Simultaneous analysis of large-scale RNAi screens for pathogen entry.

BACKGROUND: Large-scale RNAi screening has become an important technology for identifying genes involved in biological processes of interest. However, the quality of large-scale RNAi screening is often deteriorated by off-targets effects. In order to find statistically significant effector genes for pathogen entry, we systematically analyzed entry pathways in human host cells for eight pathogens using image-based kinome-wide siRNA screens with siRNAs from three vendors. We propose a Parallel Mixed Model (PMM) approach that simultaneously analyzes several non-identical screens performed with the same RNAi libraries. RESULTS: We show that PMM gains statistical power for hit detection due to parallel screening. PMM allows incorporating siRNA weights that can be assigned according to available information on RNAi quality. Moreover, PMM is able to estimate a sharedness score that can be used to focus follow-up efforts on generic or specific gene regulators. By fitting a PMM model to our data, we found several novel hit genes for most of the pathogens studied. CONCLUSIONS: Our results show parallel RNAi screening can improve the results of individual screens. This is currently particularly interesting when large-scale parallel datasets are becoming more and more publicly available. Our comprehensive siRNA dataset provides a public, freely available resource for further statistical and biological analyses in the high-content, high-throughput siRNA screening field.

iBRAIN2: automated analysis and data handling for RNAi screens.

We report on the implementation of a software suite dedicated to the management and analysis of large scale RNAi High Content Screening (HCS). We describe the requirements identified amongst our different users, the supported data flow, and the implemented software. Our system is already supporting productively three different laboratories operating in distinct IT infrastructures. The system was already used to analyze hundreds of RNAi HCS plates.

Novel inhibitors of dengue virus methyltransferase: discovery by in vitro-driven virtual screening on a desktop computer grid.

Dengue fever is a viral disease that affects 50-100 million people annually and is one of the most important emerging infectious diseases in many areas of the world. Currently, neither specific drugs nor vaccines are available. Here, we report on the discovery of new inhibitors of the viral NS5 RNA methyltransferase, a promising flavivirus drug target. We have used a multistage molecular docking approach to screen a library of more than 5 million commercially available compounds against the two binding sites of this enzyme. In 263 compounds chosen for experimental verification, we found 10 inhibitors with IC(50) values of <100 microM, of which four exhibited IC(50) values of <10 microM in in vitro assays. The initial hit list also contained 25 nonspecific aggregators. We discuss why this likely occurred for this particular target. We also describe our attempts to use aggregation prediction to further guide the study, following this finding.

The protein structure initiative structural genomics knowledgebase.

The Protein Structure Initiative Structural Genomics Knowledgebase (PSI SGKB, http://kb.psi-structuralgenomics.org) has been created to turn the products of the PSI structural genomics effort into knowledge that can be used by the biological research community to understand living systems and disease. This resource provides central access to structures in the Protein Data Bank (PDB), along with functional annotations, associated homology models, worldwide protein target tracking information, available protocols and the potential to obtain DNA materials for many of the targets. It also offers the ability to search all of the structural and methodological publications and the innovative technologies that were catalyzed by the PSI's high-throughput research efforts. In collaboration with the Nature Publishing Group, the PSI SGKB provides a research library, editorials about new research advances, news and an events calendar to present a broader view of structural biology and structural genomics. By making these resources freely available, the PSI SGKB serves as a bridge to connect the structural biology and the greater biomedical communities.

The Protein Model Portal.

