Steroidogenic factor-1 (SF-1) and liver receptor homologue-1 (LRH-1) belong to the fushi tarazu factor 1 subfamily of nuclear receptors. SF-1 is an essential factor for sex determination during development and regulates adrenal and gonadal steroidogenesis in the adult, whereas LRH-1 is a critical factor for development of endodermal tissues and regulates cholesterol and bile acid homeostasis. Regulatory ligands are unknown for SF-1 and LRH-1. A reported mouse LRH-1 structure revealed an empty pocket in a region commonly occupied by ligands in the structures of other nuclear receptors, and pocket-filling mutations did not alter the constitutive activity observed. Here we report the crystal structures of the putative ligand-binding domains of human SF-1 at 2.1-Å resolution and human LRH-1 at 2.5-Å resolution. Both structures bind a coactivator-derived peptide at the canonical activationfunction surface, thus adopting the transcriptionally activating conformation. In human LRH-1, coactivator peptide binding also occurs to a second site. We discovered in both structures a phospholipid molecule bound in a pocket of the putative ligand-binding domain. MS analysis of the protein samples used for crystallization indicated that the two proteins associate with a range of phospholipids. Mutations of the pocket-lining residues reduced the transcriptional activities of SF-1 and LRH-1 in mammalian cell transfection assays without affecting their expression levels. These results suggest that human SF-1 and LRH-1 may be ligand-binding receptors, although it remains to be seen if phospholipids or possibly other molecules regulate SF-1 or LRH-1 under physiological conditions.x-ray crystallography ͉ phospholipid ͉ nuclear receptor ͉ steroid ͉ bile S teroidogenic factor-1 (SF-1; AD4BP͞NR5A1) and liver receptor homologue-1 (LRH-1; CPF͞FTF͞NR5A2), expressed in man, are homologues of the fushi tarazu factor-1 of Drosophila (1) and FF1B of fish (2). Together, these factors constitute the NR5A subfamily of nuclear receptors (NRs) (3). SF-1 and LRH-1 function as monomers (4) to regulate genes by binding to similar response elements.SF-1 is expressed in the adrenal, testes, ovary, pituitary, hypothalamus, spleen, and skin and regulates genes that direct biosynthesis of adrenal and gonadal steroids as well as Mullerian hormone and gonadotropins (5, 6). SF-1 is essential for normal adrenal and gonadal development given that SF-1 knockout in mice causes adrenal and gonadal agenesis and impaired gonadotropin expression, resulting in postnatal death due to severe adrenal insufficiency (7,8). SF-1 knockout also causes abnormalities of the ventromedial hypothalamic nucleus, the control center for satiety and feeding, which suggests that SF-1 may have broader roles in the control of metabolism and obesity (9). In humans, partial loss-of-function mutations in SF-1 result in XY sex reversal and adrenal failure (10, 11). Although SF-1 is expressed in the ovary (12), a mutation of SF-1 was observed not to affect ovarian development; thus, SF-1 may no...
