July 1, 2011 (Vol. 31, No. 13)

Kathy Liszewski

Therapeutics in Development Aim to Enhance Selectivity and Reduce Potential Side Effects

Nuclear receptors drive many critical processes ranging from metabolism to reproduction by serving as ligand-activated transcription factors. They also represent a lucrative area for drug discovery as evidenced by current therapies utilizing glucocorticoids for asthma, estrogens for hormone-replacement therapy, and retinoids for acne.

Current efforts continue to take aim at developing compounds that improve function and target specificity while reducing untoward effects.

Last month’s “Endo 2011” meeting described emerging concepts and new studies that are helping to unravel the complex interactions of nuclear receptors and their ligands. Topics included a perspective on the state of the field, new research delineating pathways, and improved strategies for developing novel therapeutic ligands.

“Manipulation of nuclear receptors has much potential to cure diseases,” says John D. Baxter, M.D., chief of the division of endocrinology and director of the Genome Medicine Program at Methodist Hospital Research Institute. Dr. Baxter presented a perspective on the past, the present, and the future of the field.

“This has been a robust field that has probably not received the attention it deserves for its potential or for its past discoveries. At least 20 percent of all prescriptions are for drugs that bind nuclear receptors.”

Assessing the field at the present moment, Dr. Baxter reported, “We are able to manipulate nuclear receptors for a growing list of diseases. We have estrogens and androgens such as testosterone, and antagonists to these receptors.

“We use retinoids to treat skin diseases, glucocorticoids such as prednisone to treat inflammatory states, and mineralocorticoid antagonists to treat heart failure. I work with the thyroid receptor that impacts lipid disorders, obesity, diabetes, fatty liver, and atherosclerosis.”

In the past, Dr. Baxter summarized that “key discoveries were initially made in the late 1950s and early 1960s. These included demonstrating that steroid hormones could be selectively taken up in hormone-responsive tissues, an emerging recognition of the presence of receptors, and the landmark finding that these receptors served as transcription factors.

“We also learned that there are variations in receptor numbers and in their ability to act that led to the huge field of hormone resistance, important for many diseases.

“Later,” he continued, “the mosaic nature of the receptor was identified in that these are single polypeptide chain proteins with domains that serve multiple functions including promotion of binding to DNA and interactions with coregulators that connect receptors to downstream receptor actions.

“Such binding transmits information to promote regulation of transcription and cloning of coregulators, important in many diseases. This greatly enhanced our understanding of receptor function.

“Cloning of receptor genes led to realization that there are many more receptors than we thought existed and to the discovery of many additional receptors that have important roles in disease. Determinations of receptor structures at atomic resolution greatly enhanced our ability to design drugs that target receptors and to understand receptor mechanisms of action.”

For the future, Dr. Baxter sees a growing increase in using these new discoveries to better understand and treat pathologies.

“Structural modeling provides an enormous help to understand the structure/function of receptors to assist with improved drug design. This continues to be a massively important field for drug discovery. As we better determine where and how drugs act, we can develop improved drugs using combinatorial chemistry to more selectively target specific nuclear receptors.”

As a final note, Dr. Baxter said he hopes that Nobel prizes are on the horizon for the investigators that have made important discoveries in this field and the widespread impact that has resulted from them.

Many scientists maintain that a better understanding of the structure of nuclear receptors will lead to the development of new therapeutics for a number of human diseases. [Alexander Raths/Fotolia]

Orphan Receptors

The human nuclear receptor superfamily consists of 48 genes. However, three-fourths of these expressed products are orphan receptors, meaning that their natural ligands (i.e., cognate hormones) are unknown.

One unifying feature of all nuclear receptors is the presence of five functional modules. Sought after for drug discovery, the C-terminal ligand-binding domain (LBD) consists of about 250 amino acids. “Orphan nuclear binding receptors continue to be pursued as therapeutic targets,” noted Timothy M. Willson, director of chemical biology at GlaxoSmithKline.