Structural Genomics has been successful in determining the structures of many unique proteins in a high throughput manner. Still, the number of known protein sequences is much larger than the number of experimentally solved protein structures. Homology (or comparative) modeling methods make use of experimental protein structures to build models for evolutionary related proteins. Thereby, experimental structure determination efforts and homology modeling complement each other in the exploration of the protein structure space. One of the challenges in using model information effectively has been to access all models available for a specific protein in heterogeneous formats at different sites using various incompatible accession code systems. Often, structure models for hundreds of proteins can be derived from a given experimentally determined structure, using a variety of established methods. This has been done by all of the PSI centers, and by various independent modeling groups. The goal of the Protein Model Portal (PMP) is to provide a single portal which gives access to the various models that can be leveraged from PSI targets and other experimental protein structures. A single interface allows all existing pre-computed models across these various sites to be queried simultaneously, and provides links to interactive services for template selection, target-template alignment, model building, and quality assessment. The current release of the portal consists of 7.6 million model structures provided by different partner resources (CSMP, JCSG, MCSG, NESG, NYSGXRC, JCMM, ModBase, SWISS-MODEL Repository). The PMP is available at http://www.proteinmodelportal.org and from the PSI Structural Genomics Knowledgebase.

Identification of androgen-selective androgen-response elements in the human aquaporin-5 and Rad9 genes.

The AR (androgen receptor) is known to influence the expression of its target genes by binding to different sets of AREs (androgen-response elements) in the DNA. One set consists of the classical steroid-response elements which are partial palindromic repeats of the 5'-TGTTCT-3' steroid-receptor monomer-binding element. The second set contains motifs that are AR-specific and that are proposed to be partial direct repeats of the same motif. On the basis of this assumption, we used an in silico approach to identify new androgen-selective AREs in the regulatory regions of known androgen-responsive genes. We have used an extension of the NUBIScan algorithm to screen a collection of 85 known human androgen-responsive genes compiled from literature and database searches. We report the evaluation of the most promising hits resulting from this computational search by in vitro DNA-binding assays using full-size ARs and GRs (glucocorticoid receptors) as well as their isolated DBDs (DNA-binding domains). We also describe the ability of some of these motifs to confer androgen-, but not glucocorticoid-, responsiveness to reporter-gene expression. The elements found in the aquaporin-5 and the Rad9 (radiation-sensitive 9) genes showed selective AR versus GR binding in band-shift assays and a strong activity and selectivity in functional assays, both as isolated elements and in their original contexts. Our data indicate the validity of the hypothesis that selective AREs are recognizable as direct 5'-TGTTCT-3' repeats, and extend the list of currently known selective elements.

Sulfonylureas and glinides exhibit peroxisome proliferator-activated receptor gamma activity: a combined virtual screening and biological assay approach.

Most drugs currently employed in the treatment of type 2 diabetes either target the sulfonylurea receptor stimulating insulin release (sulfonylureas, glinides), or target the peroxisome proliferator-activated receptor (PPARgamma) improving insulin resistance (thiazolidinediones). Our work shows that sulfonylureas and glinides additionally bind to PPARgamma and exhibit PPARgamma agonistic activity. This activity was predicted in silico by virtual screening and confirmed in vitro in a binding assay, a transactivation assay, and by measuring the expression of PPARgamma target genes. Among the measured compounds, gliquidone and glipizide (two sulfonylureas), as well as nateglinide (a glinide), exhibit PPARgamma agonistic activity at concentrations comparable with those reached under pharmacological treatment. The most active of these compounds, gliquidone, is shown to be as potent as pioglitazone at inducing PPARgamma target gene expression. This dual mode of action of sulfonylureas and glinides may open new perspectives for the molecular pharmacology of antidiabetic drugs, because it provides evidence that drugs can be designed that target both the sulfonylurea receptor and PPARgamma. Targeting both receptors could increase pancreatic insulin secretion and improve insulin resistance. Glinides, sulfonylureas, and other acidified sulfonamides may be promising leads in the development of new PPARgamma agonists. In addition, we provide a unified concept of the PPARgamma binding ability of seemingly disparate compound classes.

Prediction of cis-regulatory elements for drug-activated transcription factors in the regulation of drug-metabolising enzymes and drug transporters.

