PPARγ is the functioning receptor for the thiazolidinedione (TZD) class of anti-diabetes drugs including rosiglitazone and pioglitazone1. These drugs are full classical agonists for this nuclear receptor, but recent data has shown that many PPARγ-based drugs have a separate biochemical activity, blocking the obesity-linked phosphorylation of PPARγ by Cdk52. Here we describe novel synthetic compounds that have a unique mode of binding to PPARγ, completely lack classical transcriptional agonism and block the Cdk5-mediated phosphorylation in cultured adipocytes and in insulin-resistant mice. Moreover, one such compound, SR1664, has potent anti-diabetic activity while not causing the fluid retention and weight gain that are serious side effects of many of the PPARγ drugs. Unlike TZDs, SR1664 also does not interfere with bone formation in culture. These data illustrate that new classes of anti-diabetes drugs can be developed by specifically targeting the Cdk5-mediated phosphorylation of PPARγ.

Abstract:The indole nucleus is an important element of many natural and synthetic molecules with significant biological activity. This review covers some of the relevant and recent achievements in the biological, chemical and pharmacological activity of important indole derivatives in the areas of drug discovery and analysis.

The retinoic acid receptor-related orphan receptors ␣ and ␥ (ROR␣ (NR1F1) and ROR␥ (NR1F3)) are orphan nuclear receptors and perform critical roles in regulation of development, metabolism, and immune function. Cholesterol and cholesterol sulfate have been suggested to be ROR␣ ligands, but the physiological significance is unclear. To date, no endogenous ROR␥ ligands have been described. Here, we demonstrate that 7-oxygenated sterols function as high affinity ligands for both ROR␣ and ROR␥ by directly binding to their ligand-binding domains In the late 1980s, as the canonical domain structure and conserved sequence of members of the nuclear hormone receptor (NHR) 3 superfamily became apparent, several laboratories began to isolate additional members of this superfamily that had no identified ligands. Many of these so-called orphan receptors still have no identified ligands. The first member of the ROR subfamily of receptors (ROR␣) was identified in the early 1990s based on sequence similarities to the retinoic acid receptor and the retinoid X receptor, hence the name "retinoic acid receptor-related orphan receptor" (1, 2). The highly similar receptors, ROR␤ and ROR␥, were identified soon after (3, 4).

Retinoic acid receptor-related orphan receptors (RORs) regulate a variety of physiological processes including hepatic gluconeogenesis, lipid metabolism, circadian rhythm, and immune function. Here we present the first high-affinity synthetic ligand for both ROR␣ and ROR␥. In a screen against all 48 human nuclear receptors, the benzenesulfonamide liver X receptor (LXR) -benzenesulfonamide (T0901317) inhibited transactivation activity of ROR␣ and ROR␥ but not ROR␤. T0901317 was found to directly bind to ROR␣ and ROR␥ with high affinity (K i ϭ 132 and 51 nM, respectively), resulting in the modulation of the receptor's ability to interact with transcriptional cofactor proteins. T0901317 repressed ROR␣/␥-dependent transactivation of ROR-responsive reporter genes and in HepG2 cells reduced recruitment of steroid receptor coactivator-2 by ROR␣ at an endogenous ROR target gene (G6Pase). Using small interference RNA, we demonstrate that repression of the gluconeogenic enzyme glucose-6-phosphatase in HepG2 cells by T0901317 is ROR-dependent and is not due to the compound's LXR activity. In summary, T0901317 represents a novel chemical probe to examine ROR␣/␥ function and an excellent starting point for the development of ROR selective modulators. More importantly, our results demonstrate that small molecules can be used to target the RORs for therapeutic intervention in metabolic and immune disorders.

The nuclear hormone receptor, REV-ERB, plays an essential role in adipogenesis. Rev-erbalpha expression is induced in 3T3-L1 cells during adipogenesis, and overexpression of this receptor leads to expression of adipogenic genes. We recently demonstrated that the porphyrin heme functions as a ligand for REV-ERB, and binding of heme is required for the receptor's activity. We therefore hypothesized that REV-ERB ligands may play a role in regulation of adipogenesis. We detected an increase intracellular heme levels during 3T3-L1 adipogenesis that correlated with induction of aminolevulinic acid synthase 1 (Alas1) expression, the rate-limiting enzyme in heme biosynthesis. If the increase in Alas1 expression was blocked, adipogenesis was severely attenuated, indicating that induction of expression of Alas1 and the increase in heme synthesis is critical for differentiation. Inhibition of heme synthesis during adipogenesis leads to decreased recruitment of nuclear receptor corepressor to the promoter of a REV-ERB target gene, suggesting alteration of REV-ERB activity. Treatment of 3T3-L1 cells with a synthetic REV-ERB ligand, SR6452, resulted in induction of adipocyte differentiation to a similar extent as treatment with the peroxisomal proliferator-activated receptor-gamma agonist, rosiglitazone. Combination of SR6452 and rosiglitazone had an additive effect on stimulation of adipocyte differentiation. These results suggest that heme, functioning as a REV-ERB ligand, is an important signaling molecule for induction of adipogenesis. Moreover, synthetic small molecule ligands for REV-ERB are effective modulators of adipogenesis and may be useful for treatment of metabolic diseases.