Objective-Epoxyeicosatrienoic acids (EETs) have been shown to have antiinflammatory effects and therefore may play a role in preventing vascular inflammatory and atherosclerotic diseases. Soluble epoxide hydrolase (s-EH) converts EETs into less bioactive dihydroxyeicosatrienoic acids. Thus, inhibition of s-EH can prevent degradation of EETs and prolong their effects. The present study aimed to test the hypothesis that inhibition of s-EH has vascular protective effects. Methods and Results-Six-month-old apolipoprotein E-deficient mice were chronically infused with angiotensin II (1.44 mg/kg/d) for 4 weeks to induce abdominal aortic aneurysm (AAA), accelerate atherosclerosis development and carotid artery ligation-induced vascular remodeling. The mice were treated with a novel s-EH inhibitor, AR9276 (1.5 g/L in drinking water) or vehicle for 4 weeks. The results demonstrated that AR9276 significantly reduced the rate of AAA formation and atherosclerotic lesion area, but had no effect on ligation-induced carotid artery remodeling. These effects were associated with a reduction of serum lipid, IL-6, murine IL-8-KC, and IL-1␣, and downregulation of gene expressions of ICAM-1, VCAM-1, and IL-6 in the arterial wall. Key Words: epoxyeicosatrienoic acids Ⅲ soluble epoxide hydrolase Ⅲ dyslipidemia Ⅲ atherosclerosis Ⅲ abdominal aorta aneurysm Ⅲ inflammatory markers A rachidonic acid can be metabolized by 3 major oxidative pathways: cyclooxygenase (COX) forming prostaglandins; lipoxygenase (LOX) forming hydroxyeicosatetraenoic acids (HETEs) and leukotrienes; and cytochrome P-450 monooxygenase forming epoxides and HETEs. 1 The COX and LOX pathways have been extensively studied, and their eicosanoid products have been shown to play important roles in a variety of biological processes such as inflammation, cell proliferation, and intracellular signaling. In contrast, less is known about the "third pathway" of arachidonic acid metabolism. Recently, epoxyeicosatrienoic acids (EETs), the cytochrome P450 metabolites of arachidonic acid, have received increasing attention for multiple beneficial biological functions, including vasodilation, antiinflammation, and inhibition of proliferation and migration of vascular smooth muscle cells. 1,2 Based on these properties, it has been postulated that EETs may exert therapeutic benefits in inflammatory vascular diseases, such as atherosclerosis. 2,3 Soluble epoxide hydrolase (s-EH) converts EETs into their corresponding dihydroxyeicosatrienoic acids (DHETs), which are generally thought to have reduced biological activity relative to EETs, and hydration of the EETs by s-EH is a dominant mechanism whereby their activity can be reduced. 4 Thus, inhibition of s-EH could be a promising therapeutic target for amplifying the beneficial effects of EETs. 4 Indeed, s-EH inhibitors have been demonstrated to lower blood pressure in hypertension, 5,6 decrease hypertension-induced renal damage 7 and cerebral ischemia injury, 8 attenuate vascular smooth muscle cell proliferation, 9 and reduce tissue...
We have developed a novel, high-throughput scintillation proximity assay to measure the membraneassociated steps (stages 2 and 3) of peptidoglycan synthesis in Escherichia coli. At least five enzymes are involved in these two stages, all of which are thought to be essential for the survival of the cell. The individual enzymes are difficult to assay since the substrates are lipidic and difficult to isolate in large quantities and analysis is done by paper chromatography. We have assayed all five enzymes in a single mixture by monitoring synthesis of cross-linked peptidoglycan, which is the final product of the pathway. E. coli membranes are incubated with the two sugar precursors, UDP-N-acetyl muramylpentapeptide and UDP-[ 3 H]-N-acetylglucosamine. The radiolabel is incorporated into peptidoglycan, which is captured using wheat germ agglutinincoated scintillation proximity assay beads. The assay monitors the activity of the translocase (MraY), the transferase (MurG), the lipid pyrophosphorylase, and the transglycosylase and transpeptidase activities of the penicillin-binding proteins. Vancomyin, tunicamycin, nisin, moenomycin, bacitracin, and penicillin inhibit the assay, and these inhibitors have been used to validate the assay. The search for new antimicrobial agents that act via the late stages of peptidoglycan biosynthesis can now be performed in high throughput in a microtiter plate.
