MedWorm.com provides a medical RSS filtering service. Over 6000 RSS medical sources are combined and output via different filters. This feed contains the latest items from the 'Journal of Biomolecular Screening' source. Thu, 09 Feb 2012 16:56:44 +0100 http://www.medworm.com/index.php?rid=5635399&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Freprint%2F17%2F2%2F275%3Frss%3D1 (Source: Journal of Biomolecular Screening) Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=5635399 Wed, 25 Jan 2012 05:00:00 +0100 5635399 http://www.medworm.com/index.php?rid=5635398&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F17%2F2%2F266%3Frss%3D1 Automated microscopes have enabled the unprecedented collection of images at a rate that precludes visual inspection. Automated image analysis is required to identify interesting samples and extract quantitative information for high-content screening (HCS). However, researchers are impeded by the lack of metrics and software tools to identify image-based aberrations that pollute data, limiting experiment quality. The authors have developed and validated approaches to identify those image acquisition artifacts that prevent optimal extraction of knowledge from high-content microscopy experiments. They have implemented these as a versatile, open-source toolbox of algorithms and metrics readily usable by biologists to improve data quality in a wide variety of biological experiments. (Source: J... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=5635398 Wed, 25 Jan 2012 05:00:00 +0100 5635398 http://www.medworm.com/index.php?rid=5635397&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F17%2F2%2F258%3Frss%3D1 Pooled short-hairpin RNA (shRNA) library screening is a powerful tool for identifying a set of genes in biological pathways that require stable expression to produce a desired phenotype. Massive parallel sequencing of half-hairpins has proven highly variable and has not given satisfactory results concerning the relative abundance of different shRNAs before and after selection. Here, the authors describe a method for quantitative comparison of half-hairpins from pooled shRNAs in the mir30-based pGIPZ vector that is analyzed by massive parallel sequencing. Introducing a multiplexing code and refining the sample preparation scheme resulted in the predicted ability to detect twofold enrichments. These improvements should permit half-hairpin sequencing to analyze either dropout screens or selec... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=5635397 Wed, 25 Jan 2012 05:00:00 +0100 5635397 http://www.medworm.com/index.php?rid=5635396&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F17%2F2%2F252%3Frss%3D1 In this study, the authors reported a microtiter plate–based colorimetric assay for RmlA enzyme activity. Using this assay, the kinetic properties of M. tuberculosis RmlA including initial velocity, optimal temperature, optimal pH, the effect of Mg2+, and kinetic parameters were determined. The establishment of the accurate and rapid colorimetric assay and kinetic analysis of M. tuberculosis RmlA will facilitate high-throughput screening of RmlA inhibitors. (Source: Journal of Biomolecular Screening) Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=5635396 Wed, 25 Jan 2012 05:00:00 +0100 5635396 http://www.medworm.com/index.php?rid=5635395&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F17%2F2%2F245%3Frss%3D1 Triglyceride lipases such as lipoprotein lipase, endothelial lipase, and hepatic lipase play key roles in controlling the levels of plasma lipoprotein. Accordingly, small-molecule modulation of these species could alter patient lipid profiles with corresponding health effects. Screening of these enzymes for small-molecule therapeutics has historically involved the use of lipid-based particles to mimic native substrates. However, particle-based artifacts can complicate the discovery of therapeutic molecules. As a simplifying solution, the authors sought to develop an approach involving a soluble and monomeric lipase substrate. Using purified bovine lipoprotein lipase as a model system, they show that the hydrolysis of resorufin butyrate can be fluorescently monitored to give a robust assay ... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=5635395 Wed, 25 Jan 2012 05:00:00 +0100 5635395 http://www.medworm.com/index.php?rid=5635394&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F17%2F2%2F237%3Frss%3D1 Methylation of arginine residues, catalyzed by protein arginine methyltransferases (PRMTs), is one important protein posttranslational modification involved in epigenetic regulation of gene expression. A fast and effective assay for PRMT can provide valuable information for dissecting the biological functions of PRMTs, as well as for screening small-molecule inhibitors of arginine methylation. Currently, among the methods used for PRMT activity measurement, many contain laborious separation procedures, which restrict the applications of these assays for high-throughput screening (HTS) in drug discovery. The authors report here a mix-and-measure method to measure PRMT activity based on the principle of scintillation proximity assay (SPA). In this assay, 3H-AdoMet was used as methyl donor, a... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=5635394 Wed, 25 Jan 2012 05:00:00 +0100 5635394 http://www.medworm.com/index.php?rid=5635393&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F17%2F2%2F225%3Frss%3D1 Identifying chemical lead matter by high-throughput screening (HTS) has been a common practice in early stage drug discovery. Evolution of small-molecule library composition to include more drug-like molecules with desirable physical chemical properties combined with improving assay technologies has vastly enhanced the capability of HTS. However, HTS campaigns can still be plagued by false positives arising from nonspecific inhibitors. The generation of assay-ready plates has permitted an incremental advancement to the speed and efficiency of HTS but has the potential to enhance the occurrence of nonspecific inhibitors. A subtle change in the order of reagent addition to the assay-ready plates can greatly alleviate false-positive inhibition. Our case studies with six different kinase and p... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=5635393 Wed, 25 Jan 2012 05:00:00 +0100 5635393 http://www.medworm.com/index.php?rid=5635392&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F17%2F2%2F216%3Frss%3D1 Flavonoids have been reported to exert multiple biological effects that include acting as pro-oxidants at very high doses. The authors determined a structural alert to identify the clastogenic activity of a series of flavonoids with pro-oxidant activity. The methodology was based on a quantitative structure–activity relationship (QSAR) study. Specifically, the authors developed a virtual screening method for a clastogenic model using the topological substructural molecular design (TOPS-MODE) approach. It represents a useful platform for the automatic generation of structural alerts, based on the calculation of spectral moments of molecular bond matrices appropriately weighted, taking into account the hydrophobic, electronic, and steric molecular features. Therefore, it was possible t... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=5635392 Wed, 25 Jan 2012 05:00:00 +0100 5635392 http://www.medworm.com/index.php?rid=5635391&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F17%2F2%2F204%3Frss%3D1 The nicotinic acetylcholine receptors (nAChRs) are a member of the ligand-gated ion channel family and play a key role in the transfer of information across neurological networks. The X-ray crystal structure of agonist-bound α7 acetylcholine binding protein (AChBP) has been recognized as the most appropriate template to model the ligand-binding domain of nAChR for studying the molecular mechanism of the receptor–ligand interactions. Virtual screening of the National Cancer Institute diversity set, a library of 1990 compounds with nonredundant pharmacophore profiles, using AutoDock against AChBPs revealed 51 potential candidates. In vitro radioligand competition assays using [3H] epibatidine against the AChBPs from the freshwater snails, Lymnaea stagnalis, and from the marine sp... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=5635391 Wed, 25 Jan 2012 05:00:00 +0100 5635391 http://www.medworm.com/index.php?rid=5635390&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F17%2F2%2F194%3Frss%3D1 The authors conducted a high-throughput screening campaign for inhibitors of SV40 large T antigen ATPase activity to identify candidate antivirals that target the replication of polyomaviruses. The primary assay was adapted to 1536-well microplates and used to screen the National Institutes of Health Molecular Libraries Probe Centers Network library of 306 015 compounds. The primary screen had an Z value of ~0.68, signal/background = 3, and a high (5%) DMSO tolerance. Two counterscreens and two secondary assays were used to prioritize hits by EC50, cytotoxicity, target specificity, and off-target effects. Hits that inhibited ATPase activity by >44% in the primary screen were tested in dose–response efficacy and eukaryotic cytotoxicity assays. After evaluation of hit cytotoxicity, ... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=5635390 Wed, 25 Jan 2012 05:00:00 +0100 5635390 http://www.medworm.com/index.php?rid=5635389&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F17%2F2%2F183%3Frss%3D1 The authors have used a surface plasmon resonance (SPR)–based biosensor approach to identify and characterize compounds with a unique binding mode to protein kinases. Biacore was used to characterize hits from an enzymatic high-throughput screen of the Tec family tyrosine kinase, IL2-inducible T cell kinase (ITK). Complex binding kinetics was observed for some compounds, which led to identification of compounds that bound simultaneously at both the adenosine triphosphate (ATP) binding site and a second, allosteric site on ITK. The presence of the second binding site was confirmed by X-ray crystallography. The second site is located in the N-terminal lobe of the protein kinase catalytic domain, adjacent to but distinct from the ATP site. To enable rapid optimization of binding propert... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=5635389 Wed, 25 Jan 2012 05:00:00 +0100 5635389 http://www.medworm.com/index.php?rid=5635388&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F17%2F2%2F177%3Frss%3D1 H+,K+-ATPase is a key enzyme in the process of gastric acid secretion, and proton pump inhibitors (PPIs) have been accepted as one of the most effective treatments for peptic ulcer and gastroesophageal reflux disease. To discover a novel class of PPIs, the authors screened a low-molecular-weight compound library and identified two prospective acid blockers that were pyrrole derivatives. Both compounds inhibited H+,K+-ATPase in a reversible and potassium-competitive manner. These compounds led to the development of TAK-438 (1-[5-(2-fluorophenyl)-1-(pyridin-3-ylsulfonyl)-1H-pyrrol-3-yl]-N-methylmethanamine monofumarate), which is currently undergoing clinical trials as a novel potassium-competitive acid blocker for the treatment of acid-related diseases. (Source: Journal of Biomolecular Scre... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=5635388 Wed, 25 Jan 2012 05:00:00 +0100 5635388 http://www.medworm.com/index.php?rid=5635387&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F17%2F2%2F163%3Frss%3D1 UBC13 is a noncanonical ubiquitin conjugating enzyme (E2) that has been implicated in a variety of cellular signaling processes due to its ability to catalyze formation of lysine 63–linked polyubiquitin chains on various substrates. In particular, UBC13 is required for signaling by a variety of receptors important in immune regulation, making it a candidate target for inflammatory diseases. UBC13 is also critical for double-strand DNA repair and thus a potential radiosensitizer and chemosensitizer target for oncology. The authors developed a high-throughput screening (HTS) assay for UBC13 based on the method of time-resolved fluorescence resonance energy transfer (TR-FRET). The TR-FRET assay combines fluorochrome (Fl)–conjugated ubiquitin (fluorescence acceptor) with terbium (T... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=5635387 Wed, 25 Jan 2012 05:00:00 +0100 5635387 http://www.medworm.com/index.php?rid=5635386&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F17%2F2%2F152%3Frss%3D1 The cancer stem cell (CSC) hypothesis proposes that a subpopulation of CSCs is frequently responsible for chemotherapy resistance and metastasis and is now a point of attack for research into the next generation of therapeutics. Although many of these agents are directed at inducing CSC apoptosis (as well as the bulk tumor), some agents may also decrease cell "stemness" possibly through induction of differentiation. Ubiquitin ligases, critical to virtually all cellular signaling systems, alter the degradation or trafficking of most proteins in the cell, and indeed broad perturbation of this system, through inhibition of the proteosome, is a successful cancer treatment. The authors examined several glioblastoma stem cell isolates pre- and postdifferentiation to elucidate the phenotypic effe... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=5635386 Wed, 25 Jan 2012 05:00:00 +0100 5635386 http://www.medworm.com/index.php?rid=5635385&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F17%2F2%2F140%3Frss%3D1 In this study, to classify a number of compounds, the authors established a gene expression–based screening system using mouse embryonic stem (ES) cells that monitored multiple parameters. ES cells were differentiated into three germ layers by embryoid body formation and then treated with the test compounds. Next, cellular changes were assessed by analyzing the expression of multiple genes with the multiplex quantitative reverse transcriptase polymerase chain reaction. By screening a library of pharmacologically active compounds with this system, the authors were able to classify 52 compounds that influenced the gene expression profile of ES cells. They also found that some compounds identified by screening could enhance osteogenic or adipogenic differentiation of human mesenchymal s... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=5635385 Wed, 25 Jan 2012 05:00:00 +0100 5635385 http://www.medworm.com/index.php?rid=5635384&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F17%2F2%2F129%3Frss%3D1 This study demonstrates that a phenotypic assay using cell type–specific antibody markers can be used for a large-scale compound screen to discover targets and pathways with impacts on differentiation of lineage-restricted precursor cells toward specific lineages. (Source: Journal of Biomolecular Screening) Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=5635384 Wed, 25 Jan 2012 05:00:00 +0100 5635384 http://www.medworm.com/index.php?rid=5568814&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Freprint%2F17%2F1%2F121%3Frss%3D1 (Source: Journal of Biomolecular Screening) Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=5568814 Thu, 05 Jan 2012 05:00:00 +0100 5568814 http://www.medworm.com/index.php?rid=5568813&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F17%2F1%2F108%3Frss%3D1 This study represents the first report of a demethylase high-content imaging assay with the ability to capture a repertoire of pharmacological tools, which are likely both to inform our mechanistic understanding of how JMJD3 is modulated and, more important, to contribute to the identification of novel therapeutic modalities for this demethylase enzyme. (Source: Journal of Biomolecular Screening) Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=5568813 Thu, 05 Jan 2012 05:00:00 +0100 5568813 http://www.medworm.com/index.php?rid=5568812&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F17%2F1%2F99%3Frss%3D1 We report herein a high-throughput DELFIA assay to quantify H3K27me3 in the prostate cancer cell line, PC3. Using a high binding MaxiSorp plate, we were able to eliminate the need for the capture antibody. We also developed an effective method, a combination of "freeze-thaw" and 0.2 N HCl, to extract histone proteins in PC3 cells cultured in a 384-well plate. To compensate for cell viability change, we normalized H3K27me3 signal to the total amount of H3 in each sample well. As a result, we show that the assay has a good dynamic range with a robust assay window. Using a methlytransferase inhibitor, DZNep, we show that the change of H3K27me3 signal is target specific. This method simplifies the logistics in screening and profiling and reduces the cost per well to an acceptable level for hig... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=5568812 Thu, 05 Jan 2012 05:00:00 +0100 5568812 http://www.medworm.com/index.php?rid=5568811&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F17%2F1%2F85%3Frss%3D1 This study demonstrates the power of virtual screening technologies for novel, therapeutically relevant epigenetics protein targets. (Source: Journal of Biomolecular Screening) Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=5568811 Thu, 05 Jan 2012 05:00:00 +0100 5568811 http://www.medworm.com/index.php?rid=5568810&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F17%2F1%2F71%3Frss%3D1 The histone methyltransferase (HMT) family of proteins consists of enzymes that methylate lysine or arginine residues on histone tails as well as other proteins. Such modifications affect chromatin structure and play a significant regulatory role in gene expression. Many HMTs have been implicated in tumorigenesis and progression of multiple malignancies and play essential roles in embryonic development and stem cell renewal. Overexpression of some HMTs has been observed and is correlated positively with various types of cancer. Here the authors report development of a continuous fluorescence-based methyltransferase assay in a 384-well format and its application in determining kinetic parameters for EHMT1, G9a, PRMT3, SETD7, and SUV39H2 as well as for screening against libraries of small mo... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=5568810 Thu, 05 Jan 2012 05:00:00 +0100 5568810 http://www.medworm.com/index.php?rid=5568809&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F17%2F1%2F59%3Frss%3D1 Methylation is a ubiquitous covalent modification used to control the function of diverse biomolecules including hormones, neurotransmitters, xenobiotics, proteins, nucleic acids, and lipids. Histone methyltransferases (HMTs) are currently of high interest as drug targets because of their role in epigenetic regulation; however, most HMT assay methods are either not amenable to a high-throughput screening (HTS) environment or are applicable to a limited number of enzymes. The authors developed a generic methyltransferase assay method using fluorescent immunodetection of adenosine monophosphate (AMP), which is formed from the MT reaction product S-adenosylhomocysteine in a dual-enzyme coupling step. The detection range of the assay; its suitability for HTS, including stability of reagents fo... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=5568809 Thu, 05 Jan 2012 05:00:00 +0100 5568809 http://www.medworm.com/index.php?rid=5568808&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F17%2F1%2F49%3Frss%3D1 Histone posttranslational modifications are among the epigenetic mechanisms that modulate chromatin structure and gene transcription. Histone methylation and demethylation are dynamic processes controlled respectively by histone methyltransferases (HMTs) and demethylases (HDMs). Several HMTs and HDMs have been implicated in cancer, inflammation, and diabetes, making them attractive targets for drug therapy. Hence, the discovery of small-molecule modulators for these two enzyme classes has drawn significant attention from the pharmaceutical industry. Herein, the authors describe the development and optimization of homogeneous LANCE Ultra and AlphaLISA antibody-based assays for measuring the catalytic activity of two epigenetic enzymes acting on lysine 4 of histone H3: SET7/9 methyltransfera... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=5568808 Thu, 05 Jan 2012 05:00:00 +0100 5568808 http://www.medworm.com/index.php?rid=5568807&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F17%2F1%2F39%3Frss%3D1 A high-throughput RapidFire mass spectrometry assay is described for the JMJD2 family of Fe2+, O2, and α-ketoglutarate-dependent histone lysine demethylases. The assay employs a short amino acid peptide substrate, corresponding to the first 15 amino acid residues of histone H3, but mutated at two positions to increase assay sensitivity. The assay monitors the direct formation of the dimethylated-Lys9 product from the trimethylated-Lys9 peptide substrate. Monitoring the formation of the monomethylated and des-methylated peptide products is also possible. The assay was validated using known inhibitors of the histone lysine demethylases, including 2,4-pyridinedicarboxylic acid and an α-ketoglutarate analogue. With a sampling rate of 7 s per well, the RapidFire technology permitted... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=5568807 Thu, 05 Jan 2012 05:00:00 +0100 5568807 http://www.medworm.com/index.php?rid=5568806&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F17%2F1%2F27%3Frss%3D1 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 sele... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=5568806 Thu, 05 Jan 2012 05:00:00 +0100 5568806 http://www.medworm.com/index.php?rid=5568805&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F17%2F1%2F18%3Frss%3D1 In the past years, a lot of attention has been given to the identification and characterization of selective and potent inhibitors of chromatin-modifying enzymes to better understand their specific role in transcriptional regulation. As aberrant histone methylation is involved in different pathological processes, the search for methyltransferase and demethylase inhibitors has emerged as a crucial issue in current medicinal chemistry research. High-throughput in vitro assays are important tools for the identification of new methyltransferase or demethylase inhibitors. These usually use oligopeptide substrates derived from histone sequences, although in many cases, they are not good substrates for these enzymes. Here, the authors report about the setup and establishment of in vitro assays th... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=5568805 Thu, 05 Jan 2012 05:00:00 +0100 5568805 http://www.medworm.com/index.php?rid=5568804&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F17%2F1%2F2%3Frss%3D1 Epigenetic modification of DNA leads to changes in gene expression. DNA methyltransferases (DNMTs) comprise a family of nuclear enzymes that catalyze the methylation of CpG dinucleotides, resulting in an epigenetic methylome distinguished between normal cells and those in disease states such as cancer. Disrupting gene expression patterns through promoter methylation has been implicated in many malignancies and supports DNMTs as attractive therapeutic targets. This review focuses on the rationale of targeting DNMTs in cancer, the historical approach to DNMT inhibition, and current marketed hypomethylating therapeutics azacytidine and decitabine. In addition, we address novel DNMT inhibitory agents emerging in development, including CP-4200 and SGI-110, analogs of azacytidine and decitabine,... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=5568804 Thu, 05 Jan 2012 05:00:00 +0100 5568804 http://www.medworm.com/index.php?rid=5568803&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Freprint%2F17%2F1%2F1%3Frss%3D1 (Source: Journal of Biomolecular Screening) Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=5568803 Thu, 05 Jan 2012 05:00:00 +0100 5568803 http://www.medworm.com/index.php?rid=5501082&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Freprint%2F16%2F10%2F1256%3Frss%3D1 (Source: Journal of Biomolecular Screening) Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=5501082 Mon, 12 Dec 2011 05:00:00 +0100 5501082 http://www.medworm.com/index.php?rid=5501081&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Freprint%2F16%2F10%2F1254%3Frss%3D1 (Source: Journal of Biomolecular Screening) Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=5501081 Mon, 12 Dec 2011 05:00:00 +0100 5501081 http://www.medworm.com/index.php?rid=5501080&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F16%2F10%2F1247%3Frss%3D1 Developing molecularly targeted therapeutics with minimal off-target effects is facilitated by an understanding of compound selectivity. However, for HDAC inhibitors, a clear understanding of specificity has been challenging. In particular, it has been suggested that use of nonspecific substrates and the presence of multiple HDAC activities in enzyme preparations may complicate interpretation of inhibitor experiments. To overcome these and other potential limitations of activity-based HDAC assays, the authors have developed an assay format based on measurement of the binding affinity of inhibitors rather than measurement of enzyme activity. One advantage of this format is that it does not require use of a substrate and thus ameliorates concerns about lack of specificity of existing substra... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=5501080 Mon, 12 Dec 2011 05:00:00 +0100 5501080 http://www.medworm.com/index.php?rid=5501079&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F16%2F10%2F1236%3Frss%3D1 In conclusion, this assay platform enables high-throughput cell-based analysis of diverse types of posttranslational modifications of Histone H3. (Source: Journal of Biomolecular Screening) Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=5501079 Mon, 12 Dec 2011 05:00:00 +0100 5501079 http://www.medworm.com/index.php?rid=5501078&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F16%2F10%2F1227%3Frss%3D1 Histone deacetylase (HDAC) enzymes modify the acetylation state of histones and other important proteins. Aberrant HDAC enzyme function has been implicated in many diseases, and the discovery and development of drugs targeting these enzymes is becoming increasingly important. In this article, the authors report the evaluation of homogeneous, single-addition, bioluminogenic HDAC enzyme activity assays that offer less assay interference by compounds in comparison to fluorescence-based formats. The authors assessed the key operational assay properties including sensitivity, scalability, reproducibility, signal stability, robustness (Z'), DMSO tolerance, and pharmacological response to standard inhibitors against HDAC-1, HDAC-3/NcoR2, HDAC-6, and SIRT-1 enzymes. These assays were successfully ... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=5501078 Mon, 12 Dec 2011 05:00:00 +0100 5501078 http://www.medworm.com/index.php?rid=5501077&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F16%2F10%2F1217%3Frss%3D1 The sirtuin enzymes, a class of NAD+-dependent histone deacetylases, are a focal point of epigenetic research because of their roles in regulating gene expression and cellular differentiation by deacetylating histones and a host of transcription factors, including p53. Here, the authors present two label-free screening methodologies to study sirtuin activity using high-throughput mass spectrometry. The first method involves the detection of native peptides and provides a platform for more detailed mechanistic studies by enabling the concurrent and direct measurement of multiple modification states. The second method obviates the need for substrate-specific assay development by measuring the O-acetyl-ADP-ribose co-product formed by sirtuin-dependent deacetylation. Both methodologies were ap... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=5501077 Mon, 12 Dec 2011 05:00:00 +0100 5501077 http://www.medworm.com/index.php?rid=5501076&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F16%2F10%2F1206%3Frss%3D1 In this study, a dual-competition assay exploiting changes in fluorescence anisotropy and lifetime was used to screen the LOPAC (Sigma-Aldrich, St Louis, MO) library against the bacterial histone deacetylase homologue HDAH from Bordetella, which shares 35% identity with the second deacetylase domain of HDAC6. The binding assay proved to be highly suitable for high-throughput screening campaigns. Several LOPAC compounds have been identified to inhibit HDAH in the lower micromolar range. Most interestingly, some of the hit compounds turned out to be weak but selective inhibitors of human class IIa and IIb HDACs. (Source: Journal of Biomolecular Screening) Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=5501076 Mon, 12 Dec 2011 05:00:00 +0100 5501076 http://www.medworm.com/index.php?rid=5501075&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F16%2F10%2F1196%3Frss%3D1 This study establishes a new high-throughput assay for HAT activity and could provide the foundation for the development of a new class of drugs for the treatment of leukemias. (Source: Journal of Biomolecular Screening) Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=5501075 Mon, 12 Dec 2011 05:00:00 +0100 5501075 http://www.medworm.com/index.php?rid=5501074&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F16%2F10%2F1186%3Frss%3D1 Histone acetyltransferases (HATs) catalyze the transfer of an acetyl group from an acetyl-coenzyme A donor molecule to specific lysine residues within proteins. The acetylation state of proteins, particularly histones, is known to modulate their intermolecular binding properties and control various cellular processes, most notably transcriptional activation. In addition, deregulation of HAT activity has been linked to the development of a number of cancers; therefore, compounds that affect these enzymes have strong potential as therapeutic agents. The research presented here demonstrates three label-free HAT screening approaches, all based on the fast and direct measurement of one or more substrate-product pairs by high-throughput mass spectrometry techniques. The first approach involves m... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=5501074 Mon, 12 Dec 2011 05:00:00 +0100 5501074 http://www.medworm.com/index.php?rid=5501073&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F16%2F10%2F1170%3Frss%3D1 Bromodomains are structurally conserved protein modules present in a large number of chromatin-associated proteins and in many nuclear histone acetyltransferases. The bromodomain functions as an acetyl-lysine binding domain and has been shown to be pivotal in regulating protein–protein interactions in chromatin-mediated cellular gene transcription, cell proliferation, and viral transcriptional activation. Structural analyses of these modules in complex with acetyl-lysine peptide ligands provide insights into the molecular basis for recognition and ligand selectivity within this epigenetic reader family. However, there are significant challenges in configuring assays to identify inhibitors of these proteins. This review focuses on the progress made in developing methods to identify pe... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=5501073 Mon, 12 Dec 2011 05:00:00 +0100 5501073 http://www.medworm.com/index.php?rid=5501072&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F16%2F10%2F1153%3Frss%3D1 The sirtuin family of NAD-dependent histone deacetylases (HDACs) consists of seven mammalian proteins, SIRT1–7. Many of the sirtuin isoforms also deacetylate nonhistone substrates, such as p53 (SIRT1) and α-tubulin (SIRT2). The sirtuin literature focuses on pharmacological activators of SIRT1 (e.g., resveratrol, SRT1720), proposed as therapeutics for diabetes, neurodegeneration, inflammation, and others. However, many of the SIRT1 activator results may have been due to artifacts in the assay methodology (i.e., use of fluorescently tagged substrates). A biological role for SIRT1 in cancer has been given less scrutiny but is no less equivocal. Although proposed initially as an oncogene, we present herein compelling data suggesting that SIRT1 is indeed a context-specific tumor sup... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=5501072 Mon, 12 Dec 2011 05:00:00 +0100 5501072 http://www.medworm.com/index.php?rid=5501071&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F16%2F10%2F1137%3Frss%3D1 Epigenetic control of the transciptome is a complex and highly coordinated cellular process. One critical mechanism involves DNA methylation, mediated by distinct but related DNA methyltransferases (DNMTs). Although several DNMT inhibitors are available, most are nonselective; selective DNMT inhibitors, therefore, could be optimal as therapeutics, as well acting as chemical probes to elucidate the fundamental biology of individual DNMTs. DNA methylation is a stable chemical modification, yet posttranslational modification of histones is transitory, with reversible effects on gene expression. Histone posttranslational modifications influence access of transcription factors to DNA target sites to affect gene activity. Histones are regulated by several enzymes, including acetylases (HATs), de... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=5501071 Mon, 12 Dec 2011 05:00:00 +0100 5501071 http://www.medworm.com/index.php?rid=5501070&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Freprint%2F16%2F10%2F1135%3Frss%3D1 (Source: Journal of Biomolecular Screening) Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=5501070 Mon, 12 Dec 2011 05:00:00 +0100 5501070 http://www.medworm.com/index.php?rid=5292885&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Freprint%2F16%2F9%2F1125%3Frss%3D1 (Source: Journal of Biomolecular Screening) Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=5292885 Tue, 04 Oct 2011 04:00:00 +0100 5292885 http://www.medworm.com/index.php?rid=5292884&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F16%2F9%2F1119%3Frss%3D1 Multicellular tumor spheroids (MCTS) are routinely employed as three-dimensional in vitro models to study tumor biology. Cultivation of MCTS in spinner flasks provides better growing conditions, especially with regard to the availability of nutrients and oxygen, when compared with microtiter plates. The main endpoint of drug response experiments is spheroid size. It is common practice to analyze spheroid size manually with a microscope and an ocular micrometer. This requires removal of some spheroids from the flask, which entails major limitations such as loss of MCTS and the risk of contamination. With this new approach, the authors present an efficient and highly reproducible method to analyze the size of complete MCTS populations in culture containers with transparent, flat bottoms. MCT... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=5292884 Tue, 04 Oct 2011 04:00:00 +0100 5292884 http://www.medworm.com/index.php?rid=5292883&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F16%2F9%2F1112%3Frss%3D1 This study evaluated the impact of temperature, exposure, and solubilization on overall compound quality for short-term storage. A small library of 25 representative compounds was evaluated over an 18-week period to monitor the change in purity and concentration by high-performance liquid chromatography with ultraviolet detection. The authors concluded that temperature had a significant impact on compound concentration, and the effects due to exposure cycles were minimal. A storage time of 12 weeks at room temperature resulted in minimal compound loss, but storage times beyond this would be unacceptable because of a >20% decrease in concentration. Finally, the acoustic solubilization protocol also increased the number of compounds at the target concentration with no impact on overall pu... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=5292883 Tue, 04 Oct 2011 04:00:00 +0100 5292883 http://www.medworm.com/index.php?rid=5292882&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F16%2F9%2F1106%3Frss%3D1 Vascular adhesion protein–1 (VAP-1), also known as semicarbazide-sensitive amine oxidase (SSAO) or copper-containing amine oxidase (AOC3, EC 1.4.3.6), catalyzes oxidative deamination of primary amines. One endogenous substrate has recently been described (Siglec 10), and although its mechanism of action in vivo is not completely understood, it is suggested to play a role in immune cell trafficking, making it a target of interest for autoimmune and inflammatory diseases. Much of the enzymology performed around this target has been conducted with absorbance, fluorescent, or radiometric formats that can have some limitations for high-throughput screening and subsequent compound profiling. The authors present the use of a bioluminescent assay, originally developed for monoamine oxidase e... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=5292882 Tue, 04 Oct 2011 04:00:00 +0100 5292882 http://www.medworm.com/index.php?rid=5292881&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F16%2F9%2F1098%3Frss%3D1 In this study, the authors used conventional and nonconventional methods to further characterize P2Y14 and its ligands. Conventional calcium mobilization and nonconventional cellular impedance functional assays revealed that UMP and UDP selectively activated HEK cells coexpressing P2Y14 and Gαqi5. In the impedance assays, the presence of exogenous Gαqi5 resulted in agonist-induced Gq signaling, whereas in the absence of exogenous Gαqi5, the signal was indicative of Gi. The authors established the first P2Y14 membrane filtration binding assay using a novel optimized expression vector and [3H]UDP as radioligand. UDP-Glc, UMP, and UDP dose dependently inhibited [3H]UDP binding in the binding assay, and saturation analysis revealed that UDP bound P2Y14 with a KD = 10 nM and a... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=5292881 Tue, 04 Oct 2011 04:00:00 +0100 5292881 http://www.medworm.com/index.php?rid=5292880&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F16%2F9%2F1089%3Frss%3D1 Inhibitors of human dimethylarginine dimethylaminohydrolase-1 (DDAH-1) are of therapeutic interest for controlling pathological nitric oxide production. Only a limited number of biologically useful inhibitors have been identified, so structurally diverse lead compounds are desired. In contrast with previous assays that do not possess adequate sensitivity for optimal screening, herein is reported a high-throughput assay that uses an alternative thiol-releasing substrate, S-methyl-L-thiocitrulline, and a thiol-reactive fluorophore, 7-diethylamino-3-(4'-maleimidylphenyl)-4-methylcoumarin, to enable continuous detection of product formation by DDAH-1. The assay is applied to query two commercial libraries totaling 4446 compounds, and two representative hits are described, including a known DDA... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=5292880 Tue, 04 Oct 2011 04:00:00 +0100 5292880 http://www.medworm.com/index.php?rid=5292879&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F16%2F9%2F1081%3Frss%3D1 Recently, the use of the Bayesian network as an alternative to existing tools for similarity-based virtual screening has received noticeable attention from researchers in the chemoinformatics field. The main aim of the Bayesian network model is to improve the retrieval effectiveness of similarity-based virtual screening. To this end, different models of the Bayesian network have been developed. In our previous works, the retrieval performance of the Bayesian network was observed to improve significantly when multiple reference structures or fragment weightings were used. In this article, the authors enhance the Bayesian inference network (BIN) using the relevance feedback information. In this approach, a few high-ranking structures of unknown activity were filtered from the outputs of BIN,... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=5292879 Tue, 04 Oct 2011 04:00:00 +0100 5292879 http://www.medworm.com/index.php?rid=5292878&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F16%2F9%2F1068%3Frss%3D1 The standard (STD) 5 x 5 hybrid median filter (HMF) was previously described as a nonparametric local backestimator of spatially arrayed microtiter plate (MTP) data. As such, the HMF is a useful tool for mitigating global and sporadic systematic error in MTP data arrays. Presented here is the first known HMF correction of a primary screen suffering from systematic error best described as gradient vectors. Application of the STD 5 x 5 HMF to the primary screen raw data reduced background signal deviation, thereby improving the assay dynamic range and hit confirmation rate. While this HMF can correct gradient vectors, it does not properly correct periodic patterns that may present in other screening campaigns. To address this issue, 1 x 7 median and a row/column 5 x 5 hybrid median filter ke... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=5292878 Tue, 04 Oct 2011 04:00:00 +0100 5292878 http://www.medworm.com/index.php?rid=5292877&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F16%2F9%2F1059%3Frss%3D1 In this study, the authors show how machine learning–based classification of individual cells outperforms those classical ratio-based techniques. Using fluorescent intensity and morphological and texture features, they evaluated how the performance of data analysis increases with increasing feature numbers. Their findings are based on a case study involving an siRNA screen monitoring nucleoplasmic and nucleolar accumulation of a fluorescently tagged reporter protein. For the analysis, they developed a complete analysis workflow incorporating image segmentation, feature extraction, cell classification, hit detection, and visualization of the results. For the classification task, the authors have established a new graphical framework, the Advanced Cell Classifier, which provides a very... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=5292877 Tue, 04 Oct 2011 04:00:00 +0100 5292877 http://www.medworm.com/index.php?rid=5292876&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F16%2F9%2F1047%3Frss%3D1 An anticough medicine, noscapine [(S)-3-((R)4-methoxy-6-methyl-5,6,7,8-tetrahydro-[1,3]dioxolo[4,5-g]isoquinolin-5-yl)-6,7-dimethoxyiso-benzofuran-1(3H)-one], was discovered in the authors’ laboratory as a novel type of tubulin-binding agent that mitigates polymerization dynamics of microtubule polymers without changing overall subunit-polymer equilibrium. To obtain systematic insight into the relationship between the structural framework of noscapine scaffold and its antitumor activity, the authors synthesized strategic derivatives (including two new ones in this article). The IC50 values of these analogs vary from 1.2 to 56.0 µM in human acute lymphoblastic leukemia cells (CEM). Geometrical optimization was performed using semiempirical quantum chemical calculations at the 3-... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=5292876 Tue, 04 Oct 2011 04:00:00 +0100 5292876 http://www.medworm.com/index.php?rid=5292875&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F16%2F9%2F1037%3Frss%3D1 In the present investigation, the authors have performed an in silico–based analysis on a series of arylthiophene derivatives for the determination of their structural features responsible for farnesyltransferase (FTase) inhibitory activity, hERG blocking activity, and toxicity by quantitative structure–activity relationship and pharmacophore analysis techniques. The statistically significant models derived through multiple linear regression analysis were validated by different validation methods. The applicability of the descriptors contributed in the selected models show that the polar and polarizable properties on the van der Waals (vdW) surface area of the molecules are important for the FTase inhibitory and hERG blocking activities, while being detrimental for the toxicity... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=5292875 Tue, 04 Oct 2011 04:00:00 +0100 5292875 http://www.medworm.com/index.php?rid=5292874&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F16%2F9%2F1027%3Frss%3D1 Identification of tick-protective antigens remains the limiting step in vaccine development. The authors have generated several B cell epitope candidates by fingerprinting Rhipicephalus (Boophilus) microplus proteins that were characterized through bioselection of random peptide phage display libraries against polyclonal antibodies antitick proteins. From 280 clones selected and sequenced, 107 distinct reactive clones were validated by dot-blot assays. Eight consensus motifs were generated, and the most frequent ones were PxxKxH, NxxKxxL, and HTS (68.2%, 65%, and 42%, respectively). The consensus sequences identified potential vaccine targets by alignment with the protein database of R. microplus, which may have putative roles in the host response. Sequences that did not align with known p... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=5292874 Tue, 04 Oct 2011 04:00:00 +0100 5292874 http://www.medworm.com/index.php?rid=5292873&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F16%2F9%2F1018%3Frss%3D1 Development and progression of colon cancer may be related to cytokines. Cytokines with diagnostic value have been identified individually but have not been implemented into clinical praxis. Using a multiplex protein array, the authors explore a panel of cytokines simultaneously and compared its performance to carcinoembryonic antigen (CEA) and carbohydrate antigen 19-9 (CA 19-9). Serum concentrations of 12 cytokines were simultaneously determined by multiplex biochip technology in 50 colon cancer patients and 50 healthy controls. Serum levels of interleukin-8 (IL-8) and CEA were significantly higher in cancer patients than in healthy controls. Areas under the receiver operating characteristic curves (AUCs) were largest for IL-8, followed by CEA, vascular endothelial growth factor (VEGF), ... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=5292873 Tue, 04 Oct 2011 04:00:00 +0100 5292873 http://www.medworm.com/index.php?rid=5292872&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F16%2F9%2F1007%3Frss%3D1 Over the past years, improvements in high-throughput screening (HTS) technology and compound libraries have resulted in a dramatic increase in the amounts of good-quality screening hits, and there is a growing need for follow-on hit profiling assays with medium throughput to further triage hits. Here the authors present such assays for the colony-stimulating factor 1 receptor (CSF1R, Fms), including tests for cellular activity and a homogeneous assay to measure affinity for inactive CSF1R. They also present a high-throughput assay to measure target residence time, which is based on competitive binding kinetics. To better fit koff rates, they present a modified mathematical model for competitive kinetics. In all assays, they profiled eight reference inhibitors (imatinib, sorafenib, sunitini... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=5292872 Tue, 04 Oct 2011 04:00:00 +0100 5292872 http://www.medworm.com/index.php?rid=5292871&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F16%2F9%2F995%3Frss%3D1 Misregulation of the Wnt pathway has been shown to be responsible for a variety of human diseases, most notably cancers. Screens for inhibitors of this pathway have been performed almost exclusively using cultured mammalian cells or with purified proteins. We have previously developed a biochemical assay using Xenopus egg extracts to recapitulate key cytoplasmic events in the Wnt pathway. Using this biochemical system, we show that a recombinant form of the Wnt coreceptor, LRP6, regulates the stability of two key components of the Wnt pathway (β-catenin and Axin) in opposing fashion. We have now fused β-catenin and Axin to firefly and Renilla luciferase, respectively, and demonstrate that the fusion proteins behave similarly as their wild-type counterparts. Using this dual lucife... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=5292871 Tue, 04 Oct 2011 04:00:00 +0100 5292871 http://www.medworm.com/index.php?rid=5292870&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F16%2F9%2F986%3Frss%3D1 This study also suggests that furanones are potentially important QS inhibitors for many LuxR-type activator proteins. (Source: Journal of Biomolecular Screening) Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=5292870 Tue, 04 Oct 2011 04:00:00 +0100 5292870 http://www.medworm.com/index.php?rid=5292869&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F16%2F9%2F974%3Frss%3D1 The molecular pathology of many protein misfolding, toxic gain-of-function diseases, such as amyotrophic lateral sclerosis (ALS), is not well understood. Although protein misfolding and aggregation are common themes in these diseases, efforts to identify cellular factors that regulate this process in an unbiased fashion and on a global scale have been lacking. Using an adapted version of an extant β-gal-based protein solubility assay, an expression screen for cellular modulators of solubility of an ALS-causing mutant SOD1 was carried out in mammalian cells. Following fluorescence-activated cell sorting enrichment of a mouse spinal cord cDNA library for gene products that increased SOD1 solubility, high-throughput screening of the cDNA pools from this enriched fraction was employed to ... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=5292869 Tue, 04 Oct 2011 04:00:00 +0100 5292869 http://www.medworm.com/index.php?rid=5292868&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F16%2F9%2F967%3Frss%3D1 This report describes the implementation of an automated work cell with commercially available hardware and software, capable of handling up to 15 separate reagents for performing 96-well or 384-well assays but with a small footprint and only a single liquid dispenser and two plate washers. Extremely flexible software was used to enable this simple work cell to perform processes that would traditionally require a much larger, more expensive automation platform. With the development of the C-Myc assays for the targets DYRK, BMX, PERK, and FAK, the authors describe a software solution to multibatch assays to run simultaneously, reducing reagent dead volume and increasing the efficiency of running multiple assays such that the time to generate data across multiple targets was significantly sh... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=5292868 Tue, 04 Oct 2011 04:00:00 +0100 5292868 http://www.medworm.com/index.php?rid=5292867&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F16%2F9%2F959%3Frss%3D1 This study investigated the use of large-scale transiently transfected cryopreserved cells for medium-throughput cellular screening. The data generated indicated that preprepared transiently transfected cryobanks can be used for cell-based assays and in fact can greatly enhance the consistency of data generated by cellular screens. In addition to this, a generic enzyme-linked immunosorbent assay method was designed that introduced a c-Myc tag to four different targets and allowed all four cell assays to be run using a standardized process. These process improvements yielded cost savings and greatly reduced the required resource, as well as reducing timelines for developing cellular assays. (Source: Journal of Biomolecular Screening) Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=5292867 Tue, 04 Oct 2011 04:00:00 +0100 5292867 http://www.medworm.com/index.php?rid=5292866&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F16%2F9%2F945%3Frss%3D1 Recent genome-wide RNAi screens have identified >842 human genes that affect the human immunodeficiency virus (HIV) cycle. The list of genes implicated in infection differs between screens, and there is minimal overlap. A reason for this variance is the interdependence of HIV infection and host cell function, producing a multitude of indirect or pleiotropic cellular effects affecting the viral infection during RNAi screening. To overcome this, the authors devised a 15-dimensional phenotypic profile to define the viral infection block induced by CD4 silencing in HeLa cells. They demonstrate that this phenotypic profile excludes nonspecific, RNAi-based side effects and viral replication defects mediated by silencing of housekeeping genes. To achieve statistical robustness, the authors use... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=5292866 Tue, 04 Oct 2011 04:00:00 +0100 5292866 http://www.medworm.com/index.php?rid=5204120&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Freprint%2F16%2F8%2F940%3Frss%3D1 (Source: Journal of Biomolecular Screening) Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=5204120 Fri, 09 Sep 2011 04:00:00 +0100 5204120 http://www.medworm.com/index.php?rid=5204119&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F16%2F8%2F932%3Frss%3D1 Genomic approaches provide enormous amounts of raw data with regard to genetic variation, the diversity of RNA species, and protein complement. High-throughput (HT) and high-content (HC) cellular screens are ideally suited to contextualize the information gathered from other "omic" approaches into networks and can be used for the identification of therapeutic targets. Current methods used for HT–HC screens are laborious, time-consuming, and prone to human error. The authors thus developed an automated high-throughput system with an integrated fluorescent imager for HC screens called the AI.CELLHOST. The implementation of user-defined culturing and assay plate setup parameters allows parallel operation of multiple screens in diverse mammalian cell types. The authors demonstrate that s... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=5204119 Fri, 09 Sep 2011 04:00:00 +0100 5204119 http://www.medworm.com/index.php?rid=5204118&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F16%2F8%2F925%3Frss%3D1 Aurora A kinase is a key regulator of mitosis, which is upregulated in several human cancers, making it a potential target for anticancer therapeutics. Consequently, robust medium- to high-throughput cell-based assays to measure Aurora A kinase activity are critical for the development of small-molecule inhibitors. Here the authors compare measurement of the phosphorylation of two Aurora A substrates previously used in high-content screening Aurora A assays, Aurora A itself and TACC3, with a novel substrate Lats2. Using antibodies directed against phosphorylated forms of Aurora A (pThr288), P-TACC3 (pSer558), and P-Lats2 (pSer83), the authors investigate their suitability in parallel for development of a cell-based assay using several reference Aurora inhibitors: MLN8054, VX680, and AZD115... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=5204118 Fri, 09 Sep 2011 04:00:00 +0100 5204118 http://www.medworm.com/index.php?rid=5204117&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F16%2F8%2F917%3Frss%3D1 This study describes the evaluation, validation, and use of contactless postcolumn fractionation of bioactive mixtures with acetylcholine binding protein (AChBP) affinity analysis with help of a spotter technology. The high-resolution fractionation tailors the fractionation frequency to the chromatographic peaks. Postcolumn reagents for AChBP bioaffinity profiling are mixed prior to droplet ejection into 1536-well plates. After an incubation step, microplate reader analysis is used to determine bioactive compounds in a mixture. For ligands tested, a good correlation was found for IC50s determined in flow injection analysis mode when compared with traditional radioligand binding assays. After the evaluation and validation, bioaffinity profiling of actual mixtures was performed. The advantag... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=5204117 Fri, 09 Sep 2011 04:00:00 +0100 5204117 http://www.medworm.com/index.php?rid=5204116&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F16%2F8%2F910%3Frss%3D1 Cardiovascular side effects are critical in drug development and have frequently led to late-stage project terminations or even drug withdrawal from the market. Physiologically relevant and predictive assays for cardiotoxicity are hence strongly demanded by the pharmaceutical industry. To identify a potential impact of test compounds on ventricular repolarization, typically a variety of ion channels in diverse heterologously expressing cells have to be investigated. Similar to primary cells, in vitro–generated stem cell–derived cardiomyocytes simultaneously express cardiac ion channels. Thus, they more accurately represent the native situation compared with cell lines overexpressing only a single type of ion channel. The aim of this study was to determine if stem cell–der... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=5204116 Fri, 09 Sep 2011 04:00:00 +0100 5204116 http://www.medworm.com/index.php?rid=5204115&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F16%2F8%2F903%3Frss%3D1 The authors report here higher throughput screening (HTS) assays for the evaluation of CYP3A4 inhibition and CYP3A4 induction in human hepatocytes using a novel CYP3A4 substrate, luciferin IPA (LIPA). Using human recombinant CYP450 isoforms, LIPA was found to be metabolized extensively by CYP3A4 but not by CYP1A2, CYP2C9, CYP2C19, CYP2D6, or CYP2E1. In the 384-well plate CYP3A4 inhibition assay, the known inhibitors 1-aminobenzotriazole, erythromycin, ketoconazole, and verapamil were found to cause extensive (maximum inhibition of >80%), dose-dependent, statistically significant inhibition of LIPA metabolism. The non-CYP3A4 inhibitors diethyldithiocarbamate, quercetin, quinidine, sulfaphenazole, ticlopidine, and tranylcypromine were found to have substantially lower (maximum inhibition ... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=5204115 Fri, 09 Sep 2011 04:00:00 +0100 5204115 http://www.medworm.com/index.php?rid=5204114&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F16%2F8%2F895%3Frss%3D1 Cytochrome P450 (CYP) enzymes are key players in drug metabolism. Therefore, it is essential to understand how these enzymes can be affected by xenobiotics with regards to induction and toxicity to avoid potential drug–drug interactions. Typically, information has been gathered by combining data from multiple experiments, which is time-consuming and labor intensive, and interassay variability may lead to misinterpretation. Monitoring CYP induction and cytotoxicity by xenobiotics using an automated, multiplexed format can decrease workload and increase data confidence. Here the authors demonstrate the ability to monitor CYP1A and CYP3A4 induction, combined with a cytotoxicity measurement, from a single microplate well using cryopreserved human hepatocytes. The assay procedure was auto... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=5204114 Fri, 09 Sep 2011 04:00:00 +0100 5204114 http://www.medworm.com/index.php?rid=5204113&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F16%2F8%2F886%3Frss%3D1 This study provides evidence that quinidine can be used as a probe substrate for ABCB1 in multiple experimental systems both in vitro and in vivo relevant to the blood–brain barrier (BBB). The combination of quinidine and PSC-833 (valspodar) is an effective tool to assess investigational drugs for interactions on ABCB1. Effects of quinidine and substrate–inhibitor interactions were tested in a membrane assay and in monolayer assays. The authors compared quinidine and digoxin as ABCB1 probes in the in vitro assays and found that quinidine was more potent and at least as specific as digoxin in ATPase and monolayer efflux assays employing MDCKII-MDR1 and the rat brain microcapillary endothelial cell system. Brain exposure to quinidine was tested in dual-/triple-probe microdialysis... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=5204113 Fri, 09 Sep 2011 04:00:00 +0100 5204113 http://www.medworm.com/index.php?rid=5204112&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F16%2F8%2F878%3Frss%3D1 The authors describe a structure-based strategy to identify therapeutically beneficial off-target effects by screening a chemical library of Food and Drug Administration (FDA)–approved small-molecule drugs matching pharmacophores defined for specific target proteins. They applied this strategy to angiotensin-converting enzyme 2 (ACE2), an enzyme that generates vasodilatory peptides and promotes protection from hypertension-associated cardiovascular disease. The conformation-based structural selection method by molecular docking using DOCK allowed them to identify a series of FDA-approved drugs that enhance catalytic efficiency of ACE2 in vitro. These data demonstrate that libraries of approved drugs can be rapidly screened to identify potential side effects due to interactions with s... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=5204112 Fri, 09 Sep 2011 04:00:00 +0100 5204112 http://www.medworm.com/index.php?rid=5204111&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F16%2F8%2F869%3Frss%3D1 This report describes the first high-throughput screen for RGS17 inhibitors, as well as a novel paradigm adaptable to many other RGS proteins, which are emerging as attractive drug targets for modulating G-protein-coupled receptor signaling. (Source: Journal of Biomolecular Screening) Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=5204111 Fri, 09 Sep 2011 04:00:00 +0100 5204111 http://www.medworm.com/index.php?rid=5204110&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F16%2F8%2F862%3Frss%3D1 Parasitic infections caused by Entamoeba histolytica are still major threats against public health, especially in developing countries. Although current therapies exist, the problems associated with parasite resistance and negative side effects make it imperative to search for new therapeutic agents. A systematic scaffold analysis reported herein of a public database containing 474 antiamoebic compounds reveals that benzimidazole is the most active scaffold reported thus far. To gain insights into the antiamoebic activity of novel compounds, the authors report herein the biological activity of 12 compounds, including benzotriazole and indazole derivatives, scaffolds not previously tested against E. histolytica. Compounds with the benzotriazole and indazole scaffolds showed low micromolar a... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=5204110 Fri, 09 Sep 2011 04:00:00 +0100 5204110 http://www.medworm.com/index.php?rid=5204109&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F16%2F8%2F852%3Frss%3D1 There are no effective antivirals currently available for the treatment of flavivirus infection in humans. As such, the identification and characterization of novel drug target sites are critical to developing new classes of antiviral drugs. The flavivirus NS5 N-terminal capping enzyme (CE) is vital for the formation of the viral RNA cap structure, which directs viral polyprotein translation and stabilizes the 5' end of the viral genome. The structure of the flavivirus CE has been solved, and a detailed understanding of the CE–guanosine triphosphate (GTP) and CE–RNA cap interactions is available. Because of the essential nature of the interaction for viral replication, disrupting CE–GTP binding is an attractive approach for drug development. The authors have previously de... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=5204109 Fri, 09 Sep 2011 04:00:00 +0100 5204109 http://www.medworm.com/index.php?rid=5204108&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F16%2F8%2F845%3Frss%3D1 In this article, the authors describe a colorimetric, high-throughput assay suitable for optimizing the activity of the recently discovered sulfotransferase LipB, by directed evolution. Crucially, LipB uses para-nitrophenol sulfate as donor in the sulfation of the nucleoside antibiotic liposidomycin B-I and other acceptor surrogates. Thus, using a robotic liquid-handling device, crude cell extracts were prepared from an Escherichia coli strain that overproduced LipB in wells of a microplate, and production of para-nitrophenol at 405 nm was monitored spectrophotometrically. Enzyme activity could be detected only in the presence of both LipB substrates and overexpressed LipB. The screen displays a suitable standard deviation for directed evolution and importantly is not limited to the natura... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=5204108 Fri, 09 Sep 2011 04:00:00 +0100 5204108 http://www.medworm.com/index.php?rid=5204107&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F16%2F8%2F836%3Frss%3D1 Tetrahydrobiopterin (BH4) is an essential cofactor for the nitric oxide (NO) synthases and the aromatic amino acid hydroxylases. Insufficient BH4 has been implicated in various cardiovascular and neurological disorders. GTP cyclohydrolase 1 (GTPCH-1) is the rate-limiting enzyme for de novo biosynthesis of BH4. The authors have recently shown that the interaction of GTPCH-1 with GTP cyclohydrolase feedback regulatory protein (GFRP) inhibits endothelial GTPCH-1 enzyme activity, BH4 levels, and NO production. They propose that agents that disrupt the GTPCH-1/GFRP interaction can increase cellular GTPCH-1 activity, BH4 levels, and NO production. They developed and optimized a novel time-resolved fluorescence resonance energy transfer (TR-FRET) assay to monitor the interaction of GTPCH-1 and GF... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=5204107 Fri, 09 Sep 2011 04:00:00 +0100 5204107 http://www.medworm.com/index.php?rid=5204106&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F16%2F8%2F825%3Frss%3D1 Despite advances toward understanding the prevention and treatment of many cancers, patients who suffer from oral squamous cell carcinoma (OSCC) confront a survival rate that has remained unimproved for more than 2 decades, indicating our ability to treat them pharmacologically has reached a plateau. In an ongoing effort to improve the clinical outlook for this disease, we previously reported that an essential component of the mechanism by which the proteasome inhibitor bortezomib (PS-341, Velcade) induced apoptosis in OSCC required the activation of a terminal unfolded protein response (UPR). Predicated on these studies, the authors hypothesized that high-throughput screening (HTS) of large diverse chemical libraries might identify more potent or selective small-molecule activators of the... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=5204106 Fri, 09 Sep 2011 04:00:00 +0100 5204106 http://www.medworm.com/index.php?rid=5204105&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F16%2F8%2F818%3Frss%3D1 The identification of compounds that specifically inhibit or kill cancer cells without affecting cells from healthy tissues is very challenging but very important for reducing the side effects of current cancer therapies. Hence, there is an urgent need for improved assays allowing the selectivity of a given compound to be monitored directly. The authors present an assay system based on the competitive co-cultivation of an excess of cancer cells with a small fraction of noncancer human indicator cells generating a fluorescence signal. In the absence of a specific anticancer compound, the cancer cells outgrow the indicator cells and abolish the fluorescence signal. In contrast, the presence of specific anticancer drugs (such as Tyrphostin-AG1478 or PLX4720) results in the selective growth of... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=5204105 Fri, 09 Sep 2011 04:00:00 +0100 5204105 http://www.medworm.com/index.php?rid=5204104&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F16%2F8%2F805%3Frss%3D1 Glioblastoma multiforme (GBM) is the most common and most aggressive type of primary brain tumor. Identification of new therapeutic regimens is urgently needed. A major challenge remains the development of a relevant in vitro model system with the necessary capacity and flexibility to profile compounds. The authors have developed and characterized a 3D culture system of brain cells (brain Hi-Spot) where GBM-derived cells can be incorporated (GBM/brain Hi-Spot). Immuno-fluorescence and electrophysiological recordings demonstrate that brain Hi-Spots recapitulate many features of brain tissue. Within this tissue, GBM-derived cell growth is monitored using a fluorescence assay. GBM-derived cells growing in Hi-Spots form tumor nodules that display properties of GBM such as 5-Ala positive staini... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=5204104 Fri, 09 Sep 2011 04:00:00 +0100 5204104 http://www.medworm.com/index.php?rid=5068560&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Freprint%2F16%2F7%2F800%3Frss%3D1 (Source: Journal of Biomolecular Screening) Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=5068560 Sun, 24 Jul 2011 23:00:00 +0100 5068560 http://www.medworm.com/index.php?rid=5068559&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Freprint%2F16%2F7%2F798%3Frss%3D1 (Source: Journal of Biomolecular Screening) Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=5068559 Sun, 24 Jul 2011 23:00:00 +0100 5068559 http://www.medworm.com/index.php?rid=5068558&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Freprint%2F16%2F7%2F794%3Frss%3D1 (Source: Journal of Biomolecular Screening) Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=5068558 Sun, 24 Jul 2011 23:00:00 +0100 5068558 http://www.medworm.com/index.php?rid=5068557&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F16%2F7%2F786%3Frss%3D1 This report describes an extension to standard luciferase reporter gene assays that enables multiple parameters to be monitored from each sample. The report describes multiplexing luciferase assays with an orthogonal readout monitoring cell viability using reduction of resazurin. In addition, this technical note shows that by using the luciferin substrate in live cells, an assay time course can be recorded. This enables the identification of nonactive or unspecific compounds that act by inhibiting luciferase, as well as compounds altering gene expression or cell growth. (Source: Journal of Biomolecular Screening) Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=5068557 Sun, 24 Jul 2011 23:00:00 +0100 5068557 http://www.medworm.com/index.php?rid=5068556&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F16%2F7%2F775%3Frss%3D1 Hit selection is the ultimate goal in many high-throughput screens. Various analytic methods are available for this purpose. Some commonly used ones are z score, z* score, strictly standardized mean difference (SSMD), SSMD*, and t statistic. It is critical to know how to use them correctly because the misusage of them can readily produce misleading results. Here, the author presents basic concepts, elaborates their commonality and difference, describes some common misusage that people should avoid, and uses simulated simple examples to illustrate how to use them correctly. (Source: Journal of Biomolecular Screening) Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=5068556 Sun, 24 Jul 2011 23:00:00 +0100 5068556 http://www.medworm.com/index.php?rid=5068555&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F16%2F7%2F765%3Frss%3D1 Frequent hitters are compounds that are detected as a "hit" in multiple high-throughput screening (HTS) assays. Such behavior is specific (e.g., target family related) or unspecific (e.g., reactive compounds) or can result from a combination of such behaviors. Detecting such hits while predicting the underlying reason behind their promiscuous behavior is desirable because it provides valuable information not only about the compounds themselves but also about the assay methodology and target classes at hand. This information can also greatly reduce cost and time during HTS hit profiling. The present study exemplifies how to mine large HTS data repositories, such as the one at Boehringer Ingelheim, to identify frequent hitters, gain further insights into the causes of promiscuous behavior, a... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=5068555 Sun, 24 Jul 2011 23:00:00 +0100 5068555 http://www.medworm.com/index.php?rid=5068554&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F16%2F7%2F755%3Frss%3D1 Many assays aimed to test the inhibitory effects of synthetic molecules, and naturally occurring products on the neuraminidase activity exploit the hydrolysis of 2'-O-(4-methylumbelliferyl)-N-acetylneuraminic acid (4-MUNANA). The amount of the released product, 4-methylumbelliferone (4-MU), is then measured fluorimetrically. The authors attempted an analysis of the inhibitory properties of 35 naturally occurring flavonoids on neuraminidase N3, where only 29 of them were sufficiently soluble in the assay medium. During the analysis, the authors noticed a strong quenching effect due to the test compounds on the fluorescence of 4-MU. The quenching constants for the flavonoids were determined according to the Stern-Volmer approach. The extent of fluorescence reduction due to quenching and the ... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=5068554 Sun, 24 Jul 2011 23:00:00 +0100 5068554 http://www.medworm.com/index.php?rid=5068553&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F16%2F7%2F744%3Frss%3D1 This study aims at generating immune chicken phage display libraries and single-chain antibodies (scFvs) specifically directed against cell surface markers of cultured peripheral blood mononuclear cells (PBMCs) that contain endothelial progenitor cells (EPCs). In contrast to previous approaches that use well-defined recombinant antigens attached to plastic surfaces that may alter the structure of the proteins, the authors describe a method that maintains the cell surface markers on live cells while providing the opportunity to rapidly screen entire libraries for antibodies that bind to unknown cell surface markers of progenitor/stem cells. Chickens immunized with live EPCs, consisting of a heterogeneous population of lymphocytes and monocytes, demonstrated a robust immune response. After t... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=5068553 Sun, 24 Jul 2011 23:00:00 +0100 5068553 http://www.medworm.com/index.php?rid=5068552&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F16%2F7%2F734%3Frss%3D1 Nicotinamide nucleotide transhydrogenase (NNT) mutant mice show glucose intolerance with impaired insulin secretion during glucose tolerance tests. Uncoupling of the β cell mitochondrial metabolism due to such mutations makes NNT a novel target for therapeutics in the treatment of pathologies such as type 2 diabetes. The authors propose that increasing NNT activity would help reduce deleterious buildup of reactive oxygen species in the inner mitochondrial matrix. They have expressed human Nnt cDNA for the first time in Saccharomyces cerevisiae, and transhydrogenase activity in mitochondria isolated from these cells is six times greater than is seen in wild-type mitochondria. The same mitochondria have partially uncoupled respiration, and the cells have slower growth rates compared to ... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=5068552 Sun, 24 Jul 2011 23:00:00 +0100 5068552 http://www.medworm.com/index.php?rid=5068551&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F16%2F7%2F724%3Frss%3D1 Transforming growth factor β (TGF-β) type I receptor (activin receptor–like kinase 5, ALK5) has been identified as a promising target for fibrotic diseases. To find a novel inhibitor of ALK5, the authors performed a high-throughput screen of a library of 420 000 compounds using dephosphorylated ALK5. From primary hits of 1521 compounds, 555 compounds were confirmed. In total, 124 compounds were then selected for follow-up based on their unique structures and other properties. Repeated concentration–response testing and final interference assays of the above compounds resulted in the discovery of a structurally novel ALK5 inhibitor (compound 8) (N-(thiophen 2-ylmethyl)-3-(3,4,5 trimethoxyphenyl)imidazo[1,2β]pyridazin 6-amine) with a low IC50 value of 0.7 µM.... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=5068551 Sun, 24 Jul 2011 23:00:00 +0100 5068551 http://www.medworm.com/index.php?rid=5068550&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F16%2F7%2F717%3Frss%3D1 GTPase-activating proteins of ADP-ribosylation factors (ARFGAPs) play key cellular roles in vesicle production and trafficking, adhesion, migration, and development. Dysfunctional regulation of ARFGAPs has been implicated in various diseases, including cancer, Alzheimer disease, and autism. Unfortunately, there are few mechanistic details describing how ARFGAPs contribute to disease states. In this regard, it would be extremely helpful to have a set of small molecules that selectively and directly modulate specific ARFGAPs as probes to dissect ARFGAP-regulated cell signaling under various conditions. Currently, such probes are lacking, and their identification is hampered by the lack of a suitable high-throughput assay to monitor ARFGAP activity. Here, the authors describe and validate a r... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=5068550 Sun, 24 Jul 2011 23:00:00 +0100 5068550 http://www.medworm.com/index.php?rid=5068549&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F16%2F7%2F706%3Frss%3D1 In conclusion, the β-arrestin recruitment assay does not fit with traditional pharmacological theory but is of great utility as the EC50 value generated is a good approximation of affinity. (Source: Journal of Biomolecular Screening) Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=5068549 Sun, 24 Jul 2011 23:00:00 +0100 5068549 http://www.medworm.com/index.php?rid=5068548&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F16%2F7%2F694%3Frss%3D1 The type II secretion (T2S) system in gram-negative bacteria comprises the Sec and Tat pathways for translocating proteins into the periplasm and an outer membrane secretin for transporting proteins into the extracellular space. To discover Sec/Tat/T2S pathway inhibitors as potential new therapeutics, the authors used a Pseudomonas aeruginosa bioluminescent reporter strain responsive to SecA depletion and inhibition to screen compound libraries and characterize the hits. The reporter strain placed a luxCDABE operon under regulation of a SecA depletion-responsive upregulated promoter in a secA deletion background complemented with an ectopic lac-regulated secA copy. Bioluminescence was indirectly proportional to the isopropyl-β-D-thiogalactopyranoside concentration and stimulated by az... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=5068548 Sun, 24 Jul 2011 23:00:00 +0100 5068548 http://www.medworm.com/index.php?rid=5068547&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F16%2F7%2F683%3Frss%3D1 The monocyte chemoattractant protein 1 (MCP-1)–driven activation of CC-type chemokine receptor 2 (CCR2) is one of the early key events to induce monocyte migration toward centers of inflammation. In this work, the authors analyzed MCP-1 internalization into primary human monocytes using partially automated liquid handling, automated fluorescence microscopic imaging, and a specific image analysis algorithm. A fluorophore-conjugated form of MCP-1 was rapidly endocytosed and retained by the monocytes. The CCR2 dependency of the MCP-1 internalization was demonstrated by the use of BMS CCR2 22, a CCR2-specific antagonist. The apparent inhibitory potencies of a series of small-molecule CCR2 antagonists were determined and compared in five assay formats, including the high-content analysis ... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=5068547 Sun, 24 Jul 2011 23:00:00 +0100 5068547 http://www.medworm.com/index.php?rid=5026808&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Freprint%2F16%2F6%2F676%3Frss%3D1 (Source: Journal of Biomolecular Screening) Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=5026808 Wed, 06 Jul 2011 23:00:00 +0100 5026808 http://www.medworm.com/index.php?rid=5026807&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F16%2F6%2F668%3Frss%3D1 Infection of certain cell types by HIV results in formation of syncytia. This process can be blocked by antibodies or compounds that prevent interaction of viral envelope protein with host cell receptors. Here the authors describe an automated imaging-based assay for inhibitors of cell-cell fusion mediated by interaction of HIV gp120 with CXCR4 coreceptor. The assay quantifies syncytia formation between U87MG astrocytoma cells constitutively expressing CD4/CXCR4 and morphologically distinct Jurkat T lymphoma cells inducibly expressing HIV env. Each cell type was differentially labeled with vital dyes. Fusion was quantified by measuring size, shape, and color of Jurkat cells and Jurkat-harboring cell syncytia. Dose–response experiments with reference inhibitors AMD 3100 and KRH-1636 y... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=5026807 Wed, 06 Jul 2011 23:00:00 +0100 5026807 http://www.medworm.com/index.php?rid=5026806&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F16%2F6%2F655%3Frss%3D1 In this study, the authors investigated gene expression changes in rat small intestine in response to heat stress. Male Sprague-Dawley rats were randomly divided into control and heat-stressed groups. Both groups were housed at 25 °C, although the heat-stressed group was also subjected to 40 °C for 2 h each day for 10 successive days. Rats were sacrificed 1, 3, 6, and 10 days after heat treatment, and sections of their small intestine epithelial tissue were excised for morphological examination and microarray analyses. The rat rectal and body surface temperatures and serum cortisol levels were all significantly increased after heat treatment (p < 0.05). The jejuna were significantly damaged by 3 days after heat treatment began. Microarray analysis showed that 422 genes were diff... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=5026806 Wed, 06 Jul 2011 23:00:00 +0100 5026806 http://www.medworm.com/index.php?rid=5026805&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F16%2F6%2F647%3Frss%3D1 This study includes performance results of a side-by-side comparison of the EpMotion 5070 LHS and conventional pipetting/dispensing systems. Critical parameters were optimized each for a given platform. Higher accuracy and reproducibility were achieved for LHS compared to manually treated samples. The practicability and accuracy of the method in a typical small laboratory setting were tested by running daily routine tasks by trained and untrained laboratory staff. In addition, advantages and disadvantages as well as the step-by-step application protocol are reported. The approach described provides a potential utility in screening biomaterials toxicity, allowing researchers to meet the needs of low- and medium-throughput laboratories. (Source: Journal of Biomolecular Screening) Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=5026805 Wed, 06 Jul 2011 23:00:00 +0100 5026805 http://www.medworm.com/index.php?rid=5026804&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F16%2F6%2F637%3Frss%3D1 A simple, optical density-based assay for inhibitors of the mevalonate-dependent pathway for isoprenoid biosynthesis was developed. The assay uses pathway-sensitized Staphylococcus aureus strains and is fully compatible with high-density screening in a 1536-well format. S. aureus strains were constructed in which genes required for mevalonate-dependent isopentenyl pyrophosphate (IPP) synthesis were regulated by an isopropyl-β-D-thiogalactopyranoside (IPTG)–inducible promoter. Inhibitors of the target enzymes displayed greater antibacterial potency in media containing low concentrations of IPTG, and therefore less induction of mevalonate pathway genes, than in media with high IPTG conditions. This differential growth phenotype was exploited to bias the cell-based screening hits t... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=5026804 Wed, 06 Jul 2011 23:00:00 +0100 5026804 http://www.medworm.com/index.php?rid=5026803&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F16%2F6%2F628%3Frss%3D1 Adiponectin is an adipokine secreted by adipocytes and plays a role in the suppression of metabolic disorders that can result in type 2 diabetes, obesity, and atherosclerosis. Several studies have shown that upregulation of adiponectin has a number of therapeutic benefits. Although peroxisome proliferator-activated receptor (PPAR) agonists are known to increase adiponectin secretion both in cultured adipocytes and humans, they have several side effects, such as weight gain, congestive heart failure, and edema. Therefore, adiponectin secretion modulators that do not possess PPAR agonistic activity seem to promising for a number of conditions. Here, the authors report on the development of a reporter-based high-throughput screening (HTS) assay using insulin-resistant-mimic 3T3-L1 adipocytes ... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=5026803 Wed, 06 Jul 2011 23:00:00 +0100 5026803 http://www.medworm.com/index.php?rid=5026802&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F16%2F6%2F618%3Frss%3D1 The thyroid hormone receptors (TR) are members of the nuclear hormone receptor (NHR) superfamily that regulate development, growth, and metabolism. Upon ligand binding, TR releases bound corepressors and recruits coactivators to modulate target gene expression. Steroid receptor coactivator 2 (SRC2) is an important coregulator that interacts with TRβ to activate gene transcription. To identify novel inhibitors of the TRβ and SRC2 interaction, the authors performed a quantitative high-throughput screen (qHTS) of a TRβ-SRC2 fluorescence polarization assay against more than 290 000 small molecules. The qHTS assayed compounds at 6 concentrations up to 92 µM to generate titration–response curves and determine the potency and efficacy of all compounds. The qHTS data set... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=5026802 Wed, 06 Jul 2011 23:00:00 +0100 5026802 http://www.medworm.com/index.php?rid=5026801&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F16%2F6%2F603%3Frss%3D1 Disentangling the complex interactions that govern stem cell fate choices of self-renewal, differentiation, or death presents a formidable challenge. Image-based phenotype-driven screening meets this challenge by providing means for rapid testing of many small molecules simultaneously. Pluripotent embryonal carcinoma (EC) cells offer a convenient substitute for embryonic stem (ES) cells in such screens because they are simpler to maintain and control. The authors developed an image-based screening assay to identify compounds that affect survival or differentiation of the human EC stem cell line NTERA2 by measuring the effect on cell number and the proportion of cells expressing a pluripotency-associated marker SSEA3. A pilot screen of 80 kinase inhibitors identified several compounds that ... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=5026801 Wed, 06 Jul 2011 23:00:00 +0100 5026801 http://www.medworm.com/index.php?rid=5026800&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F16%2F6%2F588%3Frss%3D1 Phenotypic lead generation strategies seek to identify compounds that modulate complex, physiologically relevant systems, an approach that is complementary to traditional, target-directed strategies. Unlike gene-specific assays, phenotypic assays interrogate multiple molecular targets and signaling pathways in a target "agnostic" fashion, which may reveal novel functions for well-studied proteins and discover new pathways of therapeutic value. Significantly, existing compound libraries may not have sufficient chemical diversity to fully leverage a phenotypic strategy. To address this issue, Eli Lilly and Company launched the Phenotypic Drug Discovery Initiative (PD2), a model of open innovation whereby external research groups can submit compounds for testing in a panel of Lilly phenotypic... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=5026800 Wed, 06 Jul 2011 23:00:00 +0100 5026800 http://www.medworm.com/index.php?rid=5026799&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F16%2F6%2F575%3Frss%3D1 The objective of the study was to further assess the usefulness of a real-time cell analyzer (RTCA) platform based on impedance in the context of quality control and data reproducibility. The data indicate that this technology is useful to determine the best coating and cellular density conditions for different adherent cellular models including hepatocytes, cardiomyocytes, fibroblasts, and hybrid neuroblastoma/neuronal cells. Based on 31 independent experiments, the reproducibility of cell index data generated from HepG2 cells exposed to DMSO and to Triton X-100 was satisfactory, with a coefficient of variation close to 10%. Cell index data were also well reproduced when cardiomyocytes and fibroblasts were exposed to 21 compounds three times (correlation >0.91, p < 0.0001). The data... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=5026799 Wed, 06 Jul 2011 23:00:00 +0100 5026799 http://www.medworm.com/index.php?rid=5026798&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F16%2F6%2F565%3Frss%3D1 Measuring myotube thickness is a physiological and unbiased approach for screening therapeutic compounds that prevent skeletal muscle atrophy or induce hypertrophy. However, an accurate cell thickness estimate is often quite challenging because of the extreme heterogeneity of the myotube cellular population and therefore the lack of a regular distribution of perturbed myotubes. Here the authors present a novel method to evaluate changes in myotube thickness via measuring cellular electrical impedance. They demonstrate that both qualitative and quantitative changes in electrical impedance as a function of cellular adhesion in real time correlate well with variation in myotube thickness caused by atrophy or hypertrophy agents. Conversely, pharmacologically blocking myotube hypertrophy preven... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=5026798 Wed, 06 Jul 2011 23:00:00 +0100 5026798 http://www.medworm.com/index.php?rid=4944224&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F16%2F5%2F560%3Frss%3D1 Bouck DC, Shu P, Cui J, Shelat A, Chen T. A High-Content Screen Identifies Inhibitors of Nuclear Export of Forkhead Transcription Factors. J. Biomol. Screen. 2011, 16, 394-404. (Original 10.1177/1087057110397889) In the April issue of the Journal of Biomolecular Screening, in the above-mentioned article, Figure 1 contained incorrect axis labels; Figure 3a contained an unintended artifact. Corrected figures have been posted online at http://jbx.sagepub.com/content/16/4/394/suppl/DC1. (Source: Journal of Biomolecular Screening) Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=4944224 Wed, 15 Jun 2011 23:00:00 +0100 4944224 http://www.medworm.com/index.php?rid=4944223&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Freprint%2F16%2F5%2F557%3Frss%3D1 (Source: Journal of Biomolecular Screening) Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=4944223 Wed, 15 Jun 2011 23:00:00 +0100 4944223 http://www.medworm.com/index.php?rid=4944222&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F16%2F5%2F552%3Frss%3D1 In this report, the authors show that fluorescence-based thermal shift assays could be used as prescreening methods to identify compounds with different thermodynamic profiles. This approach allows a reduction in the number of compounds to be tested in calorimetry experiments, thus favoring greater integration of thermodynamics in drug discovery. (Source: Journal of Biomolecular Screening) Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=4944222 Wed, 15 Jun 2011 23:00:00 +0100 4944222 http://www.medworm.com/index.php?rid=4944221&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F16%2F5%2F545%3Frss%3D1 The use of a high-throughput technique to perform a pilot screen for Leishmania major protein disulfide isomerase (LmPDI) inhibitors identification is reported. In eukaryotic cells, protein disulfide isomerase (PDI) plays a crucial role in protein folding by catalyzing the rearrangement of disulfide bonds in substrate proteins following their synthesis. LmPDI displays similar domain structure organization and functional properties to other PDI family members and is involved in Leishmania virulence. The authors used a method based on the enzyme-catalyzed reduction of insulin in the presence of dithiothreitol. The screen of a small library of 1920 compounds was performed in a 384-well format and led to the identification of 27 compounds with inhibitory activity against LmPDI. The authors fur... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=4944221 Wed, 15 Jun 2011 23:00:00 +0100 4944221 http://www.medworm.com/index.php?rid=4944220&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F16%2F5%2F536%3Frss%3D1 Traditionally, cell adhesion assays are performed in a manual workstation format using fluorescence-based readouts. Herein, the authors describe a label-free homogeneous assay to identify inhibitors of α4β7 integrin-mediated cell adhesion to its ligand, the mucosal addressin cell adhesion molecule (MadCAM), using the SRU BIND platform. The biosensor is optically based and comprises a subwavelength polymer grating. The assay was validated using standard compounds and an α4 blocking antibody and correlated very closely with the manual assay format when running a battery of test compounds of varying potencies. Cell adhesion was strictly dependent on the presence of divalent cations where Mg2+ was greater than Ca2+ at promoting cell adhesion. This homogeneous and label-free fo... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=4944220 Wed, 15 Jun 2011 23:00:00 +0100 4944220 http://www.medworm.com/index.php?rid=4944219&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F16%2F5%2F525%3Frss%3D1 In this study, the authors used a mammalian two-hybrid system to construct a drug screening model to obtain a small molecular inhibitor capable of interrupting the interaction between Cripto-1 and ActRII. They screened 300 natural components and identified alantolactone. Data suggested that alantolactone induced activin/SMAD3 signaling in human colon adenocarcinoma HCT-8 cells. The authors also found that alantolactone exhibited antiproliferative function specific to tumor cells, with almost no toxicity to normal cells at a concentration of 5 µg/mL. Furthermore, they proved that the antiproliferative function of alantolactone was activin/SMAD3 dependent. These results suggest that alantolactone performs its antitumor effect by interrupting the interaction between Cripto-1 and the act... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=4944219 Wed, 15 Jun 2011 23:00:00 +0100 4944219 http://www.medworm.com/index.php?rid=4944218&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F16%2F5%2F518%3Frss%3D1 The authors have devised a continuous fluorescence-based assay to measure HIV reverse transcriptase (RT) polymerase activity for both high-throughput screening (HTS) and mechanistic characterization of inhibitors. The designed substrate is composed of a recessed DNA primer annealed to a DNA template that is labeled at the 5'-terminus with a donor fluorophore (AlexaFluor 488). RT-catalyzed incorporation of an acceptor-labeled deoxyuridine (dUTP-AlexaFluor 555) at the 3'-terminus of the fully extended DNA primer juxtaposes donor and acceptor fluorophores, resulting in robust fluorescence resonance energy transfer that can be monitored kinetically in real time. The assay is sensitive, permitting the use of low enzyme concentrations (<0.5 nM), and can be miniaturized for use in 384-well HTS... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=4944218 Wed, 15 Jun 2011 23:00:00 +0100 4944218 http://www.medworm.com/index.php?rid=4944217&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F16%2F5%2F506%3Frss%3D1 Stearoyl-CoA desaturase (SCD) catalyzes the synthesis of monounsaturated fatty acids and has been implicated in a number of disease states, including obesity and diabetes. To find small-molecule inhibitor leads, a high-throughput scintillation proximity assay (SPA) was developed using the hydrophobic binding characteristics of a glass microsphere scintillant bead to capture SCD1 from a crude lysate of recombinant SCD1 in Sf9 lysate coupled with the strong binding characteristics of an azetidine compound ([3H]AZE). The SPA assay was stable over 24 h and could detect compounds with micromolar to nanomolar potencies. A robust 1536-well high-throughput screening assay was developed with good signal-to-noise ratio (10:1) and excellent Z' factor (0.8). A screening collection of 1.6 million compo... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=4944217 Wed, 15 Jun 2011 23:00:00 +0100 4944217 http://www.medworm.com/index.php?rid=4944216&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F16%2F5%2F494%3Frss%3D1 Methionine aminopeptidase (MAP) (E.C. 3.4.11.18) is a metallopeptidase that cleaves the N-terminal methionine (Met) residue from some proteins. MAP is essential for growth of several bacterial pathogens, making it a target for antibacterial drug discovery. MAP enzymes are also present in eukaryotic cells, and one is a target for antiangiogenic cancer therapy. To screen large compound libraries for MAP inhibitors as the starting point for drug discovery, a high-throughput-compatible assay is valuable. Here the authors describe a novel assay, which detects the Met product of MAP-catalyzed peptide cleavage by coupling it to adenosine triphosphate (ATP)–dependent production of S-adenosyl-L-methionine (SAM) and inorganic phosphate (Pi) by SAM synthetase (MetK) combined with inorganic pyro... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=4944216 Wed, 15 Jun 2011 23:00:00 +0100 4944216 http://www.medworm.com/index.php?rid=4944215&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F16%2F5%2F486%3Frss%3D1 This article describes a straightforward, fluorescence resonance energy transfer–based DNA ligase assay that is well suited for high-throughput screening for DNA ligase inhibitors as well as for use in enzyme kinetics studies. Its use is demonstrated for measurement of the steady-state kinetic constants of Haemophilus influenzae NAD+-dependent DNA ligase and for measurement of the potency of an inhibitor of this enzyme. (Source: Journal of Biomolecular Screening) Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=4944215 Wed, 15 Jun 2011 23:00:00 +0100 4944215 http://www.medworm.com/index.php?rid=4944214&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F16%2F5%2F476%3Frss%3D1 Protein tyrosine phosphatase– (PTP-) is a receptor-like PTP whose biological function is poorly understood. A recent mouse PTP- genetic deletion model associated the loss of PTP- gene expression with a potential antidepressant phenotype. This led the authors to screen a subset of the Bristol-Myers Squibb (BMS) compound collection to identify selective small-molecule inhibitors of receptor-like PTP- (RPTP-) for use in evaluating enzyme function in vivo. Here, they report the design of a high-throughput fluorescence resonance energy transfer (FRET) assay based on the Z'-LYTE technology to screen for inhibitors of RPTP-. A subset of the BMS diverse compound collection was screened and several compounds identified as RPTP- inhibitors in the assay. After chemical triage and clustering, co... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=4944214 Wed, 15 Jun 2011 23:00:00 +0100 4944214 http://www.medworm.com/index.php?rid=4944213&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F16%2F5%2F467%3Frss%3D1 The fatty acid binding protein 4 (FABP4) belongs to the family of lipid chaperones that control intracellular fluxes and compartmentalization of their respective ligands (e.g., fatty acids). FABP4, which is almost exclusively expressed in adipocytes and macrophages, contributes to the development of insulin resistance and atherosclerosis in mice. Lack of FABP4 protects against the development of insulin resistance associated with genetic or diet-induced obesity in mice. Furthermore, total or macrophage-specific FABP4 deficiency is protective against atherosclerosis in apolipoprotein E–deficient mice. The FABP4 small-molecule inhibitor BMS309403 has demonstrated efficacy in mouse models for type 2 diabetes mellitus and atherosclerosis, resembling phenotypes of mice with FABP4 deficien... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=4944213 Wed, 15 Jun 2011 23:00:00 +0100 4944213 http://www.medworm.com/index.php?rid=4682809&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F16%2F4%2F464%3Frss%3D1 Pang W, Wang RR, Gao YD, Yang LM, Sun Y, Huang JF, Tien P, Zheng YT. A Novel Enzyme-Linked Immunosorbent Assay for Screening HIV-1 Fusion Inhibitors Targeting HIV-1 Gp41 Core Structure. J. Biomol. Screen. 2011, 16, 221–229. In the February issue of the Journal of Biomolecular Screening, in the above-mentioned article on pages 224 and 225, the legends of Figures 1 and 2 appeared in the reverse order. (Source: Journal of Biomolecular Screening) Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=4682809 Tue, 05 Apr 2011 23:00:00 +0100 4682809 http://www.medworm.com/index.php?rid=4682808&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Freprint%2F16%2F4%2F460%3Frss%3D1 (Source: Journal of Biomolecular Screening) Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=4682808 Tue, 05 Apr 2011 23:00:00 +0100 4682808 http://www.medworm.com/index.php?rid=4682807&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F16%2F4%2F457%3Frss%3D1 RecA is a highly conserved bacterial protein that plays crucial roles in many cellular processes and hence is a potential target in the chemotherapy of bacterial infections. An understanding of the functional similarity between RecA proteins from different bacterial species should yield further insights into the biochemistry of RecA protein, along with the potential for new approaches to facilitate the improvement of RecA-targeted drugs. In this technical note, the authors present an in silico method based on tri-oligonucleotide usage correlations (TOUC) to predict the functional similarity between two RecA orthologs. The TOUC values analyzed in this study are in good agreement with the available experimental results. This method should prove useful in guiding future experimental efforts a... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=4682807 Tue, 05 Apr 2011 23:00:00 +0100 4682807 http://www.medworm.com/index.php?rid=4682806&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F16%2F4%2F450%3Frss%3D1 The p53 tumor suppressor is a potent transcription factor that regulates cell growth inhibition and apoptosis. The oncoprotein MDM2 suppresses p53 activity by direct inhibition of its transcriptional activity and enhances the degradation of p53 via the ubiquitin–proteosome pathway. Overexpression of MDM2, found in many human tumors, impairs p53-mediated cell death effectively. Inhibition of the p53–MDM2 interaction can stabilize p53 and may offer a novel strategy for cancer therapy. To search for new inhibitors of the p53–MDM2 interaction, the authors developed a cell-based high-throughput assay system based on mammalian two-hybrid technology. They also used a dual-luciferase reporter system to rule out false- positive hits due to the cytotoxic effect of compounds. Using ... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=4682806 Tue, 05 Apr 2011 23:00:00 +0100 4682806 http://www.medworm.com/index.php?rid=4682805&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F16%2F4%2F443%3Frss%3D1 JAK3 is an ideal target for the treatment of immune-related diseases and the prevention of organ allograft rejection. Several JAK3 inhibitors have been identified by biochemical enzymatic assays, but the majority display significant off-target effects on JAK2. Therefore, there is a need to develop new experimental approaches to identify compounds that specifically inhibit JAK3. Here, we show that in 32D/IL-2Rβ cells, STAT5 becomes phosphorylated by an IL-3/JAK2- or IL-2/JAK3-dependent pathway. Importantly, the selective JAK3 inhibitor CP-690,550 blocked the phosphorylation and the nuclear translocation of STAT5 following treatment of cells with IL-2 but not with IL-3. In an attempt to use the cells for large-scale chemical screens to identify JAK3 inhibitors, we established a cell lin... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=4682805 Tue, 05 Apr 2011 23:00:00 +0100 4682805 http://www.medworm.com/index.php?rid=4682804&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F16%2F4%2F436%3Frss%3D1 The ribosome-inhibiting toxin ricin binds exposed β1->4 linked galactosyls on multiple glycolipids and glycoproteins on the cell surface of most eukaryotic cells. After endocytosis, internal cell trafficking is promiscuous, with only a small proportion of ricin proceeding down a productive (cytotoxic) trafficking route to the endoplasmic reticulum (ER). Here, the catalytic ricin A chain traverses the membrane to inactivate the cytosolic ribosomes, which can be monitored by measuring reduction in protein biosynthetic capacity or cell viability. Although some markers have been discovered for the productive pathway, many molecular details are lacking. To identify a more comprehensive set of requirements for ricin intoxication, the authors have developed an RNAi screen in Drosophila S2... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=4682804 Tue, 05 Apr 2011 23:00:00 +0100 4682804 http://www.medworm.com/index.php?rid=4682803&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F16%2F4%2F427%3Frss%3D1 In this study, manual and automated studies are investigated and equivalence is demonstrated. In addition, automated applications using model probe substrates and inhibitors to assess the cholestatic potential of drugs and evaluate hepatic drug transport are examined. The successful automation of this technology provides a more reproducible and less labor-intensive approach, reducing potential operator error in complex studies and facilitating technology transfer. (Source: Journal of Biomolecular Screening) Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=4682803 Tue, 05 Apr 2011 23:00:00 +0100 4682803 http://www.medworm.com/index.php?rid=4682802&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F16%2F4%2F415%3Frss%3D1 High-throughput screening data repositories, such as PubChem, represent valuable resources for the development of small-molecule chemical probes and can serve as entry points for drug discovery programs. Although the loose data format offered by PubChem allows for great flexibility, important annotations, such as the assay format and technologies employed, are not explicitly indexed. The authors have previously developed a BioAssay Ontology (BAO) and curated more than 350 assays with standardized BAO terms. Here they describe the use of BAO annotations to analyze a large set of assays that employ luciferase- and β-lactamase–based technologies. They identified promiscuous chemotypes pertaining to different subcategories of assays and specific mechanisms by which these chemotypes ... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=4682802 Tue, 05 Apr 2011 23:00:00 +0100 4682802 http://www.medworm.com/index.php?rid=4682801&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F16%2F4%2F405%3Frss%3D1 Familial amyotrophic lateral sclerosis (fALS) accounts for 10% of ALS cases, and about 25% of fALS cases are due to mutations in superoxide dismutase 1 (SOD1). Mutant SOD1-mediated ALS is caused by a gain of toxic function of the mutant protein, and the SOD1 level in nonneuronal neighbors, including astrocytes, determines the progression of ALS (non-cell-autonomous toxicity). Therefore, the authors hypothesized that small molecules that reduce SOD1 protein levels in astrocytes might slow the progression of mutant SOD1-mediated ALS. They developed and optimized a cell-based, high-throughput assay to identify low molecular weight compounds that decrease SOD1 expression transcriptionally in human astrocyte-derived cells. Screening of a chemical library of 9600 compounds with the assay identif... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=4682801 Tue, 05 Apr 2011 23:00:00 +0100 4682801 http://www.medworm.com/index.php?rid=4682800&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F16%2F4%2F394%3Frss%3D1 Class O forkhead box (FOXO) transcription factors are downstream targets of the PI3K/AKT signaling pathway, which is upregulated in many tumors. AKT phosphorylates and inactivates FOXO1 by relocating it to the cytoplasm. Because FOXO1 functions as a tumor suppressor by negatively regulating cell cycle progression and cell survival, compounds that promote FOXO1 localization to the nucleus might have therapeutic value in oncology. Here the authors describe the identification of such compounds by using an image-based, high-content screen. Compounds that were active in retaining FOXO1 in the nucleus were tested to determine their pathway specificity and isoform specificity by using high-content assays for Rev and FOXO3, respectively. (Source: Journal of Biomolecular Screening) Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=4682800 Tue, 05 Apr 2011 23:00:00 +0100 4682800 http://www.medworm.com/index.php?rid=4682799&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F16%2F4%2F383%3Frss%3D1 The PTEN tumor suppressor gene is one of the most commonly mutated genes in human cancer. Because inactivation of PTEN is a somatic event, PTEN mutations represent an important genetic difference between cancer cells and normal cells and therefore a potential anticancer drug target. However, it remains a substantial challenge to identify compounds that target loss-of-function events such as mutations of tumor suppressors. In an effort to identify small molecules that preferentially kill cells with mutations of PTEN, the authors developed and implemented a high-throughput, paired cell-based screen composed of parental HCT116 cells and their PTEN gene-targeted derivatives. From 138 758 compounds tested, two hits were identified, and one, N'-[(1-benzyl-1H-indol-3-yl)methylene]benzenesulfonohy... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=4682799 Tue, 05 Apr 2011 23:00:00 +0100 4682799 http://www.medworm.com/index.php?rid=4549292&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Freprint%2F16%2F3%2F378%3Frss%3D1 (Source: Journal of Biomolecular Screening) Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=4549292 Fri, 04 Mar 2011 00:00:00 +0100 4549292 http://www.medworm.com/index.php?rid=4549291&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F16%2F3%2F370%3Frss%3D1 This article focuses on the application of a generic and fully automated LC/MS/MS, named Rapid and Integrated Analysis System (RIAS), as a high-throughput platform for the rapid quantification of drug-like compounds in various in vitro ADME assays. Previous efforts were dedicated to the setup and feasibility study of a workflow-integrated platform combining a modified high-throughput liquid handling LC/MS/MS system controlled by a customized software interface and a customized data-processing and reporting tool. Herein the authors present an extension of this previously developed basic application to a broad set of ADME screening campaigns, covering CYP inhibition, Caco-2, and PAMPA assays. The platform is capable of switching automatically between various ADME assays, performs MS compound... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=4549291 Fri, 04 Mar 2011 00:00:00 +0100 4549291 http://www.medworm.com/index.php?rid=4549290&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F16%2F3%2F363%3Frss%3D1 The HCV p7 protein is not involved in viral RNA replication but is essential for production of infectious virus. Based on its putative ion channel activity, p7 belongs to a family of viral proteins known as viroporins that oligomerize after insertion into a lipid membrane. To screen for compounds capable of interfering with p7 channel function, a low-throughput liposome-based fluorescent dye permeability assay was modified and converted to a robust high-throughput screening assay. Escherichia coli expressing recombinant p7 were grown in high-density fed-batch fermentation followed by a detergent-free purification using a combination of affinity and reversed-phase chromatography. The phospholipid composition of the liposomes was optimized for both p7 recognition and long-term stability. A c... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=4549290 Fri, 04 Mar 2011 00:00:00 +0100 4549290 http://www.medworm.com/index.php?rid=4549289&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F16%2F3%2F356%3Frss%3D1 G-protein–coupled receptors (GPCRs) are an important class of pharmaceutical drug targets. Functional high-throughput GPCR assays are needed to test an increasing number of synthesized novel drug compounds and their function in signal transduction processes. Measurement of changes in the cyclic adenosine monophosphate (cAMP) concentration is a widely used method to verify GPCR activation in the adenylyl cyclase pathway. Here, a single-label time-resolved fluorescence and high-throughput screening (HTS)–feasible method was developed to measure changes in cAMP levels in HEK293i cells overexpressing either β2-adrenergic or -opioid receptors. In the quenching resonance energy transfer (QRET) technique, soluble quenchers reduce the signal of unbound europium(III)-labeled cAMP i... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=4549289 Fri, 04 Mar 2011 00:00:00 +0100 4549289 http://www.medworm.com/index.php?rid=4549288&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F16%2F3%2F348%3Frss%3D1 Lipophilicity is an important parameter for any potential drug candidate. Accurate and efficient lipophilicity measurements facilitate the development of high-quality predictive in silico models that support the design of future drugs. Lipophilicity estimates derived from the traditional 1-octanol/water shake flask techniques have been the most widely employed and are therefore the best understood. This technique can be considered to give a good measure of a compound’s lipophilicity, albeit slower and more labor intensive to run compared with some other methodologies. Herein is described and validated an efficient 1-octanol/water shake flask technique that has sufficient capacity to be run as a primary screen within the drug discovery process. This is achieved by the simultaneous mea... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=4549288 Fri, 04 Mar 2011 00:00:00 +0100 4549288 http://www.medworm.com/index.php?rid=4549287&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F16%2F3%2F338%3Frss%3D1 This study comparatively evaluates unbiased approaches to reduce dimensionality as well as to summarize cell populations. To evaluate these different data-processing strategies, the prediction accuracies and the Z' factors of control compounds of a HCS cell cycle data set were monitored. As expected, dimension reduction led to a lower degree of discrimination between control samples. A high degree of classification accuracy was achieved when the cell population was summarized on well level using percentile values. As a conclusion, the generic data analysis pipeline described here enables a systematic review of alternative strategies to analyze multiparametric results from biological systems. (Source: Journal of Biomolecular Screening) Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=4549287 Fri, 04 Mar 2011 00:00:00 +0100 4549287 http://www.medworm.com/index.php?rid=4549286&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F16%2F3%2F332%3Frss%3D1 Inducible gene expression systems are particularly useful for the functional characterization of genes with putative toxic properties. In the course of studying the role of hypoxia-regulated gene expression on cell survival using the tetracycline-inducible (tet-on) system, the author noted that exposure to the inducing ligand doxycycline (dox) inhibited caspase-3 cleavage in control samples. To limit this confounding off-target effect, he devised an in vitro pulse dose, delayed-injury protocol testing both dox and a novel tetracycline analog 9-t-butyl doxycycline (9-TB). Although 9-TB induced higher transgene levels compared to matched concentrations of dox, continuous exposure to both drugs inhibited caspase-3 cleavage in hypoxic samples. Conversely, a 6-h pulse dose of 9-TB followed by a... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=4549286 Fri, 04 Mar 2011 00:00:00 +0100 4549286 http://www.medworm.com/index.php?rid=4549285&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F16%2F3%2F323%3Frss%3D1 The establishment of mammalian cell lines reliably expressing G-protein-coupled receptors (GPCRs) can be a tedious and often time-consuming process. A strategy has been developed to allow the rapid production of such cell lines. The first step of this approach was the generation of a specialized master cell line, characterized by optimized stable expression of a membrane-bound reporter protein. In the second step, this reporter gene was exchanged for that of the GPCR of interest by a DNA recombinase "cut-and-paste" engineering step. It has been demonstrated that the resulting GPCR cell lines inherit the advantages of the master cell line, expressing the GPCR in a homogeneous and stable manner. The case studies presented demonstrate the functionality of the established GPCR cell lines, and ... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=4549285 Fri, 04 Mar 2011 00:00:00 +0100 4549285 http://www.medworm.com/index.php?rid=4549284&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F16%2F3%2F313%3Frss%3D1 Strict quality control of cells is required for the standardization and interpretation of results in all areas of cell-based research, especially in drug discovery. Real-time cellular analysis using electrical impedance as a readout offers a rapid and highly reproducible method for quality control as it provides a quantitative measure of overall cell morphology and growth. In a case study, the authors demonstrate that samples of a single cell line obtained from several different labs show clear differences in their impedance profiles when compared with the corresponding standard cell line. A number of kinetic parameters were derived from the impedance profiles and used to quantify the differences among these cell lines. Our findings indicate that this methodology can detect cell line diffe... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=4549284 Fri, 04 Mar 2011 00:00:00 +0100 4549284 http://www.medworm.com/index.php?rid=4549283&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F16%2F3%2F303%3Frss%3D1 1-deoxy-D-xylulose 5-phosphate reductoisomerase (Dxr) is involved in the synthesis of isoprenoids by the methylerythritol phosphate pathway. Dxr is essential in Mycobacterium tuberculosis (Mtu), absent in humans and amenable to structure-aided design. To further assess the druggability of the enzyme, the energetics of binding of fosmidomycin to Mtu Dxr was studied by isothermal calorimetry. Binding was enhanced by nicotinamide adenine dinucleotide phosphate hydrogen (NADPH) and driven by enthalpy (H –10.2 kcal/mol, S 1.1 cal mol–1K–1). This suggests the possibility of finding novel inhibitors that bind enthalpically, making Dxr an attractive target. The cost of the Dxr substrate, 1-deoxy-D-xylulose-5-phosphate, for high-throughput screening (HTS) is prohibitive. Hence, an... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=4549283 Fri, 04 Mar 2011 00:00:00 +0100 4549283 http://www.medworm.com/index.php?rid=4549282&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F16%2F3%2F295%3Frss%3D1 Tissue factor (TF), the primary initiator of the coagulation cascade, plays a critical role in hemostasis and thrombosis, and inhibition of TF activity appears to be an attractive target for the treatment of cardiovascular diseases. However, few selective small-molecule inhibitors of TF are available, and the present assays for measuring TF activity are relatively expensive and complex. The authors present a simple and high-throughput chromogenic assay for screening TF inhibitors based on using commercial human prothrombin complex instead of purified coagulation factors, reducing the dosage, and performing with a one-stage procedure. In the optimized assay, <45 µL cell lysates was incubated with Tris-CaCl2 buffer (pH 7.3) containing human prothrombin complex at 37°C for 15 min... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=4549282 Fri, 04 Mar 2011 00:00:00 +0100 4549282 http://www.medworm.com/index.php?rid=4549281&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F16%2F3%2F285%3Frss%3D1 Proteases are industrially important enzymes but often have to be improved for their catalytic efficiency and stabilities to suit applications. Flow cytometry screening technology based on in vitro compartmentalization in double emulsion had been developed and applied on directed evolution of paraoxonase and β-galactosidase. Further advancements of flow cytometry–based screening technologies will enable an ultra-high throughput of variants offering novel opportunities in directed enzyme evolution under high mutational loads. For the industrially important enzyme class of proteases, a first flow cytometry–based screening system for directed protease evolution has been developed based on an extracellular protease-deficient Bacillus subtilis strain (WB800N), a model protease ... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=4549281 Fri, 04 Mar 2011 00:00:00 +0100 4549281 http://www.medworm.com/index.php?rid=4438814&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Freprint%2F16%2F2%2F278%3Frss%3D1 (Source: Journal of Biomolecular Screening) Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=4438814 Fri, 04 Feb 2011 00:00:00 +0100 4438814 http://www.medworm.com/index.php?rid=4438813&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F16%2F2%2F272%3Frss%3D1 To facilitate discovery of compounds modulating sphingosine-1-phosphate (S1P) signaling, the authors used high-throughput mass spectrometry technology to measure S1P formation in human whole blood. Since blood contains endogenous sphingosine (SPH) and S1P, mass spectrometry was chosen to detect the conversion of an exogenously added 17-carbon-long variant of sphingosine, C17SPH, into C17S1P. The authors developed procedures to achieve homogeneous mixing of whole blood in 384-well plates and for a method requiring minimal manipulations to extract S1P from blood in 96- and 384-well plates prior to analyses using the RapidFire® mass spectrometry system. (Source: Journal of Biomolecular Screening) Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=4438813 Fri, 04 Feb 2011 00:00:00 +0100 4438813 http://www.medworm.com/index.php?rid=4438812&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F16%2F2%2F266%3Frss%3D1 The protective antigen (PA) of Bacillus anthracis is a secreted protein that functions as a critical virulence factor. Protective antigen has been selected as a biomarker in detecting bacterial infection. The in vitro selection method, systematic evolution of ligands by exponential enrichment (SELEX), was used to find single-stranded DNAs that were tightly bound to PA. After 8 rounds of the SELEX process with PA, 4 different oligonucleotides (referred to as aptamers) that contain a 30-residue ssDNA sequence were identified. Dissociation constant (Kd) values with Cy3-attached aptamers were determined via fluorophotometry to be within a nanomolar range. The authors attempted to visualize the detection of PA using an aptamer-based enzyme-linked immunosorbent assay method, which has proven to ... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=4438812 Fri, 04 Feb 2011 00:00:00 +0100 4438812 http://www.medworm.com/index.php?rid=4438811&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F16%2F2%2F259%3Frss%3D1 The cellular response to DNA damage is emerging as a promising target for cancer therapy. In the present study, the authors exploited the relationship between the level of the phosphorylated form of histone H2AX (H2AX) and the extent of DNA damage and developed a quantitative, cell-based, high-content screening system for measuring the DNA damage response (DDR). In this system, the authors quantified the level of H2AX by measuring DNA damage–induced H2AX nuclear foci using an automated cell imager. They found that the total area of H2AX foci per cell exhibited a good correlation with the concentration of DNA damage–inducing agents, including etoposide. The effects of 2 well-known inhibitors of DNA damage could be quantified using this system, suggesting the suitability of the H... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=4438811 Fri, 04 Feb 2011 00:00:00 +0100 4438811 http://www.medworm.com/index.php?rid=4438810&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F16%2F2%2F251%3Frss%3D1 We describe methods for automated analysis of POD formation using high throughput microscopy (HTM) to localize PML immunofluorescence in conjunction with image analysis software for POD quantification. Using this HCS assay in 384 well format, we performed pilot screens of a small synthetic chemical library and mixture-based combinatorial libraries, demonstrating the robust performance of the assay. HCS counter-screening assays were also developed for hit characterization, based on immunofluorescence analyses of the subcellular location of phosphorylated H2AX or phosphorylated CHK1, which increase in a punctate nuclear pattern in response to DNA damage. Thus, the HCS assay devised here represents a high throughput screen that can be utilized to discover POD-inducing compounds that may resto... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=4438810 Fri, 04 Feb 2011 00:00:00 +0100 4438810 http://www.medworm.com/index.php?rid=4438809&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F16%2F2%2F239%3Frss%3D1 In conclusion, the developed FIA EAD approach is suitable to screen for diversity within BM3 mutants and this alternative screening technology offers new perspectives for rapid and sensitive screening of compound libraries towards BM3 mutants. (Source: Journal of Biomolecular Screening) Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=4438809 Fri, 04 Feb 2011 00:00:00 +0100 4438809 http://www.medworm.com/index.php?rid=4438808&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F16%2F2%2F230%3Frss%3D1 DNA gyrase, a type II topoisomerase that introduces negative supercoils into DNA, is a validated antibacterial drug target. The holoenzyme is composed of 2 subunits, gyrase A (GyrA) and gyrase B (GyrB), which form a functional A2B2 heterotetramer required for bacterial viability. A novel fluorescence polarization (FP) assay has been developed and optimized to detect inhibitors that bind to the adenosine triphosphate (ATP) binding domain of GyrB. Guided by the crystal structure of the natural product novobiocin bound to GyrB, a novel novobiocin–Texas Red probe (Novo-TRX) was designed and synthesized for use in a high-throughput FP assay. The binding kinetics of the interaction of Novo-TRX with GyrB from Francisella tularensis has been characterized, as well as the effect of common buf... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=4438808 Fri, 04 Feb 2011 00:00:00 +0100 4438808 http://www.medworm.com/index.php?rid=4438807&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F16%2F2%2F221%3Frss%3D1 The gp41 subunit of the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein mediates the fusion of viral and host cell membranes. As the HIV-1 enters the host cells, the 2 helical regions, HR1 and HR2, in the ectodomain of gp41 can form a 6-helix bundle, which brings the viral and target cell membranes to close proximity and serves as an attractive target for developing HIV-1 fusion inhibitors. Now, there are several cell- and molecule-based assays to identify potential HIV-1 fusion inhibitors targeting gp41. However, these assays cannot be used universally because they are time-consuming, inconvenient, and expensive. In the present study, the authors expressed and purified GST-HR121 and C43-30a proteins that were derived from the HIV-1 gp41 ectodomain region. GST-HR121 has a... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=4438807 Fri, 04 Feb 2011 00:00:00 +0100 4438807 http://www.medworm.com/index.php?rid=4438806&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F16%2F2%2F211%3Frss%3D1 Hepatitis C virus (HCV) is a considerable global health problem for which new classes of therapeutics are needed. The authors developed a high-throughput assay to identify compounds that selectively block translation initiation from the HCV internal ribosome entry site (HCV IRES). Rabbit reticulocyte lysate conditions were optimized to faithfully report on authentic HCV IRES-dependent translation relative to a 5' capped mRNA control. The authors screened a library of ~430,000 small molecules for IRES inhibition, leading to ~1700 initial hits. After secondary counterscreening, the vast majority of hits proved to be luciferase and general translation inhibitors. Despite well-optimized in vitro translation conditions, in the end, the authors found no selective HCV IRES inhibitors but did disc... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=4438806 Fri, 04 Feb 2011 00:00:00 +0100 4438806 http://www.medworm.com/index.php?rid=4438805&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F16%2F2%2F201%3Frss%3D1 The flaviviral RNA-dependent RNA polymerase (RdRp) is an attractive drug target. To discover new inhibitors of dengue virus RdRp, the authors have developed a fluorescence-based alkaline phosphatase–coupled polymerase assay (FAPA) for high-throughput screening (HTS). A modified nucleotide analogue (2'-[2-benzothiazoyl]-6'-hydroxybenzothiazole) conjugated adenosine triphosphate (BBT-ATP) and 3'UTR-U30 RNA were used as substrates. After the polymerase reaction, treatment with alkaline phosphatase liberates the BBT fluorophore from the polymerase reaction by-product, BBTPPi, which can be detected at excitation and emission wavelengths of 422 and 566 nm, respectively. The assay was evaluated by examining the time dependency, assay reagent effects, reaction kinetics, and signal stability ... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=4438805 Fri, 04 Feb 2011 00:00:00 +0100 4438805 http://www.medworm.com/index.php?rid=4438804&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F16%2F2%2F192%3Frss%3D1 Specific viral proteins enter the nucleus of infected cells to perform essential functions, as part of the viral life cycle. The integrase (IN) molecule of human immunodeficiency virus (HIV)–1 is of particular interest in this context due to its integral role in integrating the HIV genome into that of the infected host cell. Most IN-based antiviral compounds target the IN/DNA interaction, but since IN must first enter the nucleus before it can perform these critical functions, nuclear transport of IN is also an attractive target for therapeutic intervention. Here the authors describe a novel high-throughput screening assay for identifying inhibitors of nuclear import, particularly IN, based on amplified luminescent proximity homogeneous assay (AlphaScreen®) technology, which is h... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=4438804 Fri, 04 Feb 2011 00:00:00 +0100 4438804 http://www.medworm.com/index.php?rid=4438803&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F16%2F2%2F183%3Frss%3D1 The retinoid acid receptor–related orphan receptors (RORs) represent important targets for the treatment of metabolic and immune disorders. Here the authors describe the application of AlphaScreen® technology to develop a high-throughput screening (HTS)–compatible assay to facilitate the discovery of ROR modulators. Using the ligand binding domain (LBD) of ROR and a peptide derived from the NR1 box of the nuclear receptor coactivator PGC-1, a 384-well format assay was developed exhibiting high sensitivity, requiring only low nanomolar concentration of reagents. Recently, it was shown that oxysterols such as 7-hydroxycholesterol (7-OHC) function as modulators of the RORs. In this assay, 7-OHC produced a concentration-response curve with an EC50 of 162 nM, a Z' factor of 0.6,... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=4438803 Fri, 04 Feb 2011 00:00:00 +0100 4438803