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Scientific Report 2007

Scripps Florida

Molecular Therapeutics

Mechanism of Activation of Nuclear Receptors

P.R. Griffin, S.A. Busby, M.J. Chalmers, S. Prasad, S.Y. Dai

We use a wide range of technologies to study ligand activation of nuclear receptors. Previously, we focused heavily on peroxisome proliferator-activated receptor γ (PPARγ; see following), but we are beginning to study several orphan nuclear receptors that are implicated in a variety of disorders. Of particular interest are the orphan receptors liver receptor homolog-1 and steroidogenic factor-1. We are building the appropriate tools to begin comprehensive mechanistic studies of these 2 receptors.

Mechanistic Studies of Ligand Activation of PPARγ

PPARγ is a multidomain ligand-dependent transcription factor. Activation of PPARγ is regulated by binding of ligands to the receptor's ligand-binding domain, which induces a change in the conformational dynamics of the domain-mediated dissociation of corepressor molecules and forms suitable neoepitopes for binding coactivator molecules. Ligands of PPARγ are characterized as full or partial agonists on the basis of their ability to transactivate PPARγ target genes. Full agonists have maximal transcriptional activity, whereas partial agonists have moderate activity. Although the structural and molecular determinants of full-agonist regulation of PPARγ have been studied in detail, the determinants for partial agonists have not been completely characterized. We are using structural, biochemical, and cell-based techniques to examine the mechanism of regulation of PPARγ transcriptional activity by partial agonists.

Hydrogen-deuterium exchange (HDX) coupled with mass spectrometry was used to characterize ligand-dependent structural and dynamic changes in PPARγ. Ligand-induced protection of amide exchange in a transcriptional complex composed of PPARγ, retinoic X receptor α, and either steroid receptor coactivator-1 or steroid receptor coactivator-3 were probed by using automated solution-phase HDX. We used fluorescence-based assays to determine recruitment of coactivators and to measure the affinity of the receptor heterodimer composed of PPARγ and retinoic X receptor α.

A mammalian 2-hybrid genetic screen was used to probe the molecular determinants of ligand-dependent coactivator selectivity.

We found that the magnitude of PPARγ agonism is regulated by coactivator recruitment selectivity of p160 coactivators. The functional relevance of the genetic screen was further confirmed in a 3T3-L1 preadipocyte differentiation assay as an in vitro model of adipogenesis. We found that conversion of the full-agonist phenotype to the partial-agonist phenotype and vice versa was a function of the availability of specific p160 coactivators.

Using combined proteomic and genomic differential analysis, we have extended these studies to examine the molecular components of PPARγ-mediated insulin sensitization and adipogenesis after treatment of preadipocyte cells with full and partial agonists of PPARγ. Our goal is to determine unique components of the insulin sensitivity pathway and dissociate them from components of the proadipogenic pathways that lead to the adverse side effects common after therapy with PPARγ full agonists. To complete this study, we are developing new strategies to improve the detection of membrane- and lipid-associated proteins, and we are using new mass spectrometry methods to measure the samples.

In other studies, we are using coactivators as chemical tools to generate desired functional responses and distinguish beneficial functions from adverse functions, a novel therapeutic avenue for treating insulin resistance that has not yet been exploited. Our goals are to determine the structure-activity relationships between PPARγ ligands and their coactivator recruitment selectivity and to obtain PPARγ ligands with preferences for specific coactivators. To this end, we have developed a validated homogenous time-resolved fluorescence assay for ligand-dependent recruitment of the coactivator to PPARγ for a large-scale high-throughput screen to identify coactivator-selective agonists of the receptor. We will do 3 different primary screenings of the National Institutes of Health small-molecule chemical library. In each screen, we will target PPARγ association with a different coactivator to obtain coactivator-specific agonists. The results obtained from this research will provide molecular insight into how recruitment of coactivators modulates activation of PPARγ and will shed light on the role of specific coactivators in the pharmacologic behavior of PPARγ modulators.

Mechanistic Studies of Ligand Activation of the Estrogen Receptor

In collaboration with scientists at Eli Lilly and Company, Indianapolis, Indiana, we used HDX to characterize the estrogen receptor. As drug targets, estrogen receptors play important roles in the treatment of multiple diseases, including breast cancer and osteoporosis. Tamoxifen and raloxifene, modulators of estrogen receptors approved by the Food and Drug Administration, have intriguing mixed agonism and antagonism effects depending on the target tissue. Structural studies have revealed differences between complexes consisting of the estrogen receptor and an agonist and complexes consisting of the receptor and an antagonist and have provided insight into how these ligands interact with the receptor. We have used HDX to examine an array of chemical compounds that have different degrees of agonism/antagonism. We found an excellent correlation between HDX profiles and pharmacologic properties, and we were able to classify estrogen receptor ligands on the basis of HDX signatures. Discoveries derived from this study will help in understanding tissue-specific activities of drugs targeted to estrogen receptors and will facilitate future drug development programs.


Busby, S.A., Chalmers, M.J., Griffin, P.R. Improving digestion efficiency under H/D exchange conditions with activated pepsinogen columns. Int. J. Mass Spectrom. 259:130, 2007.

Pascal, B.D., Chalmers, M.J., Busby, S.A., Mader, C.C., Southern, M.R., Tsinoremas, N.F., Griffin, P.R. The Deuterator: software for the determination of backbone amide deuterium levels from H/D exchange MS data. BMC Bioinformatics 8:156, 2007.

Quint, P., Ayala, I., Busby, S.A., Chalmers, M.J., Griffin, P.R., Rocca, J., Nick, H.S., Silverman, D.N. Structural mobility in human manganese superoxide dismutase. Biochemistry 45:8209, 2006.


Patrick R. Griffin, Ph.D.
Florida Campus

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