In a search for more effective anti-diabetic treatment, we used a process coupling low-affinity biochemical screening with highthroughput co-crystallography in the design of a series of compounds that selectively modulate the activities of all three peroxisome proliferator-activated receptors (PPARs), PPAR␣, PPAR␥, and PPAR␦. Transcriptional transactivation assays were used to select compounds from this chemical series with a bias toward partial agonism toward PPAR␥, to circumvent the clinically observed side effects of full PPAR␥ agonists. Co-crystallographic characterization of the lead molecule, indeglitazar, in complex with each of the 3 PPARs revealed the structural basis for its PPAR pan-activity and its partial agonistic response toward PPAR␥. Compared with full PPAR␥-agonists, indeglitazar is less potent in promoting adipocyte differentiation and only partially effective in stimulating adiponectin gene expression. Evaluation of the compound in vivo confirmed the reduced adiponectin response in animal models of obesity and diabetes while revealing strong beneficial effects on glucose, triglycerides, cholesterol, body weight, and other metabolic parameters. Indeglitazar has now progressed to Phase II clinical evaluations for Type 2 diabetes mellitus (T2DM).adiponectin ͉ diabetes ͉ partial agonist ͉ PPAR pan-agonist ͉ Scaffold-based drug discovery T herapeutic approaches to Type 2 diabetes mellitus (T2DM), which currently affects Ϸ6% of adults in the United States (US Department of Health and Human Services, Centers for Disease Control and Prevention, Atlanta, GA; 2005), are generally polypharmaceutical in nature, targeting effects on insulin sensitivity and elements of the coincident dyslipidemia and cardiovascular diseases (1). However, polypharmacy in these treatment regimens has been cited as a potential additional risk factor (2), with many patients on 4 or more concomitant medications. A more effective strategy would be to use a single agent that possesses combined benefits from simultaneous inhibition or stimulation of several related targets, without the risks associated with combination therapy. However, optimizing activities against several targets is a complex design problem that necessitates judicious choice of targets and requires new ways in which therapeutic agents are generated.Two classes of marketed therapeutics, the fibrates (as lipidlowering agents) and the glitazones (as insulin-sensitizing drugs) target related receptors known as PPAR␣ and PPAR␥, respectively, whereas a third member of the subfamily, PPAR␦, has been the target of intense preclinical interest as an avenue for treatment of dyslipidemia (3). A pan-agonist, capable of stimulating the 3 peroxisome proliferator-activated receptors (PPARs) as a group, would be expected to be particularly useful in the treatment of T2DM from the standpoints of both efficacy and reduction in the additional risk factors associated with polypharmacy. Despite the close structural relationship between these 3 receptors, the search for compounds whi...
1-(1-Acetyl-piperidin-4-yl)-3-adamantan-1-yl-urea 14a (AR9281), a potent and selective soluble epoxide hydrolase inhibitor, was recently tested in a phase 2a clinical setting for its effectiveness in reducing blood pressure and improving insulin-resistance in pre-diabetic patients. In a mouse model of diet induced obesity, AR9281 attenuated the enhanced glucose excursion following an intraperitoneal glucose tolerance test. AR9281 also attenuated the increase in blood pressure in angiotensin-II-induced hypertension in rats. These effects were dose-dependent and well correlated with inhibition of the sEH activity in whole blood, consistent with a role of sEH in the observed pharmacology in rodents.
Penicillin binding protein (PBP) 1b of Escherichia coli has both transglycosylase and transpeptidase activities, which are attractive targets for the discovery of new antibacterial agents. A high-throughput assay that detects inhibitors of the PBPs was described previously, but it cannot distinguish them from inhibitors of the MraY, MurG, and lipid pyrophosphorylase. We report on a method that distinguishes inhibitors of both activities of the PBPs from those of the other three enzymes. Radioactive peptidoglycan was synthesized by using E. coli membranes. Following termination of the reaction the products were analyzed in three ways. Wheat germ agglutinin (WGA)-coated scintillation proximity assay (SPA) beads were added to one set, and the same beads together with a detergent were added to a second set. Type A polyethylenimine-coated WGA-coated SPA beads were added to a third set. By comparison of the results of assays run in parallel under the first two conditions, inhibitors of the transpeptidase and transglycosylase could be distinguished from inhibitors of the other enzymes, as the inhibitors of the other enzymes showed similar inhibitory concentrations (IC 50 s) under both conditions but the inhibitors of the PBPs showed insignificant inhibition in the absence of detergent. Furthermore, comparison of the results of assays run under conditions two and three enabled the distinction of transpeptidase inhibitors. Penicillin and other -lactams showed insignificant inhibition with type A beads compared with that shown with WGA-coated SPA beads plus detergent. However, inhibitors of the other four enzymes (tunicamycin, nisin, bacitracin, and moenomycin) showed similar IC 50 s under both conditions. We show that the main PBP being measured under these conditions is PBP 1b. This screen can be used to find novel transglycosylase or transpeptidase inhibitors.