The retinoic acid receptor-related orphan receptor α and γ (RORα [NR1F1] and RORγ [NR1F3]) are members of the nuclear hormone receptor superfamily. These 2 receptors regulate many physiological processes including development, metabolism and immunity. We recently found that certain oxysterols, namely the 7-substituted oxysterols, bound to the ligand binding domains (LBDs) of RORα and RORγ with high affinity, altered the LBD conformation and reduced coactivator binding resulting in suppression of the constitutive transcriptional activity of these two receptors. Here, we show that another oxysterol, 24S-hydroxycholesterol (24S-OHC), is also a high affinity ligand for RORα and RORγ (K i ∼ 25 nM). 24S-OHC is also known as cerebrosterol due to its high level in the brain where it plays an essential role as an intermediate in cholesterol elimination from the CNS. 24S-OHC functions as a RORα/γ inverse agonist suppressing the constitutive transcriptional activity of these receptors in cotransfection assays. Additionally, 24S-OHC suppressed the expression of several RORα target genes including BMAL1 and REV-ERBα in a RORdependent manner. We also demonstrate that 24S-OHC decreases the ability of RORα to recruit the coactivator SRC-2 when bound to the BMAL1 promoter. We also noted that 24(S), 25-epoxycholesterol selectively suppressed the activity of RORγ. These data indicate that RORα and RORγ may serve as sensors of oxsterols. Thus, RORα and RORγ display an overlapping ligand preference with another class of oxysterol nuclear receptors, the liver X receptors (LXRα [NR1H3] and LXRβ [NR1H2]).

The retinoic acid receptor-related receptors (RORs) are members of the nuclear receptor (NR) superfamily of transcription factors. Several NRs are still characterized as orphan receptors since ligands have not yet been identified for these proteins. Here, we describe the identification of a synthetic RORα/RORγ ligand, SR1078. SR1078 modulates the conformation of RORγ in a biochemical assay and activates RORα and RORγ driven transcription. Furthermore, SR1078 stimulates expression of endogenous ROR target genes in HepG2 cells that express both RORα and RORγ. Pharmacokinetic studies indicate that SR1078 displays reasonable exposure following injection into mice and consistent with SR1078 functioning as a RORα/RORγ agonist, expression of two ROR target genes, glucose-6-phosphatase and fibroblast growth factor 21, were stimulated in the liver. Thus, we have identified the first synthetic RORα/γ agonist and this compound can be utilized as a chemical tool to probe the function of these receptors both in vitro and in vivo.

OBJECTIVEWe examined the effect of β-adrenergic receptor (βAR) activation and cAMP-elevating agents on respiration and mitochondrial uncoupling in human adipocytes and probed the underlying molecular mechanisms.RESEARCH DESIGN AND METHODSOxygen consumption rate (OCR, aerobic respiration) and extracellular acidification rate (ECAR, anaerobic respiration) were examined in response to isoproterenol (ISO), forskolin (FSK), and dibutyryl-cAMP (DB), coupled with measurements of mitochondrial depolarization, lipolysis, kinase activities, and gene targeting or knock-down approaches.RESULTSISO, FSK, or DB rapidly increased oxidative and glycolytic respiration together with mitochondrial depolarization in human and mouse white adipocytes. The increase in OCR was oligomycin-insensitive and contingent on cAMP-dependent protein kinase A (PKA)-induced lipolysis. This increased respiration and the uncoupling were blocked by inhibiting the mitochondrial permeability transition pore (PTP) and its regulator, BAX. Interestingly, compared with lean individuals, adipocytes from obese subjects exhibited reduced OCR and uncoupling capacity in response to ISO.CONCLUSIONSLipolysis stimulated by βAR activation or other maneuvers that increase cAMP levels in white adipocytes acutely induces mitochondrial uncoupling and cellular energetics, which are amplified in the absence of scavenging BSA. The increase in OCR is dependent on PKA-induced lipolysis and is mediated by the PTP and BAX. Because this effect is reduced with obesity, further exploration of this uncoupling mechanism will be needed to determine its cause and consequences.