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The WD-repeat protein FAN was originally identified as mediator of signal transduction events originating from the 55-kDa tumor necrosis factor-receptor (TNF-R55). This function is conveyed by direct interaction of the FAN C-terminal region with the receptor protein. By binding to the neutral sphingomy-elinase domain (NSD) of TNF-R55, FAN couples TNF-R55 to the TNF-mediated transient activation of neutral sphingomyelinase (N-SMase). Ultimately, FAN is thought to initiate signal transduction events that induce inflammatory and proliferative responses to TNF stimulation. FAN is identified as a member of the WD-repeat protein family, most of which have regulatory or signal transducing functions. Moreover, like the gene for the lysosomal trafficking regulator LYST, which is responsible for Chediak-Higashi-Syndrome, FAN contains a BEACH domain.

Inflammatory bowel disease (IBD) is associated with a loss of intestinal barrier function and dysregulated immune responses. It has been shown that short chain fatty acids (SCFAs) are protective in IBD and that GPR43 mediates the protective effects of SCFAs. In this study, we investigated the effects of SCFAs in comparison to highly specific GPR43 agonists on human intestinal epithelial and immune cells. Our results confirm that SCFAs are enhancers of barrier function in intestinal epithelial cells. Additionally, SCFAs also displayed potent immunoregulatory properties based upon the ability to inhibit LPS-induced cytokine production in PBMC, and human T cell proliferation and cytokine production. Unexpectedly, and in contrast to the current belief, specific GPR43 agonists failed to exhibit similar barrier enhancing and anti-inflammatory properties. These findings demonstrate that SCFA possess broad protective functions in IBD and agonizing GPR43 alone is unlikely to be beneficial in patients.

Off-target effects of drugs on nuclear hormone receptors (NHRs) may result in adverse effects in multiple organs/physiological processes. Reliable assessments of the NHR activities for drug candidates are therefore crucial for drug development. However, the highly permissive structures of NHRs for vastly different ligands make it challenging to predict interactions by examining the chemical structures of the ligands. Here, we report a detailed investigation on the agonistic and antagonistic activities of 615 known drugs or drug candidates against a panel of 6 NHRs: androgen, progesterone, estrogen α/β, and thyroid hormone α/β receptors. Our study revealed that 4.7 and 12.4% compounds have agonistic and antagonistic activities, respectively, against this panel of NHRs. Nonetheless, potent, unintended NHR hits are relatively rare among the known drugs, indicating that such interactions are perhaps not tolerated during drug development. However, we uncovered examples of compounds that unintentionally agonize or antagonize NHRs. In addition, a number of compounds showed multi-NHR activities, suggesting that the cross-talk between multiple NHRs co-operate to elicit in vivo effects. These data highlight the merits of counter screening drug candidate against NHRs during drug discovery/development., (© The Author 2015. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

FFA1 (GPR40) and GPR120 are G-protein-coupled receptors activated by long-chain fatty acids. FFA1 is expressed in pancreatic β-cells, where it regulates glucose-dependent insulin secretion, and GPR120 has been implicated in mediating GLP-1 secretion. We show here that FFA1 co-localizes with GLP-1 in enteroendocrine cells and plays a critical role in glucose management by mediating GLP-1 secretion in vivo. Corn oil induces GLP-1 secretion in wild type mice and in GPR120-/- mice, but not in FFA1-/- mice. α-Linolenic acid, an endogenous ligand of FFA1, induces GLP-1 secretion in GLUTag cells and in primary fetal mouse intestinal cells. Synthetic partial FFA1 agonists do not stimulate GLP-1 secretion in mice, but partial and full agonists combined function cooperatively to enhance receptor activation and GLP-1 secretion both in vitro and in vivo. We conclude that allosterism at FFA1 can contribute to postprandial glucose management by stimulating insulin secretion via an extrapancreatic mechanism of action, and that GPR120 in GLP-1 secretion requires further investigation., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)