The expression of drug-metabolising enzymes is affected by many endogenous and exogenous factors, including sex, age, diet and exposure to xenobiotics and drugs. To understand fully how the organism metabolises a drug, these alterations in gene expression must be taken into account. The central process, the definition of likely regulatory elements in the genes coding for enzymes and transporters involved in drug disposition, can be vastly accelerated using existing and emerging bioinformatics methods to unravel the regulatory networks causing drug-mediated induction of genes. Here, various approaches to predict transcription factor interactions with regulatory DNA elements are reviewed.

LXR deficiency and cholesterol feeding affect the expression and phenobarbital-mediated induction of cytochromes P450 in mouse liver.

Metabolic transformation by the superfamily of cytochromes P450 (CYPs) plays an important role in the detoxification of xenobiotics such as drugs, environmental pollutants, and food additives. Endogenous substrates of CYPs include fatty acids, sterols, steroids, and bile acids. Induction of CYPs via transcriptional activation by substrates and other xenobiotics is an important adaptive mechanism that increases the organism's defense capability against toxicity. Numerous in vivo and in vitro data have highlighted the concept that the molecular mechanism of hepatic drug induction is linked to endogenous regulatory pathways. In particular, in vitro data suggest that oxysterols via the liver X receptor (LXR) inhibit phenobarbital (PB)-mediated induction of CYPs. To study the link between LXR, cholesterol homeostasis, and drug induction in vivo, we designed experiments in wild-type, LXRalpha-, LXRbeta-, and LXRalpha/beta-deficient mice. Our data expose differential regulatory patterns for Cyp2b10 and Cyp3a11 dependent on the expression of LXR isoforms and on challenge of cholesterol homeostasis by excess dietary cholesterol. Our results suggest that, in the mouse, liver cholesterol status significantly alters the pattern of expression of Cyp3a11, whereas the absence of LXR leads to an increase in PB-mediated activation of Cyp2b10.

Rapid, accurate and non-invasive detection of cerebrospinal fluid leakage using combined determination of beta-trace protein in secretion and serum.

BACKGROUND: beta-Trace protein (Btp) has been proposed as a valuable marker of cerebrospinal fluid (CSF) leakage overcoming the drawbacks of beta-2-transferrin (B-2Tr) determination. However, there is still controversy about the appropriate cut-offs to be used (range 0.35-6 mg/L). The aim of the study was to evaluate cut-offs of Btp determination for detection CSF leakage. Further, we assessed whether the Btp secretion to serum ratio (Btp-sec/ser-ratio) would add diagnostic value. METHODS: Prospective study in patients with suspected CSF leakage. Quantitative determination of Btp in secretion and serum (Dade-Behring) and qualitative measurement of B-2-Tr in secretion and serum. Results were assessed in view of clinical data. Cut-offs and diagnostic characteristics were determined by ROC analysis. RESULTS: A total of 176 samples were assessed originating from 105 patients. In 43 samples CSF leakage could be confirmed. Sensitivity of B-2-Tr was 84%, specificity amounted to 100%. The area under the curve (AUC) for Btp-measurement in secretion was 0.98. At a cut-off of 0.68 mg/L, sensitivity was 100% and specificity 91%. At a cut-off of 1.11 mg/L, the specificity was 100% with a sensitivity of 93%. The Btp-sec/ser-ratio has an AUC of 0.99. Combining a 0.68 mg/L cut-off in secretion with a Btp-sec/ser-ratio cut-off of 4.9 reveals a sensitivity of 99% and a specificity of 100%. CONCLUSIONS: Btp is a rapid and accurate marker for the presence of CSF leakage. Combining measurement of Btp in secretion together with determination of the Btp-sec/ser-ratio enhances the diagnostic characteristics of the Btp assay. Determination of Btp in both serum and secretion is thus recommended.

The evolution of drug-activated nuclear receptors: one ancestral gene diverged into two xenosensor genes in mammals.

