The peroxisome proliferator-activated receptor-gamma (PPAR-gamma) is a ligand-dependent transcription factor that is important in adipocyte differentiation and glucose homeostasis and which depends on interactions with co-activators, including steroid receptor co-activating factor-1 (SRC-1). Here we present the X-ray crystal structure of the human apo-PPAR-gamma ligand-binding domain (LBD), at 2.2 A resolution; this structure reveals a large binding pocket, which may explain the diversity of ligands for PPAR-gamma. We also describe the ternary complex containing the PPAR-gamma LBD, the antidiabetic ligand rosiglitazone (BRL49653), and 88 amino acids of human SRC-1 at 2.3 A resolution. Glutamate and lysine residues that are highly conserved in LBDs of nuclear receptors form a 'charge clamp' that contacts backbone atoms of the LXXLL helices of SRC-1. These results, together with the observation that two consecutive LXXLL motifs of SRC-1 make identical contacts with both subunits of a PPAR-gamma homodimer, suggest a general mechanism for the assembly of nuclear receptors with co-activators.

Repression of gene transcription by nuclear receptors is mediated by interactions with co-repressor proteins such as SMRT and N-CoR, which in turn recruit histone deacetylases to the chromatin. Aberrant interactions between nuclear receptors and co-repressors contribute towards acute promyelocytic leukaemia and thyroid hormone resistance syndrome. The binding of co-repressors to nuclear receptors occurs in the unliganded state, and can be stabilized by antagonists. Here we report the crystal structure of a ternary complex containing the peroxisome proliferator-activated receptor-alpha ligand-binding domain bound to the antagonist GW6471 and a SMRT co-repressor motif. In this structure, the co-repressor motif adopts a three-turn alpha-helix that prevents the carboxy-terminal activation helix (AF-2) of the receptor from assuming the active conformation. Binding of the co-repressor motif is further reinforced by the antagonist, which blocks the AF-2 helix from adopting the active position. Biochemical analyses and structure-based mutagenesis indicate that this mode of co-repressor binding is highly conserved across nuclear receptors.

In the course of a high throughput screen to search for ligands of peroxisome proliferator activated receptor-gamma (PPARgamma), we identified GW9662 using a competition binding assay against the human ligand binding domain. GW9662 had nanomolar IC(50) versus PPARgamma and was 10- and 600-fold less potent in binding experiments using PPARalpha and PPARdelta, respectively. Pretreatment of all three PPARs with GW9662 resulted in the irreversible loss of ligand binding as assessed by scintillation proximity assay. Incubation of PPAR with GW9662 resulted in a change in the absorbance spectra of the receptors consistent with covalent modification. Mass spectrometric analysis of the PPARgamma ligand binding domain treated with GW9662 established Cys(285) as the site of covalent modification. This cysteine is conserved among all three PPARs. In cell-based reporter assays, GW9662 was a potent and selective antagonist of full-length PPARgamma. The functional activity of GW9662 as an antagonist of PPARgamma was confirmed in an assay of adipocyte differentiation. GW9662 showed essentially no effect on transcription when tested using both full-length PPARdelta and PPARalpha. Time-resolved fluorescence assays of ligand-modulated receptor heterodimerization, coactivator binding, and corepressor binding were consistent with the effects observed in the reporter gene assays. Control activators increased PPAR:RXR heterodimer formation and coactivator binding to both PPARgamma and PPARdelta. Corepressor binding was decreased. In the case of PPARalpha, GW9662 treatment did not significantly increase heterodimerization and coactivator binding or decrease corepressor binding. The experimental data indicate that GW9662 modification of each of the three PPARs results in different functional consequences. The selective and irreversible nature of GW9662 treatment, and the observation that activity is maintained in cell culture experiments, suggests that this compound may be a useful tool for elucidation of the role of PPARgamma in biological processes.

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The peroxisome proliferator-activated receptors (PPARs) are nuclear hormone receptors that regulate glucose and lipid homeostasis. The PPAR␥ subtype plays a central role in the regulation of adipogenesis and is the molecular target for the 2,4-thiazolidinedione class of antidiabetic drugs. Structural studies have revealed that agonist ligands activate the PPARs through direct interactions with the C-terminal region of the ligand-binding domain, which includes the activation function 2 helix. GW0072 was identified as a high-affinity PPAR␥ ligand that was a weak partial agonist of PPAR␥ transactivation. X-ray crystallography revealed that GW0072 occupied the ligand-binding pocket by using different epitopes than the known PPAR agonists and did not interact with the activation function 2 helix. In cell culture, GW0072 was a potent antagonist of adipocyte differentiation. These results establish an approach to the design of PPAR ligands with modified biological activities.The nuclear hormone receptors are ligand-activated transcription factors that regulate target genes essential for mammalian physiology and development (1). The peroxisome proliferatoractivated receptors (PPARs) are nuclear receptors activated by fatty acids and their eicosanoids metabolites, which regulate genes involved in the biosynthesis, storage, and metabolism of these ligands (2). The pharmacology of synthetic PPAR ligands demonstrated the role of these receptors in regulating glucose and lipid homeostasis and established their utility as molecular targets for the development of drugs for the treatment of diabetes and cardiovascular disease (3).Biochemical and structural studies with several nuclear receptors revealed that hormone binding induces allosteric changes in the conformation of the ligand-binding domain, which promote recruitment of transcriptional coactivator proteins such as steroid receptor coactivator 1 (SRC1) (4) and CREB binding protein (CBP) (5). We recently reported x-ray crystallographic analysis of the ternary complex of PPAR␥ with the 2,4-thiazolidinedione (TZD) rosiglitazone (Fig. 1A) and the coactivator SRC1 (6), as well as the complexes of PPAR␦ with either the fibrate GW2433 or the essential fatty acid eicosapentaenoic acid (7). Despite differences in their gross chemical structure, all of these small molecule PPAR agonists share a common binding mode, in which the acidic head groups form a network of hydrogen bonds with Y473, H449, and H323 within the ligand-binding pocket. These interactions stabilize a charge clamp (6) between the Cterminal activation function 2 (AF-2) helix and a conserved lysine residue on the surface of the receptor, through which coactivator proteins are recruited to the receptor.

