In the World of Agonists and Antagonists, Something Inverse Comes Our Way

By Eric Sauter

For anyone comfortable with the Manichean concept of dualism the very idea of an inverse agonist comes as something of a challenge. The functions of agonists and antagonists are well known – a compound that activates a receptor is an agonist and one that blocks it is an antagonist.

But what does an inverse agonist do?

Patrick Griffin, Scripps Research chair of the Department of Molecular Therapeutics and director of the Translational Research Institute at Scripps Florida, explains: "Some receptors have an endogenous or intrinsic activity that doesn't need the presence of a ligand. An inverse agonist binds to the receptor and induces the opposite action of an agonist of that receptor, which is obviously different than just turning its activity off."

Now, in a pair of studies, Griffin, along with Thomas Burris, a professor in the Department of Molecular Therapeutics, and their colleagues, have for the first time identified high affinity synthetic inverse agonists as well as some potent natural ligands of a pair of human nuclear receptors that regulate a variety of physiological processes, including fat metabolism, liver glucose production, circadian rhythm, and immune function.

Their discoveries, published in Molecular Pharmacology and The Journal of Biological Chemistry, could potentially lead to the development of selective modulators of these key actors, which raises the possibility that small molecules can be used to target these same receptors in the treatment of a wide range of metabolic and immune disorders.

Key Players

The receptors in question are retinoic acid receptor-related orphan receptors or RORs, members of the nuclear receptor superfamily, a large group of proteins that regulate the expression of key genes involved in a wide range of physiological processes, such as carbohydrate and fat metabolism, often in response to ligands such as steroid hormones, lipids, or sterols; in this context, ligands bind to proteins and trigger a biological response.

"In recent years, the RORs have gained a lot of interest because they play key roles in metabolic disorders and autoimmune disease," Griffin said. "For instance, one recent study showed that a mouse model with an inability to produce one of the RORs, ROR alpha [RORα], recapitulated von Gierke's disease, a common glycogen storage disorder—patients lack an enzyme that releases glucose from glycogen, which causes an abnormal build up of glycogen. So, repressing RORα would have a role in inhibiting glucose production in the liver in diabetes."

The first ROR (RORα) was discovered in the early 1990s. Similarities to the retinoic acid receptor (RAR) and the retinoid X receptor (RXR) resulted in the name "retinoic acid receptor-related orphan receptor." The orphan moniker stems from the fact that the RORs, unlike the RARs and RXRs, have not yet had a widely accepted natural ligand identified.

RORα is expressed in the liver, skeletal muscle, skin, lungs, adipose tissue, kidney, thymus, and brain, while RORγ is highly expressed in the thymus; however, significant expression is also found in the liver, skeletal muscle, adipose tissue, and kidney.

Both RORα and RORγ regulate key physiological pathways and are also involved in pathogenic processes. RORα regulates lipid and glucose metabolism and is believed to play a role in protection against development of atherosclerosis. This receptor also is critical for normal function of the mammalian clock.

The most prominent role for RORγ is regulation of immune function, especially in development of cells that are believed to play an important role in autoimmunity, thus knocking down RORγ activity with synthetic ligands should suppress the immune response in autoimmune disease. RORγ also helps coordinate lipid and glucose metabolism in concert with RORα. RORα and RORγ have also been implicated in bone development and cancer.

Productive Screens

For the Molecular Pharmacology study, Griffin, Burris, and colleagues used a screen against all 48 known human nuclear receptors and found that a potent LXR (liver X receptor) agonist known as T0901317 also directly binds to RORα and RORγ resulting in modulation of the receptors' activity. The liver X receptors are nuclear receptors that play a role in maintaining cholesterol and fatty acids homeostasis in the liver.

"The discovery of this novelty began with a genomic screening program that already existed as part of the Scripps National Screening Center grant," Griffin said. "Since we had the genomic screening platform at Scripps Florida, we thought, 'Why not build a library of all known human nuclear receptors and use that to profile ligand selectivity? Maybe we'll find common structures of these ligands that jump from one receptor to the other.'"

Juliana Conkright, a staff scientist who works in Scripps Florida's Translational Research Institute, worked with Griffin to first clone a library of all 48 human nuclear receptors, and then optimize an assay to test a small set of about 65 compounds that were known ligands for nuclear receptors against that library.

"Our screen did what we expected, the compounds we tested activated a lot of the receptors," Griffin said, "but what was surprising was finding a ligand that repressed the intrinsic activity of the RORs—thus functioning as inverse agonists."

This ligand was T0901317, a well characterized agonist of the LXRs. Through careful examination it was determined that T0901317 binds directly to the RORS and represses their activity and functions as a potent inverse agonist of both RORα and RORγ.

Natural Ligands for the RORs

In the Journal of Biological Chemistry study, Griffin, Burris, and colleagues showed that 7-oxygenated sterols were also natural, high affinity ligands for both RORα and RORγ that suppressed their transcriptional activities.

Sterols are organic compounds found in plants and animals that play key roles in signaling; cholesterol is an animal sterol. One of the 7-oxygenated sterols functions as intermediate of bile acid metabolism while others are key fats in atherosclerosis. These sterols may regulate the transcriptional output of the active RORs in both liver glucose production and circadian rhythms.

"These were orphan receptors in both these studies—meaning that their ligands were unknown," Tom Burris said. "We managed to de-orphanize them. For the Journal of Biological Chemistry study, the fact that we discovered endogenous or natural ligands gives us a better idea of what these receptors are doing physiologically. In fact, using this information we were able to show that RORs provide a link between the regulation of bile acid metabolism and glucose metabolism. Additionally, the fact that one of these sterols is found in atherosclerotic plaque and suppresses RORα activity is consistent with previous genetic studies indicating that loss of RORα predisposes animals to development of atherosclerosis."

What Burris and Griffin suggest is that these natural ligands that are found in atherosclerotic plaque may, in fact, be turning off the RORs and advancing cardiovascular disease. This new finding, Burris added, could point the way toward the development of novel synthetic treatments (drugs) for the disease.

As in the other study, these findings also validate these nuclear receptors as viable drug targets, he said.

The first author of the Molecular Pharmacology study, "The Benzenesulfoamide T0901317 is a Novel RORα/γ Inverse Agonist," is Naresh Kumar of The Scripps Research Institute. Other authors in addition to Griffin and Burris include Laura A. Solt, Juliana J. Conkright, Yongjun Wang, Monica A. Istrate, Scott A. Busby, and Ruben D. Garcia-Ordonez of The Scripps Research Institute. For more information, see http://molpharm.aspetjournals.org/content/early/2009/11/10/mol.109.060905.

The first author of the The Journal of Biological Chemistry study, "Modulation Of Rorα And Rorγ Activity By 7-Oxygenated Sterol Ligands," is Yongjun Wang of The Scripps Research Institute. Other authors include, Naresh Kumar, Laura A. Solt, Christine Crumbley, Ruben D. Garcia-Ordonez, Xi Zhang, Scott Novick and Michael J. Chalmers of The Scripps Research Institute; and Timothy I. Richardson, Leah M. Helvering and Keith R. Stayrook of Eli Lilly & Company. For more information see http://www.jbc.org/cgi/doi/10.1074/jbc.M109.080614.

Both studies were supported by the National Institutes of Health.

Send comments to: mikaono[at]scripps.edu

"In recent years, the RORs have gained a lot of interest because they play key roles in metabolic disorders and autoimmune disease," says Patrick Griffin, chair of the Department of Molecular Therapeutics.


"These were orphan receptors in both these studies—meaning that their ligands were unknown," Tom Burris says. "We managed to de-orphanize them."