BACKGROUND: Drugs and other xenobiotics alter gene expression of cytochromes P450 (CYP) by activating the pregnane X receptor (PXR) and constitutive androstane receptor (CAR) in mammals. In non-mammalian species, only one xenosensor gene has been found. Using chicken as a model organism, the aim of our study was to elucidate whether non-mammalian species only have one or two xenosensors like mammals. RESULTS: To explore the evolutionary aspect of this divergence, we tried to identify additional xenobiotic sensing nuclear receptors in chicken using various experimental approaches. However, none of those revealed novel candidates. Ablation of chicken xenobiotic receptor (CXR) function by RNAi or dominant-negative alleles drastically reduced drug-induction in a chicken hepatoma cell line. Subsequently, we functionally and structurally characterized CXR and compared our results to PXR and CAR. Despite the high similarity in their amino acid sequence, PXR and CAR have very distinct modes of activation. Some aspects of CXR function, e.g. direct ligand activation and high promiscuity are very reminiscent of PXR. On the other hand, cellular localization studies revealed common characteristics of CXR and CAR in terms of cytoplasmic-nuclear distribution. Finally, CXR has unique properties regarding its regulation in comparison to PXR and CAR. CONCLUSION: Our finding thus strongly suggest that CXR constitutes an ancestral gene which has evolved into PXR and CAR in mammals. Future studies should elucidate the reason for this divergence in mammalian versus non-mammalian species.

Identification of the xenosensors regulating human 5-aminolevulinate synthase.

Heme is an essential component of numerous hemoproteins with functions including oxygen transport, energy metabolism, and drug biotransformation. In nonerythropoietic cells, 5-aminolevulinate synthase (ALAS1) is the rate-limiting enzyme in heme biosynthesis. Upon exposure to drugs that induce cytochromes P450 and other drug-metabolizing enzymes, ALAS1 is transcriptionally up-regulated, increasing the rate of heme biosynthesis to provide heme for cytochrome P450 hemoproteins. We used a combined in silico-in vitro approach to identify sequences in the ALAS1 gene that mediate direct transcriptional response to xenobiotic challenge. We have characterized two enhancer elements, located 20 and 16 kb upstream of the transcriptional start site. Both elements respond to prototypic inducer drugs and interact with the human pregnane X receptor NR1I2 and the human constitutive androstane receptor NR1I3. Our results suggest that the fundamental mechanism of drug induction is the same for cytochromes P450 and ALAS1. Transcriptional activation of the ALAS1 gene is the first step in the coordinated up-regulation of apoprotein and heme synthesis in response to exogenous and endogenous signals controlling heme levels. Understanding the direct effects of drugs on heme synthesis is of clinical interest, particularly in patients with hepatic porphyrias.

The estrogen-related receptor alpha (ERRalpha) functions in PPARgamma coactivator 1alpha (PGC-1alpha)-induced mitochondrial biogenesis.

Estrogen-related receptor alpha (ERRalpha) is one of the first orphan nuclear receptors to be identified, yet its physiological functions are still unclear. We show here that ERRalpha is an effector of the transcriptional coactivator PGC-1alpha [peroxisome proliferator-activated receptor gamma (PPARgamma) coactivator 1alpha], and that it regulates the expression of genes involved in oxidative phosphorylation and mitochondrial biogenesis. Inhibition of ERRalpha compromises the ability of PGC-1alpha to induce the expression of genes encoding mitochondrial proteins and to increase mitochondrial DNA content. A constitutively active form of ERRalpha is sufficient to elicit both responses. ERRalpha binding sites are present in the transcriptional control regions of ERRalpha/PGC-1alpha-induced genes and contribute to the transcriptional response to PGC-1alpha. The ERRalpha-regulated genes described here have been reported to be expressed at reduced levels in humans that are insulin-resistant. Thus, changes in ERRalpha activity could be linked to pathological changes in metabolic disease, such as diabetes.

In silico approaches, and in vitro and in vivo experiments to predict induction of drug metabolism.