We have identified a novel series of antidiabetic N-(2-benzoylphenyl)-L-tyrosine derivatives which are potent, selective PPARgamma agonists. Through the use of in vitro PPARgamma binding and functional assays (2S)-3-(4-(benzyloxy)phenyl)-2-((1-methyl-3-oxo-3-phenylpropenyl)+ ++amin o)propionic acid (2) was identified as a structurally novel PPARgamma agonist. Structure-activity relationships identified the 2-aminobenzophenone moiety as a suitable isostere for the chemically labile enaminone moiety in compound 2, affording 2-((2-benzoylphenyl)amino)-3-(4-(benzyloxy)phenyl)propionic acid (9). Replacement of the benzyl group in 9 with substituents known to confer in vivo potency in the thiazolidinedione (TZD) class of antidiabetic agents provided a dramatic increase in the in vitro functional potency and affinity at PPARgamma, affording a series of potent and selective PPARgamma agonists exemplified by (2S)-((2-benzoylphenyl)amino)-3-¿4-[2-(methylpyridin-2-ylamino+ ++)ethoxy ]phenyl¿propionic acid (18), 3-¿4-[2-(benzoxazol-2-ylmethylamino)ethoxy]phenyl¿-(2S)-((2- benzoylph enyl)amino)propanoic acid (19), and (2S)-((2-benzoylphenyl)amino)-3-¿4-[2-(5-methyl-2-phenyloxazol-4-y l)e thoxy]phenyl¿propanoic acid (20). Compounds 18 and 20 show potent antihyperglycemic and antihyperlipidemic activity when given orally in two rodent models of type 2 diabetes. In addition, these analogues are readily prepared in chiral nonracemic fashion from L-tyrosine and do not show a propensity to undergo racemization in vitro. The increased potency of these PPARgamma agonists relative to troglitazone may translate into superior clinical efficacy for the treatment of type 2 diabetes.

The discovery that peroxisome proliferator-activated receptor (PPAR)-gamma was the molecular target of the thiazolidinedione class of antidiabetic agents suggested a key role for PPAR-gamma in the regulation of carbohydrate and lipid metabolism. Through the use of high-throughput biochemical assays, GW1929, a novel N-aryl tyrosine activator of human PPAR-gamma, was identified. Chronic oral administration of GW1929 or troglitazone to Zucker diabetic fatty (ZDF) rats resulted in dose-dependent decreases in daily glucose, free fatty acid, and triglyceride exposure compared with pretreatment values, as well as significant decreases in glycosylated hemoglobin. Whole body insulin sensitivity, as determined by the euglycemic-hyperinsulinemic clamp technique, was significantly increased in treated animals. Comparison of the magnitude of glucose lowering as a function of serum drug concentrations showed that GW1929 was 2 orders of magnitude more potent than troglitazone in vivo. These data were consistent with the relative in vitro potencies of GW1929 and troglitazone. Isolated perfused pancreas studies performed at the end of the study confirmed that pancreata from vehicle-treated rats showed no increase in insulin secretion in response to a step change in glucose from 3 to 10 mmol/l. In contrast, pancreata from animals treated with GW1929 showed a first- and second-phase insulin secretion pattern. Consistent with the functional data from the perfusion experiments, animals treated with the PPAR-gamma agonist had more normal islet architecture with preserved insulin staining compared with vehicle-treated ZDF rats. This is the first demonstration of in vivo efficacy of a novel nonthiazolidinedione identified as a high-affinity ligand for human PPAR-gamma. The increased potency of GW1929 compared with troglitazone both in vitro and in vivo may translate into improved clinical efficacy when used as monotherapy in type 2 diabetic patients. In addition, the significant improvement in daily meal tolerance may impact cardiovascular risk factor management in these patients.

Background: Insulin resistance (IR) can precede the dysglycemic states of prediabetes and type 2 diabetes mellitus (T2DM) by a number of years and is an early marker of risk for metabolic and cardiovascular disease. There is an unmet need for a simple method to measure IR that can be used for routine screening, prospective study, risk assessment, and therapeutic monitoring. We have reported several metabolites whose fasting plasma levels correlated with insulin sensitivity. These metabolites were used in the development of a novel test for IR and prediabetes. Methods: Data from the Relationship between Insulin Sensitivity and Cardiovascular Disease Study were used in an iterative process of algorithm development to define the best combination of metabolites for predicting the M value derived from the hyperinsulinemic euglycemic clamp, the gold standard measure of IR. Subjects were divided into a training set and a test set for algorithm development and validation. The resulting calculated M score, MQ, was utilized to predict IR and the risk of progressing from normal glucose tolerance to impaired glucose tolerance (IGT) over a 3 year period. Results: MQ correlated with actual M values, with an r value of 0.66. In addition, the test detects IR and predicts 3 year IGT progression with areas under the curve of 0.79 and 0.70, respectively, outperforming other simple measures such as fasting insulin, fasting glucose, homeostatic model assessment of IR, or body mass index. Conclusions: The result, Quantose™, is a simple test for IR based on a single fasting blood sample and may have value as an early indicator of risk for the development of prediabetes and T2DM.