Lysine demethylase 1 (LSD1) and Jumonji C domain-containing oxygenase D2C (JMJD2C) participate in regulating the methylation status of histone H3 lysine residues. In some contexts, LSD1 and JMJD2C activity causes enhanced cellular proliferation, which may lead to tumorigenesis. The authors explored the utility of time-resolved fluorescence resonance energy transfer (TR-FRET) immunoassays, which employed peptides consisting of the first 21 amino acids of histone H3 in which lysine 4 (H3K4) or lysine 9 (H3K9) was methylated (me) to quantify LSD1 and JMJD2C activity. The LSD1 assay monitored demethylation of the H3K4me1 peptide using an antibody that recognizes H3K4me1 but not the unmethylated peptide product. The JMJD2C assay measured demethylation of H3K9me3 with an antibody that selectively recognizes H3K9me2. The optimized conditions resulted in robust assays (Z' > 0.7) that required only 3 to 6 nM of enzyme in a reaction volume of 6 to 10 µL. These assays were used to compare the activity of different LSD1 constructs and to determine the apparent K(m) of each JMJD2C substrate. Finally, both assays were used in a high-throughput setting for identifying demethylase inhibitors. Compounds discovered by these TR-FRET methods may lead to powerful tools for ascertaining the roles of demethylases in a cellular context and ultimately for potential cancer treatments.

Despite the prevalence of KRAS mutations in human cancers, there remain no targeted therapies for treatment. The serine-threonine kinase STK33 has been proposed to be required for the survival of mutant KRAS-dependent cell lines, suggesting that small molecule kinase inhibitors of STK33 may be useful to treat KRAS-dependent tumors. In this study, we investigated the role of STK33 in mutant KRAS human cancer cells using RNA interference, dominant mutant overexpression, and small molecule inhibitors. As expected, KRAS downregulation decreased the survival of KRAS-dependent cells. In contrast, STK33 downregulation or dominant mutant overexpression had no effect on KRAS signaling or survival of these cells. Similarly, a synthetic lethal siRNA screen conducted in a broad panel of KRAS wild-type or mutant cells identified KRAS but not STK33 as essential for survival. We also obtained similar negative results using small molecule inhibitors of the STK33 kinase identified by high-throughput screening. Taken together, our findings refute earlier proposals that STK33 inhibition may be a useful therapeutic approach to target human KRAS mutant tumors., (©2011 AACR.)

FFA2 (GPR43) is a receptor for short-chain fatty acids (SCFAs), acetate, and propionate. FFA2 is predominantly expressed in islets, a subset of immune cells, adipocytes, and the gastrointestinal tract which suggest a possible role in inflammatory and metabolic conditions. We have previously described the identification and characterization of novel phenylacetamides as allosteric agonists of FFA2. In the current study, we have investigated the molecular determinants contributing to receptor activation with the endogenous and synthetic ligands as well as allosteric interactions between these two sites. The mutational analysis revealed previously unidentified sites that may allosterically regulate orthosteric ligand's function as well as residues potentially important for the interactions between orthosteric and allosteric binding sites., (Copyright © 2011 Elsevier Inc. All rights reserved.)

FFAR2 (GPR43) is a receptor for short-chain fatty acids (SCFAs), acetate and propionate. In the current study, we investigate the molecular determinants contributing to receptor activation by endogenous ligands. Mutational analysis revealed several important residues located in transmembrane domains (TM) 3, 4, 5, 6, and 7 for acetate binding. Interestingly, mutations that abolished acetate activity, including the mutation in the well-conserved D(E)RY motif, could be rescued by a recently identified synthetic allosteric agonist. These findings provide additional insight into agonist binding and activation which may aid in designing allosteric ligands for targeting receptor function in various diseases., (Copyright © 2010 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

Free fatty acid receptor 2 (FFA2) is a G-protein coupled receptor for which only short-chain fatty acids (SCFAs) have been reported as endogenous ligands. We describe the discovery and optimization of phenylacetamides as allosteric agonists of FFA2. These novel ligands can suppress adipocyte lipolysis in vitro and reduce plasma FFA levels in vivo, suggesting that these allosteric modulators can serve as pharmacological tools for exploring the potential function of FFA2 in various disease conditions., (Copyright 2009 Elsevier Ltd. All rights reserved.)

