. You didn't know if what you were going to get was going to be useful."

Moreover, the amount of energy going into the process of deorphanizing GPCRs and then figuring out how they worked, not to mention developing drugs against them, became much too costly, says Walker. In general, "a deorphanization project will cost more in the long run because by definition you are further back in the biology pipeline, knowing less about the function and potential disease relevance before you commit resources," she says. Now, Lundbeck researchers use gene arrays and gene expression to identify other factors in addition to deorphanized GPCRs that are altered in disease states. "People's thinking has matured," Walker says, "because other technologies are able to give us [the targets] we needed."

Johnson & Johnson, Wyeth, and Takeda Pharmaceuticals declined comment on their orphan GPCR drug-discovery programs.

"So far there aren't many drugs that have come through against deorphanized receptors," says Milligan. "That partially may be a timeline [issue], but a number of easy-to-deorphanize receptors don't have important roles in controlling physiology; they don't have big effects in the way a beta blocker or antihistamine does in the industry."

Nevertheless, progress in the field of once-orphaned GPCR drugs may not be as disappointing as it seems. According to a recent review by Civelli, drugs targeted to at least 14 deorphanized GPCRs are in pharmaceutical development, with some moving into Phase III clinical trials.⁵ One late-stage project includes drugs that target the deorphanized orexin receptor, involved in appetite signaling. Although the orexin receptor was deorphanized 10 years ago, it has taken some time to develop an understanding of the receptor's function, which adds time to the search for compounds for clinical trials, says Milligan.

More than a dozen of Lundbeck's drugs in the pipeline target GPCRs that were once orphans, says Walker. In the past few years, several promising former orphan targets have moved from the bench to beside, including Pfizer's Maraviroc, and propranolol, a beta blocker that several companies market. Propranolol blocks the action of epinephrine at the beta 2AR and is primarily used to treat hypertension (See chart for some of the clinical progress).

If one field might be able to accelerate the pace of GPCR drug development, it is structural biology. At the start of 2007, Brian Kobilka's group at Stanford University successfully solved the structure of the beta 2AR.⁶ Before that structure was available, researchers could model their receptors on only the rhodopsin structure, which was solved at high resolution in 2000. In June, the beta1-adrenergic receptor structure was published,⁷ and researchers are hopeful that structural information will quickly advance work on GPCRs. Also in June, researchers successfully solved the structure of opsin, the unbound version of rhodopsin.⁸ "I think it will reduce the need to carry out very extensive screens, and in many cases that's a major expense," Davenport says.

Benovic warns, however, that the active conformation of these structures is still unknown. In vivo, these receptors are constantly mobile, changing conformation. To crystallize them, researchers have come up with ways to keep them locked, which deactivates the molecules.

Still, "I'm very enthusiastic," says Davenport. "I think big pharmaceutical companies are reeling from 10 years of not being good at making" compounds that can block or stimulate ligand-receptor activity. That's likely to change, he adds, as those same big companies commandeer some proof-of-concept technologies, such as allosteric-binding approaches that manipulate receptors at different sites from where their natural ligands bind. These technologies have been developed at academic or small biotech labs such as Addex, 7TM, and Predix. "I still think lots of new targets are out there; GPCRs will undergo a renaissance in the pharma industry."

Two members in Civelli's lab still try to deorphanize a handful of receptors, but the work hasn't produced any findings for more than three years. Meanwhile, he focuses mainly on studying the melanin-concentrating hormone receptor, which he deorphanized in 1999, and which has implications in anxiety, depression, and obesity. Civelli's group has developed a small-molecule antagonist to block the reward response for cocaine self-administration in rodents at the melanin-concentrating hormone receptor. The results are promising. "It's not as clear-cut as when you get a [homologous] sequence in front of your eyes," Civelli says, referring to his discovery of the D2 receptor more than 20 years ago, "but we have big hopes."

Correction: The original version of this story incorrectly referred to haloperidol as a D2 receptor agonist. Haloperidol is a D2 receptor antagonist. The change has been made and The Scientist regrets the error.

this article is mind-numbingly boring!

Propanolol is hardly a new compound. In the 1970s it was used to quantify and to characterize the binding behavior of adrenergic receptors. (You don't need DNA or protein sequence/structure to study a receptor's behavior.) Now the pharmaceutical companies are going to market propanolol as a new anti-hypertension drug? I wonder if they'll have to come up with a novel delivery system, e.g., time-release pills, or if it can be patented because it's "new" as a pharmaceutical - even though it's been around for at least a quarter-century.

The underlying assumption in "the hunt" is that the ligand is always a protein. What if it's not a protein? When an approach stops working, it's time to think outside the box!

Without knowing the ligand or defined function, could we call the protein a receptor? A protein should be named receptor on the basis of "functional homology". A beter approach, a more economical approach to finiding ligands would be to use immuno-localization techniques. Most of the receptors as noted in the article are mobile ; upon binding the ligand they trigger cellular events such as trans-location of the receptor-ligand complexes from the original site to another site within the cell. This event can be studied by immuno-localization techniques using atibody probes to the targeted receptor. Technology is available for rapid screening of a variety of compunds to study the receptors in the tissue in their natural state. This approach could shed light on possible ligand/s and function of these "receptors".

Haloperidol is actually a D2 antagonist not an agonist (typo in article). Otherwise an interesting read.

The D2 receptor as an orphan receptor? Give me a break - Civelli might not have know what he had but this was probably the best charaterized brain receptor in 1988 - this was the first cloning but it was hardly an orphan.