“We know that nuclear receptors, including the 36 orphan receptors, serve to regulate mRNA production. But we have many unanswered questions. Do they all have ligands? What is the biological function and structure of each receptor? Which nuclear receptors are appropriate targets for drug discovery?”

Dr. Willson described GSK’s approach. “We’ve spent more than 10 years working on orphan receptors as drug discovery targets. We initially took a chemical approach in screening receptors with synthetic ligands and assessing their biology. We found many that are ligand regulated, but also a number that are not.

“Next, we undertook a biochemical approach to express receptors, purify the proteins, and identify their captured ligands by mass spectroscopy. We found that each one has a unique story.”

One example is the company’s work on the multifunctional farnesoid X receptor (FXR) that binds bile acid, regulates bile acid biosynthesis, bile acid enterohepatic circulation, and triglyceride metabolism. “FXR is a fascinating protein implicated in healthy liver function. We helped develop a small molecule ligand that is currently in Phase II clinical trials for the treatment of liver disorders.”

As nuclear receptors are better delineated, Dr. Willson expects the identification of many other novel ligands. “This is an emerging field capable of providing unique resources that will allow us not only to better understand how these receptors function in health, but also new treatments for human diseases. Achieving greater knowledge will especially help drug discovery groups focus on what diseases to target.”

Metabolic Syndrome

Metabolic syndrome is becoming as widespread as the common cold, afflicting a staggering one in six (~47 million Americans), according to experts. However it is not a disease, but rather a pooling of risk factors that includes obesity, elevated levels of blood sugar (insulin resistance) and cholesterol, and hypertension. Individuals with this cluster of conditions have a fivefold higher chance of developing type 2 diabetes and twice the risk for blood vessel and heart disease.

“Excessive body fat is the nucleus of metabolic syndrome,” reported Bruce M. Spiegelman, Ph.D., Korsmeyer professor of cell biology and medicine, department of cancer biology and division of metabolism and chronic disease, Dana-Farber Cancer Institute, Harvard Medical School.

“One of the key players controlling fat metabolism is the peroxisome proliferator-activated receptor gamma (PPARγ), a target of type 2 diabetes drugs such as the thiazolidinediones. Engagement of PPARγ by these drugs helps ameliorate insulin resistance but has long-term adverse effects such as increased risk of heart failure.”

Dr. Spiegelman’s group found that PPARγ activators seem to have a second role. “In doing structural studies on PPARγ, we noticed the presence of a consensus site for phosphorylation by the cyclin-dependent kinase 5 (Cdk5) and decided to study this. In in vitro studies, we found that Cdk5 in the presence of its cofactor, p35, phosphorylates PPARγ.

“We also found that Cdk5 phosphorylates PPARγ in mice fed a high-fat diet and that this subsequently causes the dysregulated expression of a set of genes important for metabolism, such as adipsin, which is a fat cell-selective gene altered in obesity, and adiponectin, which regulates insulin sensitivity.”

However, the team found that antidiabetic drugs could block phosphorylation of PPARγ. This could be important for designing future therapeutics. “Our studies demonstrated that antidiabetic drugs can inhibit obesity-linked phosphorylation of PPARγ by Cdk5. It appears that two separate activities are occurring. Thus, only part of the side effects seen with these drugs may be elicited via classical agonist actions.

“There is also a separate activity of blocking phosphorylation by Cdk5. Since partial agonists can alter protein phosphorylation in some nuclear receptors, it seems reasonable to believe that this could allow identification of new and better drugs for metabolic syndromes and other nonadipose tissue, since the adverse effects of obesity extend to cancer and even neurodegeneration.”

Dr. Spiegelman is now collaborating to produce ligands with these separate activities. “By examining known PPARγ ligands, we’ve begun extensive chemical modification of scaffolds to create higher affinity ligands. We’ve created a number of promising candidates. Our hope is to produce improved drugs for metabolic syndrome.”