FFA2 (GPR43) has been identified as a receptor for short-chain fatty acids (SCFAs) that include acetate and propionate. FFA2 is highly expressed in islets, a subset of immune cells, and adipocytes. Although the potential roles of FFA2 activation in these tissues have previously been described, the physiological functions are still unclear. The potency for SCFAs on FFA2 is low, in the high micromolar to millimolar concentrations. To identify better pharmacological tools to study receptor function, we used high-throughput screening (HTS) to discover a series of small molecule phenylacetamides as novel and more potent FFA2 agonists. This series is specific for FFA2 over FFA1 (GPR40) and FFA3 (GPR41), and it is able to activate both the Galpha(q) and Galpha(i) pathways in vitro on Chinese hamster ovary cells stably expressing FFA2. Treatment of adipocytes with these compounds also resulted in Galpha(i)-dependent inhibition of lipolysis similar to that of endogenous ligands (SCFAs). It is noteworthy that these compounds not only acted as FFA2 agonists but also exhibited positive cooperativity with acetate or propionate. The observed allosteric modulation was consistent in all the functional assays that we have explored, including cAMP, calcium mobilization, guanosine 5'-[gamma-thio]triphosphate binding, and lipolysis. Molecular modeling analysis of FFA2 based on human beta(2)-adrenergic receptor structure revealed potential nonoverlapping binding sites for the endogenous and synthetic ligands, further providing insight into the binding pocket for the allosteric interactions. This is the first report describing the identification of novel allosteric modulators with agonist activity for FFA2, and these compounds may serve as tools for further unraveling the physiological functions of the receptor and its involvement in various diseases.

GPCRs had significant representation in the drug discovery portfolios of most major commercial drug discovery organizations for many years. This is due in part to the diverse biological roles mediated by GPCRs as a class, as well as the empirical discovery that they have proven relatively tractable to the development of small molecule therapeutics. Publication of the human genome sequence in 2001 confirmed GPCRs as the largest single gene superfamily with more than 700 members, furthering the already strong appeal of addressing this target class using efficient and highly parallelized platform approaches. The GPCR research platform implemented at Amgen is used as a case study to review the evolution and implementation of available assays and technologies applicable to GPCR drug discovery. The strengths, weaknesses, and applications of assay technologies applicable to G alpha s, G alpha i and G alpha q-coupled receptors are described and their relative merits evaluated. Particular consideration is made of the role and practice of "de-orphaning" and signaling pathway characterization as a pre-requisite to establishing effective screens. In silico and in vitro methodology developed for rapid, parallel high throughput hit characterization and prioritization is also discussed extensively.

The LPA(2) protein is overexpressed in many tumor cells. We report the optimization of a series of LPA(2) antagonists using calcium mobilization assay (aequorin assay) that led to the discovery of the first reported inhibitors selective for LPA(2). Key compounds were evaluated in vitro for inhibition of LPA(2) mediated Erk activation and proliferation of HCT-116 cells. These compounds could be used to evaluate the benefits of LPA(2) inhibition both in vitro and in vivo.

Hyperactivation of immune cells by bacterial products through toll-like receptors (TLRs) is thought of as a causative mechanism of septic shock pathology. Infections with Gram-negative or Gram-positive bacteria provide TLR2-specific agonists and are the major cause of severe sepsis. In order to intervene in TLR2-driven toxemia, we raised mAb's against the extracellular domain of TLR2. Surface plasmon resonance analysis showed direct and specific interaction of TLR2 and immunostimulatory lipopeptide, which was blocked by T2.5 in a dose-dependent manner. Application of mAb T2.5 inhibited cell activation in experimental murine models of infection. T2.5 also antagonized TLR2-specific activation of primary human macrophages. TLR2 surface expression by murine macrophages was surprisingly weak, while both intra- and extracellular expression increased upon systemic microbial challenge. Systemic application of T2.5 upon lipopeptide challenge inhibited release of inflammatory mediators such as TNF-alpha and prevented lethal shock-like syndrome in mice. Twenty milligrams per kilogram of T2.5 was sufficient to protect mice, and administration of 40 mg/kg of T2.5 was protective even 3 hours after the start of otherwise lethal challenge with Bacillus subtilis. These results indicate that epitope-specific binding of exogenous ligands precedes specific TLR signaling and suggest therapeutic application of a neutralizing anti-TLR2 antibody in acute infection.