Despite being described more than 40 years ago, the molecular mechanism that regulates hepatic induction of cytochromes P450 and other drug-metabolizing enzymes and drug transporters by xenobiotics has remained enigmatic until recently. A major breakthrough was the discovery of the orphan nuclear receptors pregnane X receptor and constitutive androstane receptor playing key roles as species-specific xenosensors in this induction response. Using this newly acquired knowledge, the human induction response can now be more accurately predicted. This is of considerable clinical importance, since induction of cytochrome P450s and other enzymes can lead to unwanted drug-drug interactions, adverse drug reactions and drug toxicity. In this review, in vitro, in vivo and in silico techniques are discussed that can identify troublesome compounds at an early stage and that can help to design new, safer medicines faster.

Human prion diseases: epidemiology and integrated risk assessment.

Human prion diseases are devastating and incurable, but are very rare. Fears that the bovine spongiform encephalopathy epizootic would lead to a large epidemic of its presumed human counterpart, variant Creutzfeldt-Jakob disease (vCJD), have not been realised. Yet a feeling of uncertainty prevails in the general public and in the biomedical world. The lack of data on the prevalence of asymptomatic carriers of vCJD compounds this uncertainty. In addition to this problem, Switzerland is currently faced with another issue of major public concern: a recent rise in the incidence of CJD. Here we examine the plausibility of several scenarios that may account for the increase in CJD incidence, including ascertainment bias due to improved reporting of CJD, iatrogenic transmission, and transmission of a prion zoonosis. In addition, we present the design and current status of a Swiss population-wide study of subclinical vCJD prevalence.

Molecular cloning and characterization of chicken orphan nuclear receptor cTR2.

Orphan nuclear receptors belong to the nuclear receptor superfamily of liganded transcription factors, whose ligands either do not exist or remain to be identified. We report here the cloning and characterization of the chicken orphan nuclear receptor, cTR2 (chicken testicular receptor 2). The cTR2 gene encodes a protein of 569 amino acids which shows approximately 72% overall identity with TR2 (NR2C1) and 95% identity in the DNA-binding domain (DBD). The cTR2 gene is expressed in almost all adult tissues and embryonic stages examined unlike its mammalian relative TR2, which is specifically expressed in testis. Electrophoretic mobility shift assays demonstrate that cTR2 binds the canonical direct repeat DNA recognition sequences spaced by one, four, and five nucleotides (DR1, DR4, and DR5), and in consistence with the results with canonical DNA-binding sequences, cTR2 forms specific DNA-protein complex with chicken phenobarbital response elements containing DR4 motifs. Both in vitro and in vivo interaction studies demonstrate that cTR2 forms homodimer. Moreover, transient transfection studies reveal its capability to transactivate canonical DR1, DR4, and DR5 sequences and the constitutive activity of cTR2 is mapped to the N-terminal region of this orphan receptor. Finally, cTR2 represses transactivation of estrogen receptor in a dose-dependent manner.

Cytochrome P450 CYP1A1 accumulates in the cytosol of kidney and brain and is activated by heme.

Cytochrome P450 CYP1A1 is expressed in most tissues. In brain and kidney, its function remains unclear because its enzymatic activity is barely measurable. Here, we report on the localization of CYP1A1 in the cytosol of kidney and brain, as revealed by immunoblotting with anti-CYP1A1 antibodies and by 7-ethoxyresorufin deethylation (EROD). Hematin (8 microM) added in vitro to cytosol increased the EROD-activity 10-fold in brain olfactory bulb and 7-fold in kidney, presumably by reconstitution of apocytochrome. Succinylacetone, an inhibitor of heme biosynthesis, increased the ratio of cytosolic to microsomal EROD activity of transiently expressed CYP1A1 in COS-1 cells from 1:1 to nearly 6:1. This indicates a strong decrease of microsomal activity with increasing succinylacetone concentration. CYP1A1 activities correlated with CYP1A1 protein assessed by immunoblotting. We conclude that the availability of heme is a limiting factor of P450 function in extrahepatic tissue. Our data further suggest that reduced availability of heme limits the incorporation of P450s into brain endoplasmic reticulum. These observations are important when assessing the function of P450s in extrahepatic tissue.