The host structural maintenance of chromosomes 5/6 complex (Smc5/6) suppresses hepatitis B virus (HBV) transcription. HBV counters this restriction by expressing the X protein (HBx), which redirects the cellular DNA damage-binding protein 1 (DDB1)-containing E3 ubiquitin ligase to target Smc5/6 for degradation. However, the details of how HBx modulates the interaction between DDB1 and Smc5/6 remain to be determined. In this study, we performed biophysical analyses of recombinant HBx and functional analysis of HBx mutants in HBV-infected primary human hepatocytes (PHH) to identify key regions and residues that are required for HBx function. We determined that recombinant HBx is soluble and exhibits stoichiometric zinc binding when expressed in the presence of DDB1. Mass spectrometry-based hydrogen-deuterium exchange and cysteine-specific chemical footprinting of the HBx:DDB1 complex identified several HBx cysteine residues (located between amino acids 61 and 137) that are likely involved in zinc binding. These cysteine residues did not form disulfide bonds in HBx expressed in human cells. In line with the biophysical data, functional analysis demonstrated that HBx amino acids 45 to 140 are required for Smc6 degradation and HBV transcription in PHH. Furthermore, site-directed mutagenesis determined that C61, C69, C137, and H139 are necessary for HBx function, although they are likely not essential for DDB1 binding. This CCCH motif is highly conserved in HBV as well as in the X proteins from various mammalian hepadnaviruses. Collectively, our data indicate that the essential HBx cysteine and histidine residues form a zinc-binding motif that is required for HBx function. IMPORTANCE The structural maintenance of chromosomes 5/6 complex (Smc5/6) is a host restriction factor that suppresses HBV transcription. HBV counters this restriction by expressing HBV X protein (HBx), which redirects a host ubiquitin ligase to target Smc5/6 for degradation. Despite this recent advance in understanding HBx function, the key regions and residues of HBx required for Smc5/6 degradation have not been determined. In the present study, we performed biochemical, biophysical, and cell-based analyses of HBx. By doing so, we mapped the minimal functional region of HBx and identified a highly conserved CCCH motif in HBx that is likely responsible for coordinating zinc and is essential for HBx function. We also developed a method to produce soluble recombinant HBx protein that likely adopts a physiologically relevant conformation. Collectively, this study provides new insights into the HBx structure-function relationship and suggests a new approach for structural studies of this enigmatic viral regulatory protein.
Monoclonal antibodies (mAbs) are an important class of biotherapeutics. Successful development of a mAb depends not only on its biological activity but also on its physicochemical properties, such as homogeneity and stability. mAb stability is affected by its formulation. Among the many techniques used to study the stability of mAbs, differential scanning fluorimetry (DSF) offers both excellent throughput and minimal material consumption. DSF measures the temperature of the protein unfolding transition (Tm) based on the change in fluorescence intensity of the environmentally sensitive dye SYPRO Orange. With DSF adapted to a 96-well plate format, we have shown that low-pH or high-salt concentrations decrease the thermal stability of mAb1, whereas some excipients, such as sucrose, polysorbate 80, and sodium phosphate, increase its stability. The basal fluorescence of SYPRO Orange was enhanced by the presence of detergents, limiting the use of this approach to diluted detergent solutions. Throughput of DSF can be increased further with the use of a 384-well plate. DSF thermograms are in good agreement with the melting profiles obtained by differential scanning calorimetry (DSC). The Tms determined by DSF and DSC were well correlated, with the former being on average lower by 3 °C.
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