Signaling through its widely distributed cell surface receptor, interleukin (IL)-17 enhances the transcription of genes encoding proinflammatory molecules. Although it has been well documented that IL-17 activates the transcription factor nuclear factor (NF)-kappaB and c-Jun NH(2)-terminal kinase (JNK), the upstream signaling events are largely unknown. Here we report the requirement of tumor necrosis factor receptor-associated factor (TRAF)6 in IL-17-induced NF-kappaB and JNK activation. In embryonic fibroblasts (EFs) derived from TRAF6 knockout mice, IL-17 failed to activate the IkappaB kinases (IKKs) and JNK. Consequently, IL-17-induced IL-6 and intercellular adhesion molecule 1 expression in the TRAF6-deficient cells was abolished. Lack of TRAF6 appeared to be the sole defect responsible for the observed failure to respond to IL-17, because transient transfection of TRAF6 expression plasmid into the TRAF6-deficient cells restored IL-17-induced NF-kappaB activation in a luciferase reporter assay. Furthermore, the levels of IL-17 receptor (IL-17R) on the TRAF6-deficient EFs were comparable to those on the wild-type control cells. Defect in IL-17 response was not observed in TRAF2-deficient EFs. Moreover, when TRAF6 and IL-17R were coexpressed in 293 cells, TRAF6 coimmunoprecipitated with IL-17R. Together, these results indicate that TRAF6, but not TRAF2, is a crucial component in the IL-17 signaling pathway leading to proinflammatory responses.

Ceramide has been recognized as a common intracellular second messenger for various cytokines, growth factors and other stimuli, such as CD95, chemotherapeutic drugs and stress factors. To understand the role of ceramide during apoptosis and other cellular responses, it is critically important to characterize direct targets of ceramide action. In this paper, we show that ceramide specifically binds to and activates the endosomal acidic aspartate protease cathepsin D. Direct interaction of ceramide with cathepsin D results in autocatalytic proteolysis of the 52 kDa pre-pro cathepsin D to form the enzymatically active 48/32 kDa isoforms of cathepsin D. Acid sphingomyelinase (A-SMase)-deficient cells show decreased cathepsin D activity, which could be reconstituted by transfection with A-SMase cDNA. The results of our study identify cathepsin D as the first endosomal ceramide target that colocalizes with and may mediate downstream signaling effects of A-SMase.

The agent of Lyme disease, Borrelia burgdorferi, produces membrane lipoproteins possessing potent inflammatory properties linked to disease pathology. The recent association of toll-like receptors (TLR) 2 and 4 with LPS responses prompted the examination of TLR involvement in lipoprotein signaling. The ability of human cell lines to respond to lipoproteins was correlated with the expression of TLR2. Transfection of TLR2 into cell lines conferred responsiveness to lipoproteins, lipopeptides, and sonicated B. burgdorferi, as measured by nuclear translocation of NF-kappaB and cytokine production. The physiological importance of this interaction was demonstrated by the 10-fold greater sensitivity of TLR2-transfected cells to lipoproteins than LPS. Futhermore, TLR2-dependent signaling by lipoproteins was facilitated by CD14. These data indicate that TLR2 facilitates the inflammatory events associated with Lyme arthritis. In addition, the widespread expression of lipoproteins by other bacterial species suggests that this interaction may have broad implications in microbial inflammation and pathogenesis.