Novel Ligands

The proinflammatory T helper type 17 (TH17) cells are an important part of the immune armamentarium, but they also have been linked to development of autoimmune diseases such as multiple sclerosis, rheumatoid disease, systemic lupus erythematosus, and inflammatory bowel disease.

The discovery that two nuclear receptors play an important role for the development of this special subset of T cells opened up the possibility that the receptors could be utilized for development of focused therapeutics that inhibit inflammation in general and TH17 cells in particular.

Thomas P. Burris, Ph.D., professor at Scripps Research Institute, and colleagues are studying the retinoic-acid-receptor-related orphan receptors. They developed a high-affinity synthetic ligand to this nuclear receptor that inhibits differentiation and function of TH17 cells.

“TH17 produce several proinflammatory cytokines. Targeting TH17 directly has the advantage of shutting down inflammation rather than attempting to block each individual cytokine. This approach also has an advantage in that it does not interfere with natural T regulatory cells.”

Animal studies performed in Dr. Burris’ lab found that the synthetic compound SR1001 delayed onset and severity in a murine model of autoimmune encephalomyelitis. “These studies have created a lot of excitement in the field. We continue to work with this compound and also have developed others with improved activity that we are studying.”

Dr. Burris is also developing novel ligands for nuclear receptors important for metabolism. “Nuclear receptors have been clearly linked to lipid homeostasis as well as circadian rhythms. We identified and then modified ligands for the REV-ERB family that regulates lipid and glucose metabolism. We found ligands that not only modulated metabolism but also resulted in weight loss. We are looking to see if this could be a treatment for metabolic disorders.”

One serendipitous finding of these studies was that some synthetic compounds also had an effect on behavior. “We found ligands for the REV-ERB family that regulates sleep patterns and others that reduce anxiety. The association of clock genes with these types of behavior may provide new targets for developing novel therapeutics. We are currently looking for partners to license our compounds for clinical development.”

Versatile Estrogen Receptors

Nuclear receptors can be promiscuous, that is, can react with multiple ligands. “I prefer to call that eclectic behavior,” said John A. Katzenellenbogen, professor, department of chemistry, University of Illinois at Urbana-Champaign.

“The estrogen receptor is an example of a receptor that plays diverse roles. Estrogen receptors are rather remarkable in being expressed not only in reproductive tissues such as the uterus and breast but also in the cardiovascular system, the lung, and the brain. This can be helpful or deleterious with regard to therapies. The breast cancer inhibitor tamoxifen blocks the estrogen receptor but unfortunately also stimulates the uterus.

“Our knowledge of the structure and function of nuclear receptors continues to grow. The old lock-and-key concept has evolved to a more sophisticated level in that we are now looking at the cross-talk among receptors and their exquisite ability to accommodate different ligand structures. Using medicinal chemistry approaches, we have found that rather unusual structures can surprisingly result in enhanced selectivity.”

Dr. Katzenellenbogen was recently part of a collaboration with Christopher K. Glass of the University of California School of Medicine, San Diego, that found ligands capable of exerting neuroprotective effects in a subset of estrogen receptors in microglia and astrocytes.

“These cells are found primarily in neural tissue and play a role in the maintenance of homeostasis and responses to infection and cellular injury. We provided a set of our ligands and saw that different ligands affected inflammation differently.

“Aside from in vitro assays, these neuroprotective effects extended to an animal model of multiple sclerosis—i.e., murine experimental autoimmune encephalomyelitis. We hope to continue studies with these ligands that we feel have a potential for therapeutic development.”

Dr. Katzenellenbogen cautioned that “overall, these studies show how specific chemical modifications in ligands can have different effects for the better or for the worse. Ultimately, as we better understand how ligands fit into their various receptors, we hope to be able to design improved drugs with reduced untoward effects.”

Researchers from the University of Illinois and the University of California, San Diego recently found ligands capable of exerting neuroprotective effects in a subset of estrogen receptors in microglia and astrocytes.[University of Illinois]

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