Drugs mediate the transcriptional activation of the 5-aminolevulinic acid synthase (ALAS1) gene via the chicken xenobiotic-sensing nuclear receptor (CXR).

Heme is an essential component in oxygen transport and metabolism in living systems. In non-erythropoietic cells, 5-aminolevulinate synthase (ALAS1) is the first and rate-limiting enzyme in the heme biosynthesis pathway. ALAS1 expression and heme levels are increased in vivo by drugs and other chemical inducers of cytochrome P450 hemoproteins through mechanisms that are poorly understood. In the present studies, a chicken genomic cosmid library was employed to isolate a major portion of the ALAS1 gene. Two drug-responsive enhancer sequences, 176 and 167 base pairs in length, were identified in the 5'-flanking region of the gene in reporter gene assays in the hepatoma cell line LMH. The relative potency of inducers to activate these enhancers corresponds to induction of ALAS1 mRNA levels in LMH cells. Analysis of putative transcription factor binding sites within the enhancers revealed DR5 and DR4 type recognition sequences for nuclear receptors. Drug activation of the enhancer elements was reduced at least 60% after mutagenesis of individual nuclear receptor binding sites and was virtually eliminated following alteration of both recognition sites within the respective elements. Electrophoretic mobility shift assays and transactivation studies demonstrate direct interactions between the nuclear receptor binding sites and the recently described chicken xenobiotic-sensing receptor, (CXR) implicating drug activation mechanisms for ALAS1 similar to those found in inducible cytochrome(s) P450. This is the first report describing direct transcriptional activation of ALAS1 by drugs via drug-responsive enhancer sequences.

NUBIScan, an in silico approach for prediction of nuclear receptor response elements.

Nuclear receptors (NRs) are transcription factors activated by a multitude of hormones, other endogenous substances, and exogenous molecules. These proteins modulate the regulation of target genes by contacting their promoter or enhancer sequences at specific recognition sites. The identification of these response elements is the first step toward detailed insight into the regulatory mechanisms affecting a gene. We have developed NUBIScan, a computer algorithm to predict DNA recognition sites for NRs in the regulatory regions of genes. The algorithm is based on weighted nucleotide distribution matrices and combines scores from both half-sites necessary for NR dimer binding. It provides more specific identification of functional sites than previous in silico approaches, as evidenced by scanning published regulatory regions of drug-inducible genes and comparing the obtained predictions with experimental results. In prospective analyses, NUBIScan consistently identified new functional NR binding sites in sets of large sequences, which had eluded previous analyses. This is exemplified by the detailed functional analysis of the flanking region of two genes. This approach therefore facilitates the selection of likely sites of gene regulation for subsequent experimental analysis.

Cholesterol and bile acids regulate xenosensor signaling in drug-mediated induction of cytochromes P450.

Cytochromes P450 (CYP) constitute the major enzymatic system for metabolism of xenobiotics. Here we demonstrate that transcriptional activation of CYPs by the drug-sensing nuclear receptors pregnane X receptor, constitutive androstane receptor, and the chicken xenobiotic receptor (CXR) can be modulated by endogenous cholesterol and bile acids. Bile acids induce the chicken drug-activated CYP2H1 via CXR, whereas the hydroxylated metabolites of bile acids and oxysterols inhibit drug induction. The cholesterol-sensing liver X receptor competes with CXR, pregnane X receptor, or constitutive androstane receptor for regulation of drug-responsive enhancers from chicken CYP2H1, human CYP3A4, or human CYP2B6, respectively. Thus, not only cholesterol 7 alpha-hydroxylase (CYP7A1), but also drug-inducible CYPs, are diametrically affected by these receptors. Our findings reveal new insights into the increasingly complex network of nuclear receptors regulating lipid homeostasis and drug metabolism.