Activation of cytosolic phospholipase A2 (cPLA2) is an essential step in the initiation of the cascade of enzymatic reactions leading to the generation of proinflammatory lipid mediators. Hence, the regulation of cPLA2 is a key event in the induction of inflammatory responses. cPLA2 is activated, in part, by apoptotic stimuli such as TNF or Fas ligand. Apoptosis, however, does not provoke an inflammatory response. Here, we demonstrate that cPLA2 is cleaved by caspase-3 and/or a related caspase in HeLa cells undergoing apoptosis. Mutation of a predicted caspase-3 cleavage site abolishes cPLA2 processing both in vitro and in intact cells. The 70-kDa cleavage product of cPLA2 itself has no catalytic function, while inhibition of cleavage results in an increased enzymatic activity. Additionally, overexpression of the 70-kDa fragment appears to produce a dominant negative effect on endogenous cPLA2 activity. In HeLa cells, cPLA2 activity was dispensable for the course of apoptosis. We cannot rule out, however, that cPLA2 activity is involved in the induction of apoptosis in other cell types. Taken together, our results suggest that the enzymatic activity of cPLA2 is specifically inhibited by caspase-mediated cleavage during apoptosis. The inactivation of cPLA2 represents a previously unrecognized mechanism for avoiding an inflammatory reaction against apoptotic cells.

Ceramide, generated by the enzymatic function of sphingomyelinases (SMases) has emerged as an important signaling pathway transducing diverse biological effects of various cytokine receptors. The 55-kDa receptor for tumor necrosis factor (TNF-R55) activates two types of SMases through distinct cytoplasmic domains. The death domain that is responsible for the initiation of the apoptotic pathway also signals for the activation of an acid SMase (A-SMase). The adapter protein TRADD binds to TNF-R55 in a ligand-dependent manner and serves as anchor for the subsequent recruitment of other proteins into the signaling complex that directly lead to cell death or nuclear factor-kappaB (NF-kappaB) induction. Notably, the two pro-apoptotic adapter proteins TRADD and FADD are also involved in the activation of A-SMase. In contrast, the NF-kappaB-inducing adapters TRAF2 and RIP do not signal for A-SMase. Thus, activation of A-SMase appears to belong to signals leading to TNF-induced cell death. A second signaling domain (NSD) is located upstream of the death domain and directly links the TNF-R55 to the activation of a neutral SMase (N-SMase). A novel adapter protein, FAN, has been identified that specifically binds to the NSD. FAN contains five WD repeats at its carboxy terminus, while it shows significant sequence homology with the mouse beige protein and its human homolog, the CHS protein, in the center portion of the protein. Overexpression of full-length FAN enhanced N-SMase activity in TNF-treated cells, whereas truncated mutants of FAN produced dominant negative effects. FAN, however, did not interfere with any of the TNF responses signaled for by the death domain. Taken together, our data suggest that distinct cytoplasmic domains of TNF-R55 initiate independent signaling pathways by binding different adapter proteins.

It is reported of 81 patients (average age: 86.1 years) with fractures of the proximal femur who have been treated with Ender-nails. The results of the preoperative state justify the immediate operation. Local complications were found in 29.6% out of the cases, mainly outcracks of the corticalis in 14.8%. The hospital letality was 19.8%. In 35 patients a follow-up examination was done after an average period of 20.2 months. Before operation about 75% of patients had a very good or good degree of mobility, in follow-up examination the rate was about 50%.

The frequency of chest deformities is 1:100; operative treatment, however, is necessary in only 1:1000 patients with such a deformity. The most frequent deformity--in 91%--is the funnel chest. Chicken breast, combined types, and fissures of the sternum are seldom seen. Indications for operative treatment are: deformity of more than 25% of the normal sagittal diameter of the chest; heart failure and the degree of pathologic signs on the EKG; alterations of the vertebral column; psychologic aspects. Our operative procedure consists of double cartilage incision and stabilization of the chest with a metal strut.