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. Fri, 19 Mar 2010 17:24:11 +0100 http://www.medworm.com/index.php?rid=3345965&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Freprint%2F15%2F3%2F340%3Frss%3D1 (Source: Journal of Biomolecular Screening) Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=3345965 Mon, 08 Mar 2010 17:34:28 +0100 3345965 http://www.medworm.com/index.php?rid=3345964&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Freprint%2F15%2F3%2F336%3Frss%3D1 (Source: Journal of Biomolecular Screening) Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=3345964 Mon, 08 Mar 2010 17:34:28 +0100 3345964 http://www.medworm.com/index.php?rid=3345963&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F15%2F3%2F327%3Frss%3D1 The authors recently reported the development and application of multiple LanthaScreen® cellular assays to interrogate specific steps within the PI3K/Akt pathway. The importance of this signaling cascade in regulating fundamental aspects of cell growth and survival, as well as in the progression of cancer, underscores the need for portable cell-based assays for compound profiling in multiple disease-relevant cell backgrounds. To meet this need, the authors have now expanded their LanthaScreen® assay platform across a variety of cell types using a gene delivery technology known as BacMam. Here, they have demonstrated the successful detection of Akt-dependent phosphorylation of PRAS40 at Thr246 in 10 different cell lines harboring mutations known to activate the PI3K/Akt pathway. In ... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=3345963 Mon, 08 Mar 2010 17:34:28 +0100 3345963 http://www.medworm.com/index.php?rid=3345962&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F15%2F3%2F321%3Frss%3D1 Richard Feynman postulated in 1948 that the path of an electron can be best described by the sum or functional integral of all possible trajectories rather than by the notion of a single, unique trajectory. As a consequence, the position of an electron does not harbor any information about the paths that contributed to this position. This observation constitutes a classical endpoint observation. The endpoint assay is the desired type of experiment for high-throughput screening applications, mainly because of limitations in data acquisition and handling. Quite contrary to electrons, it is possible to extract information about the path of a protein using endpoint assays, and these types of applications are reviewed in this article. (Source: Journal of Biomolecular Screening) Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=3345962 Mon, 08 Mar 2010 17:34:28 +0100 3345962 http://www.medworm.com/index.php?rid=3345961&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F15%2F3%2F314%3Frss%3D1 Protein stabilization upon ligand binding has frequently been used to identify ligands for soluble proteins. Methods such as differential scanning fluorimetry (DSF) and differential static light scattering (DSLS) have been employed in the 384-well format and have been useful in identifying ligands that promote crystallization and 3D structure determination of proteins. However, finding a generic method that is applicable to membrane proteins has been a challenge as the high hydrophobicity of membrane proteins and the presence of detergents essential for their solubilization interfere with fluorescence-based detections. Here the authors used MsbA (an adenosine triphosphate binding cassette transporter), CorA (a Mg++ channel), and CpxA (a histidine kinase) as model proteins and show that DSL... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=3345961 Mon, 08 Mar 2010 17:34:28 +0100 3345961 http://www.medworm.com/index.php?rid=3345960&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F15%2F3%2F308%3Frss%3D1 Several monoclonal antibodies (McAbs) have been developed that show high sensitivity and specificity to gastric cancer and colorectal cancer. However, few of the antigens recognized by these antibodies have been identified. The authors now report the selection of anti-idiotype (anti-id) antibodies of MGb1 McAb against gastric cancer and MC5 McAb against colorectal cancer using phage-displayed single-chain variable fragment (ScFv) libraries. After purification, the anti-id antibodies were approximately 30 kd and could be recognized by MGb1/MC5 McAb. Anti-id antibodies significantly blocked the binding of MGb1 and MC5 to gastric cancer/colorectal cancer cells, respectively, suggesting that the antibodies were specific to MGb1 and MC5. Antibodies against gastric and colorectal cancer could be... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=3345960 Mon, 08 Mar 2010 17:34:28 +0100 3345960 http://www.medworm.com/index.php?rid=3345959&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F15%2F3%2F302%3Frss%3D1 A chip-based automated patch-clamp technique provides an attractive biophysical tool to quantify solute permeation through membrane channels. Proteo–giant unilamellar vesicles (proteo-GUVs) were used to form a stable lipid bilayer across a micrometer-sized hole. Because of the small size and hence low capacitance of the bilayer, single-channel recordings were achieved with very low background noise. The latter allowed the characterization of the influx of 2 major classes of antibiotics—cephalosporins and fluoroquinolones—through the major Escherichia coli porins OmpF and OmpC. Analyzing the ion current fluctuations in the presence of antibiotics revealed transport properties that allowed the authors to determine the mode of permeation. The chip-based setup allows rapid so... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=3345959 Mon, 08 Mar 2010 17:34:28 +0100 3345959 http://www.medworm.com/index.php?rid=3345958&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F15%2F3%2F297%3Frss%3D1 In this study, the effects of dopamine or a dopamine reaction product on yeast growth and survival have been explored. Yeast is ideal for such a study because, unlike mammalian cells, yeast cells can be grown even when respiratory function is totally absent. Indeed, dopamine was found to be inhibitory to yeast growth in media where respiratory function was required and cytotoxic to yeast cells suspended in water. The inhibitory effects of dopamine were reduced greatly by the antioxidants ascorbate and glutathione, suggesting the involvement of reactive oxygen species in dopamine-mediated toxicity. It would appear that yeast may offer a convenient model to perform screens for further compounds that may provide protection against dopamine-mediated growth inhibition and toxicity. (Source: Jou... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=3345958 Mon, 08 Mar 2010 17:34:28 +0100 3345958 http://www.medworm.com/index.php?rid=3345957&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F15%2F3%2F287%3Frss%3D1 The development of multidrug resistance (MDR) remains a significant obstacle in treating cancer patients with chemotherapy. To identify small-molecule compounds that can reverse MDR, the authors used a cell-based screening assay with an MDR ovarian cancer cell line. Incubating MDR cells with a sublethal concentration of paclitaxel in combination with each of 2000 small-molecule compounds from the National Cancer Institute Diversity Set Library, they identified NSC77037. The cytotoxic activity of NSC77037 and the duration of its effect were evaluated in vitro using a panel of cancer cell lines expressing permeability glycoprotein (Pgp), multiple drug resistance protein 1 (MRP 1), and breast cancer resistance protein (BCRP). The mechanism of its effects was further analyzed by assessing the ... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=3345957 Mon, 08 Mar 2010 17:34:28 +0100 3345957 http://www.medworm.com/index.php?rid=3345956&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F15%2F3%2F279%3Frss%3D1 The molecular chaperone heat shock protein 90 (Hsp90) is required for the correct folding and stability of a number of client proteins that are important for the growth and maintenance of cancer cells. Heat shock protein 72 (Hsp72), a co-chaperone of Hsp90, is also emerging as an attractive cancer drug target. Both proteins bind and hydrolyze adenosine triphosphate (ATP), and ATPase activity is essential for their function. Inhibition of Hsp90 ATPase activity leads to the degradation of client proteins, resulting in cell growth inhibition and apoptosis. Several small-molecule inhibitors of the ATPase activity of Hsp90 have been described and are currently being evaluated clinically for the treatment of cancer. A number of methods for the measurement of ATPase activity have been previously ... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=3345956 Mon, 08 Mar 2010 17:34:28 +0100 3345956 http://www.medworm.com/index.php?rid=3345955&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F15%2F3%2F268%3Frss%3D1 The human estrogen receptors (hER) are members of the nuclear hormone receptor (NHR) superfamily and represent important drug targets for the pharmaceutical industry. Initially, ligand binding assays were used to identify novel ligands using receptors purified from native tissues. With the advent of molecular cloning techniques, cell-based transactivation assays have been the gold standard for many years of drug discovery. With the elucidation of the structural mechanisms underlying the activation of NHRs, cell-free assays with purified receptors have become important tools to directly assess different binding sites (e.g., the hormone binding site or the cofactor binding site). The available cell-free assays have so far facilitated the study of one binding site at a time. With the introduc... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=3345955 Mon, 08 Mar 2010 17:34:28 +0100 3345955 http://www.medworm.com/index.php?rid=3345954&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F15%2F3%2F261%3Frss%3D1 Functional cell signaling assays have become important tools for measuring ligand-induced receptor activation in cell-based biomolecular screening. Guanosine-5'-triphosphate (GTP) is a generic signaling marker responsible for the first intracellular signaling event of the G-protein-coupled receptors (GPCRs). [35S]GTPS binding assay is the classical well-established method for measuring agonist-induced G-protein activation requiring a separation of free and bound fractions prior to measurement. Here a novel, separation-free, time-resolved fluorescence GTP binding assay has been developed based on a non–fluorescence resonance energy transfer (FRET) single-label approach and quenching of a nonbound europium-labeled, nonhydrolyzable GTP analog (Eu-GTP). The quenching resonance energy tra... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=3345954 Mon, 08 Mar 2010 17:34:28 +0100 3345954 http://www.medworm.com/index.php?rid=3345953&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F15%2F3%2F251%3Frss%3D1 Many G-protein-coupled receptors (GPCRs) have been shown to form heteromeric complexes primarily by biochemical methods, including competitive radioligand binding assays or measurements of changes in second-messenger concentration in lysed cells. These results are often cell line specific, and the expression of other cell surface proteins makes it difficult to detect potential functional consequences of GPCR interaction. Here, 2-electrode voltage clamping in Xenopus oocytes was used as a bioassay to explore heterodimerization of bradykinin type 2 receptor (Bk2R) and beta 2 adrenergic receptor (β2AR), using chloride channels as outputs for receptor activation. The data show for the first time that these 2 receptors heterodimerize with functional consequences. Stimulation with bradykini... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=3345953 Mon, 08 Mar 2010 17:34:28 +0100 3345953 http://www.medworm.com/index.php?rid=3345952&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F15%2F3%2F239%3Frss%3D1 CD36, a member of the class B scavenger receptor, is a high-affinity receptor for oxidatively modified low-density lipoprotein (oxLDL). Extensive evidence points to a significant role of CD36 in atherosclerosis and suggests that CD36 could be a potential target for treatment of atherosclerosis. Here, the extracellular domain of human CD36 (Gly30-Asn439) was expressed in Escherichia coli as His6-tagged soluble CD36 (sCD36), which could bind oxLDL specifically and effectively inhibit the uptake of oxLDL by murine macrophage RAW 264.7 cells. An enzyme-linked immunosorbent assay (ELISA)–like high-throughput screening (HTS) assay was developed for the discovery of CD36 antagonists, based on the competition of sCD36 binding to immobilized oxLDL and detection with a monoclonal antibody agai... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=3345952 Mon, 08 Mar 2010 17:34:27 +0100 3345952 http://www.medworm.com/index.php?rid=3243176&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Freprint%2F15%2F2%2F233%3Frss%3D1 (Source: Journal of Biomolecular Screening) Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=3243176 Thu, 04 Feb 2010 23:56:24 +0100 3243176 http://www.medworm.com/index.php?rid=3243175&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Freprint%2F15%2F2%2F230%3Frss%3D1 (Source: Journal of Biomolecular Screening) Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=3243175 Thu, 04 Feb 2010 23:56:24 +0100 3243175 http://www.medworm.com/index.php?rid=3243174&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F15%2F2%2F224%3Frss%3D1 A cell-based fluorescent protein reporter assay for proteinase activity amenable to high-throughput applications was developed. This assay is based on Förster resonance energy transfer (FRET) between 2 variants of the green fluorescent protein connected by a short cleavable linker and expressed in Escherichia coli as tagged proteins. A library to assay proteinase specificity was generated by randomizing a portion of the linker using PCR. The library could be grown in microplates, allowing cells to be lysed in situ and substrate cleavage to be monitored through loss of FRET signal using a plate reader. Progress curves were generated to estimate cleavage efficiency, facilitating the identification of well-cleaved substrates. The polyhistidine-tagged fluorescent substrates could then be ... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=3243174 Thu, 04 Feb 2010 23:56:24 +0100 3243174 http://www.medworm.com/index.php?rid=3243173&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F15%2F2%2F218%3Frss%3D1 Recently, it has been shown that 2-photon fluorescence correlation spectroscopy of single glycosylated 20-nm fluorescent spheres allows measurement of the relative carbohydrate binding affinities of unlabeled proteins and that these modified spheres can mimic the glycocalix of cell or virus surfaces. An especially useful extension would be the analysis of mixtures of nanospheres that each contain different fluorescent labels and are thus differentially "encoded." If the surfaces of these encoded nanospheres are modified with various receptors, many different biomolecule-surface interactions and concurrent reactions can be measured quickly and simultaneously in a single-reaction vessel. An essential prerequisite for this general assay principle is the ability to identify with an accuracy of... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=3243173 Thu, 04 Feb 2010 23:56:24 +0100 3243173 http://www.medworm.com/index.php?rid=3243172&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F15%2F2%2F213%3Frss%3D1 In this study, the authors developed an efficient and low-cost chitinase assay using colloidal chitin azure. The assay feasibility is evaluated and compared with traditional assays employing colloidal chitin and chitin azure. The authors found that the optimum pH for determining chitinase activity using colloid chitin azure was pH 5 or 8. The method was sensitive, and the assay was complete within 30 min. When the assay was used to measure chitinase activities produced by 2 strains of chitinolytic bacteria, BCTS (an Escherichia coli BL21 [DE3] expressing a secretory recombinant chitinase) and AS1 (a chitinolytic bacterium with low levels of chitinase), it was shown that cultivation in Bushnell-Haas selection medium caused AS1 to secrete a higher level of chitinase than was secreted when th... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=3243172 Thu, 04 Feb 2010 23:56:24 +0100 3243172 http://www.medworm.com/index.php?rid=3243171&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F15%2F2%2F206%3Frss%3D1 In this study, the authors have developed a novel high-throughput assay, with which they have identified 5 mTGase mutants that are highly specific for conjugating PEG to Q141 of hGH. In this scintillation proximity assay (SPA)–based method, the authors have (1) achieved a high expression level of active mTGase, which is toxic to the living cell, directly from Escherichia coli (0.2 U/mL/OD600) by in vivo activation; (2) developed a high-throughput affinity purification method to eliminate the strong interference of cellular protein to mTGase reaction; and (3) used therapeutic protein as the substrate. This method is highly sensitive, is easily automated, and could be generally applied to screening mTGases with desired specificity targeting on different therapeutic proteins. (Source: J... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=3243171 Thu, 04 Feb 2010 23:56:24 +0100 3243171 http://www.medworm.com/index.php?rid=3243170&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F15%2F2%2F196%3Frss%3D1 A high-throughput (HT) agar-based halo assay is described, which allows for rapid screening of chemical libraries for bioactivity in microorganisms such as yeast and bacteria. A pattern recognition algorithm was developed to identify halo-like shapes in plate reader optical density (OD) measurements. The authors find that the total growth inhibition within a detected halo provides an accurate estimate of a compound’s potency measured in terms of its EC50. The new halo recognition method performs significantly better than an earlier method based on single-point OD readings. An assay based on the halo algorithm was used to screen a 21,120-member library of drug-like compounds in Saccharomyces cerevisiae, leading to the identification of novel bioactive scaffolds containing derivatives ... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=3243170 Thu, 04 Feb 2010 23:56:24 +0100 3243170 http://www.medworm.com/index.php?rid=3243169&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F15%2F2%2F185%3Frss%3D1 Voltage-gated K+ channels are potential drug targets for an increasing number of disease indications. Searching for compounds that modulate K+ channel activities by high-throughput screening (HTS) is becoming a standard approach in the drug discovery effort. Here the authors report an improved fluorometric imaging plate reader (FLIPR) membrane potential assay for Kv1.3 K+ channel HTS. They have found that the Chinese hamster ovary (CHO) cells have endogenous membrane electrogenic transporters that contribute to maintaining membrane potential. Blocking the recombinant K+ channels in the overexpressing CHO cell line hardly changed the membrane potential. Inhibition of the endogenous transporters is essential to achieve the required assay robustness. The authors identified the optimal assay c... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=3243169 Thu, 04 Feb 2010 23:56:24 +0100 3243169 http://www.medworm.com/index.php?rid=3243168&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F15%2F2%2F177%3Frss%3D1 KCC2, potassium chloride cotransporter 2, is expressed exclusively in the CNS (on inhibitory neurons) and plays a major role in maintaining appropriately low intracellular chloride levels that ensure inhibitory actions of GABAA and glycine receptors. As such, it plays a pivotal role in inhibitory mechanisms that control neuronal excitation in the CNS. KCC2 downregulation has been implicated in various excitatory disorders, such as epilepsy and neuropathic pain. Positive modulators of KCC2 expression or activity may thus provide effective therapy for these disorders. However, the identification of such agents is hindered by the lack of a high-throughput screening method. Here the authors report the development of a fluorescence-based thallium (Tl+) transport assay using a Fluorometric Imagi... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=3243168 Thu, 04 Feb 2010 23:56:24 +0100 3243168 http://www.medworm.com/index.php?rid=3243167&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F15%2F2%2F169%3Frss%3D1 A multiplexed cell assay has been optimized to measure the activities of fatty acyl-CoA elongase, delta-5 desaturase (5D), delta-6 desaturase (6D), and delta-9 desaturase (9D) together using 14C-labeled tracers in HepG2 cells, which express the human stearoyl-CoA desaturase-1 isoform (SCD1) exclusively. The 5 and 9 desaturase activities are indexed by the efficient conversion of [1-14C]-eicosatrienoic acid (C20:3, cis-8,11,14) to 14C-arachidonic acid (C20:4, cis-5,8,11,14) and the conversion of [1-14C]-stearic acid to 14C-oleic acid (C18:1, cis-9), respectively. CP-74006 potently blocks the 5D activity with an IC50 value of 20 nM and simplifies the metabolism of [1-14C]--linolenate (C18:3, cis-9,12,15) by accumulating 14C-eicosatetraenoic acid (C20:4, cis-8,11,14,17) as the major 14C-eicos... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=3243167 Thu, 04 Feb 2010 23:56:24 +0100 3243167 http://www.medworm.com/index.php?rid=3243166&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F15%2F2%2F159%3Frss%3D1 The self-renewal and phenotypic properties of neural stem cells make them an abundant and more physiologically relevant alternative to recombinant cell lines for drug screens to identify ligands acting at neural targets. Here, the authors use high-throughput phenotypic and signaling assays to test the ability of neural stem cells isolated from postnatal mouse hippocampus (mNSCs) to deliver high-content and physiologically relevant data on native peptide receptor activity. The authors find that mNSCs express PAC1 but not the related VPAC1 and VPAC2 receptors. PAC1 promotes both the proliferation of mNSCs and their differentiation into neuronal-like cells. In addition, the authors show that PAC1 stimulates markedly different extracellular signal-regulated kinase signals in mNSCs than in reco... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=3243166 Thu, 04 Feb 2010 23:56:24 +0100 3243166 http://www.medworm.com/index.php?rid=3243165&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F15%2F2%2F148%3Frss%3D1 mGluR1 antagonists have been postulated to be novel CNS drugs, including antipsychotics. Toward this end, the authors developed a β-lactamase reporter assay to identify mGluR1 antagonists. β-Lactamase has several interesting features for high-throughput screening, including very high sensitivity and less well-to-well variation than other reporter enzymes. mGluR1-expressing Chinese hamster ovary (CHO) cells with the β-lactamase gene under control of the nuclear factor of activated T cells (NFAT) promoter (CHO-NFAT-bla-hmGluR1b) exhibited very high basal activity, resulting in an inadequate signal-to-basal (S/B) ratio. Coexpression of glutamate/aspartate transporter (GLAST) with mGluR1 in the cell line (CHO-NFAT-bla-hmGluR1b-GLAST) dramatically decreased basal activity and imp... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=3243165 Thu, 04 Feb 2010 23:56:24 +0100 3243165 http://www.medworm.com/index.php?rid=3243164&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F15%2F2%2F138%3Frss%3D1 Rapid mixing in microplates is still an underappreciated challenge in screening assay development, particularly with the use of noncontact nanoliter liquid handlers. In high-content/throughput screening (HC/TS), fast and efficient mixing between compounds and cell culture medium is even more critical as biological kinetics dictates speed of mixing, usually within a few minutes. Moreover, mixing in HC/TS should be gentle enough to avoid any negative disruption in cell layer. Here the authors introduce a method to accurately quantify drop diffusion into a microplate well, independently of buffer, liquid handler, or dispensing protocol. This method was used to determine the effect of various mixing methods on the diffusion of a nanoliter drop of pure DMSO in aqueous buffer in 384-well plates.... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=3243164 Thu, 04 Feb 2010 23:56:24 +0100 3243164 http://www.medworm.com/index.php?rid=3243163&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F15%2F2%2F131%3Frss%3D1 Integrins play critical roles in the process of angiogenesis and are attractive targets for anticancer therapies. It is desirable to develop new types of small-molecule inhibitors of integrin. Herein, the binding features of several inhibitors to integrin vβ3 have been studied by surface plasmon resonance (SPR) biosensor technology and molecular docking analyses. The SPR results indicated that the equilibrium dissociation constant (KD) values are evaluated for the inhibitors and showed that the KD value of cyclopeptide c-Lys is much lower than the reference molecule. In addition, the 3D structural model of integrin vβ3 was generated according to the crystal structure of the integrin vβ3 complex, and the molecular docking simulation analyses revealed that the predicted bindin... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=3243163 Thu, 04 Feb 2010 23:56:24 +0100 3243163 http://www.medworm.com/index.php?rid=3243162&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F15%2F2%2F119%3Frss%3D1 This article discusses the unique features of allosteric ligands as drugs as well as the special conditions that should be considered to optimize a high-throughput screen toward the detection of allosteric drugs. Finally, the likelihood of detecting allosteric ligands that have direct effects on cells (either conventional agonism or functionally selective effects) is discussed as well as the optimization of detection of such ligands in screening assays. (Source: Journal of Biomolecular Screening) Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=3243162 Thu, 04 Feb 2010 23:56:24 +0100 3243162 http://www.medworm.com/index.php?rid=3178446&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Freprint%2F15%2F1%2F112%3Frss%3D1 (Source: Journal of Biomolecular Screening) Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=3178446 Fri, 15 Jan 2010 21:54:46 +0100 3178446 http://www.medworm.com/index.php?rid=3178445&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Freprint%2F15%2F1%2F108%3Frss%3D1 (Source: Journal of Biomolecular Screening) Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=3178445 Fri, 15 Jan 2010 21:54:46 +0100 3178445 http://www.medworm.com/index.php?rid=3178444&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F15%2F1%2F102%3Frss%3D1 Microfluidic devices have become invaluable tools in recent years to model biological phenomena. Here, the authors present a well plate microfluidic (WPM) device for conducting cell biology assays under shear flow. Physiological shear flow conditions of cell-cell and cell-ligand adhesion within this device produce results with higher biological significance than conventional well plates. The WPM format also produced significant work flow advantages such as faster liquid handling compared to static well plate assays. The authors used the VLA-4–VCAM-1 cell adhesion model as the basis for a rapid, higher throughput adhesion inhibition screen of monoclonal antibodies against VLA-4. Using the WPM device, they generated IC50 dose-response curves 96 times faster than conventional flow cells... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=3178444 Fri, 15 Jan 2010 21:54:46 +0100 3178444 http://www.medworm.com/index.php?rid=3178443&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F15%2F1%2F95%3Frss%3D1 This report suggests an extension of the Z' factor, which integrates multiple readouts for assay quality assessment. Using linear projections, multiple readouts are condensed to a single parameter, based on which the assay quality is monitored. The authors illustrate and evaluate this approach using simulated data and real-world data from a high-content screen. The suggested approach is applicable during assay development, to optimize the image analysis, as well as during screening to monitor assay robustness. Furthermore, data sets from high-content imaging assays and other state-of-the-art multiparametric screening technologies, such as flow cytometry or transcript analysis, could be analyzed. (Source: Journal of Biomolecular Screening) Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=3178443 Fri, 15 Jan 2010 21:54:46 +0100 3178443 http://www.medworm.com/index.php?rid=3178442&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F15%2F1%2F86%3Frss%3D1 More accurate dose-response curves can be constructed by eliminating aqueous serial dilution of compounds. Traditional serial dilutions that use aqueous diluents can result in errors in dose-response values of up to 4 orders of magnitude for a significant percentage of a compound library. When DMSO is used as the diluent, the errors are reduced but not eliminated. The authors use acoustic drop ejection (ADE) to transfer different volumes of model library compounds, directly creating a concentration gradient series in the receiver assay plate. Sample losses and contamination associated with compound handling are therefore avoided or minimized, particularly in the case of less water-soluble compounds. ADE is particularly well suited for assay miniaturization, but gradient volume dispensing i... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=3178442 Fri, 15 Jan 2010 21:54:46 +0100 3178442 http://www.medworm.com/index.php?rid=3178441&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F15%2F1%2F80%3Frss%3D1 Formalin-fixed, paraffin-embedded tissues are widely available for gene expression analysis using TaqMan® PCR. Five methods, including 4 commercial kits, for recovering RNA from paraffin-embedded renal tumor tissue were compared. The MasterPureTM kit from Epicentre produced the highest RNA yield. However, the difference in RNA yield between the kit from Epicenter and Invitrogen’s TRIzol method was not significant. Using the top 3 RNA isolation methods, the manufacturers’ protocols were modified to include an overnight Proteinase K digestion. Overnight protein digestion resulted in a significant increase in RNA yield. To optimize the reverse transcription reaction, conventional reverse transcription with random oligonucleotide primers was compared to reverse transcription us... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=3178441 Fri, 15 Jan 2010 21:54:46 +0100 3178441 http://www.medworm.com/index.php?rid=3178440&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F15%2F1%2F72%3Frss%3D1 Deoxycytidine kinase (dCK) phosphorylates deoxycytidine, deoxyguanosine, and deoxyadenosine and plays an important role in the salvage pathway of nucleoside metabolism. dCK is also required for the phosphorylation of several antiviral and anticancer nucleoside drugs, with resistance to these agents often being associated with a loss or decrease in dCK activity. Data also indicate a role for dCK in immune function, and dCK inhibitors may provide treatment for immune disorders. To identify novel dCK inhibitors, the authors evaluated 2 existing biochemical assays, adapted both to high-throughput screening, and identified several series of hits. They also compared the potency of the hits between purified recombinant and endogenous enzyme. Meanwhile, they also developed a novel cell-based assay... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=3178440 Fri, 15 Jan 2010 21:54:46 +0100 3178440 http://www.medworm.com/index.php?rid=3178439&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F15%2F1%2F62%3Frss%3D1 The histone code comprises many posttranslational modifications that occur mainly in histone tail peptides. The identity and location of these marks are read by a variety of histone-binding proteins that are emerging as important regulators of cellular differentiation and development and are increasingly being implicated in numerous disease states. The authors describe the development of the first high-throughput screening assay for the discovery of inhibitors of methyl-lysine binding proteins that will be used to initiate a full-scale discovery effort for this broad target class. They focus on the development of an AlphaScreenTM-based assay for malignant brain tumor (MBT) domain-containing proteins, which bind to the lower methylation states of lysine residues present in histone tail pept... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=3178439 Fri, 15 Jan 2010 21:54:46 +0100 3178439 http://www.medworm.com/index.php?rid=3178438&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F15%2F1%2F52%3Frss%3D1 A high-throughput mass spectrometry assay to measure the catalytic activity of UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase, LpxC, is described. This reaction is essential in the biosynthesis of lipopolysaccharide (LPS) of gram-negative bacteria and is an attractive target for the development of new antibacterial agents. The assay uses the RapidFireTM mass spectrometry platform to measure the native LpxC substrate and the reaction product and thereby generates a ratiometric readout with minimal artifacts due to detection interference. The assay was robust in a high-throughput screen of a library of more than 700,000 compounds arrayed as orthogonal mixtures, with a median Z' factor of 0.74. Selected novel inhibitors from the screening campaign were confirmed as binding to ... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=3178438 Fri, 15 Jan 2010 21:54:46 +0100 3178438 http://www.medworm.com/index.php?rid=3178437&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F15%2F1%2F42%3Frss%3D1 In this study, the authors developed novel BoNT serotype A (BoNT/A) cleavage-sensitive (BACS) antibodies that only interact with full-length SNAP-25 (synaptosomal-associated protein of 25 kDa), the molecular target of the BoNT/A serotype. These antibodies exhibit high specificity for full-length SNAP-25, allowing the BoNT/A-mediated proteolysis of this protein to be measured in diverse assay formats, including several variations of enzyme-linked immunosorbent assay and multiple immunofluorescence methods. Assays built around the BACS antibodies displayed excellent sensitivity, had excellent reproducibility, and were amenable to multiwell formats. Importantly, these assays provided novel methods for evaluating BoNT/A activity in cellular models of intoxication and allowed for the high-throu... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=3178437 Fri, 15 Jan 2010 21:54:46 +0100 3178437 http://www.medworm.com/index.php?rid=3178436&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F15%2F1%2F30%3Frss%3D1 This study presents the implementation and optimization of 3 cell-based assays on a TECAN Genesis workstation—the Caspase-Glo® 3/7 and sulforhodamine B (SRB) screening assays and the mechanistic Caco-2 permeability protocol—and evaluates their feasibility for automation. During implementation, the dispensing speed to add drug solutions and fixative trichloroacetic acid and the aspiration speed to remove the supernatant immediately after fixation were optimized. Decontamination steps for cleaning the tips and pipetting tubing were also added. The automated Caspase-Glo® 3/7 screen was successfully optimized with Caco-2 cells (Z' 0.7, signal-to-base ratio [S/B] 1.7) but not with DU-145 cells. In contrast, the automated SRB screen was successfully optimized with the DU-145 ... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=3178436 Fri, 15 Jan 2010 21:54:46 +0100 3178436 http://www.medworm.com/index.php?rid=3178435&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F15%2F1%2F21%3Frss%3D1 The novel tumor suppressor Pdcd4 affects tumorigenesis by inhibiting translation. Pdcd4 is phosphorylated and subsequently lost by proteasomal degradation in response to tumor-promoting conditions. Here, the authors describe the development of a reporter cell system to monitor the stability of Pdcd4. The phosphorylation-dependent degradation domain ("target") or an adjacent ("off-target") region of Pdcd4 was cloned into a luciferase expression system. The target constructs were responsive to Pdcd4 degrading conditions (e.g., TPA, p70S6K1 overactivation), whereas the off-target constructs remained stable. The system was optimized for and shown to be reliable in a high-throughput compatible 384-well format. Screening of 15,275 pure compounds resulted in a hit rate of 0.30% (>50% inhibitio... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=3178435 Fri, 15 Jan 2010 21:54:46 +0100 3178435 http://www.medworm.com/index.php?rid=3178434&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F15%2F1%2F10%3Frss%3D1 Small GTPases are key regulators of cellular activity and represent novel targets for the treatment of human diseases using small-molecule inhibitors. The authors describe a multiplex, flow cytometry bead-based assay for the identification and characterization of inhibitors or activators of small GTPases. Six different glutathione-S-transferase (GST)–tagged small GTPases were bound to glutathione beads, each labeled with a different red fluorescence intensity. Subsequently, beads bearing different GTPase were mixed and dispensed into 384-well plates with test compounds, and fluorescent–guanosine triphosphate (GTP) binding was used as the readout. This novel multiplex assay allowed the authors to screen a library of almost 200,000 compounds and identify more than 1200 positive c... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=3178434 Fri, 15 Jan 2010 21:54:46 +0100 3178434 http://www.medworm.com/index.php?rid=3178433&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F15%2F1%2F1%3Frss%3D1 In the past decade, high-content screening has become a highly developed approach to obtaining richly descriptive quantitative phenotypic data using automated microscopy. From early use in drug screening, the technique has evolved to embrace a diverse range of applications in both academic and industrial sectors and is now widely recognized as providing an efficient and effective approach to large-scale programs investigating cell biology in situ and in context. (Source: Journal of Biomolecular Screening) Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=3178433 Fri, 15 Jan 2010 21:54:46 +0100 3178433 http://www.medworm.com/index.php?rid=3082033&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Freprint%2F14%2F10%2F1275%3Frss%3D1 (Source: Journal of Biomolecular Screening) Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=3082033 Sat, 12 Dec 2009 00:18:48 +0100 3082033 http://www.medworm.com/index.php?rid=3082032&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Freprint%2F14%2F10%2F1270%3Frss%3D1 (Source: Journal of Biomolecular Screening) Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=3082032 Sat, 12 Dec 2009 00:18:48 +0100 3082032 http://www.medworm.com/index.php?rid=3082031&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F14%2F10%2F1263%3Frss%3D1 Using a commercially available time-resolved fluorescence resonance energy transfer (TR-FRET)—based assay for IKKβ, the authors have automated the assay procedure on a high-throughput screening station to carry out screening campaigns on multiwell plates. They have determined the Z' factor and optimized volumes, times, and time-resolved fluorescence parameters. They have also compared 2 kinases with different fusion tags, the influence of different enzyme/substrate ratios and of DMSO presence at different concentration. The authors found that glutathione S-transferase (GST)—fused IKKβ shows better signal-to-noise (S/N) ratios over the poly-histidine-tagged variant. The substrate can be used at 50 nM with optimal performances when the enzyme is used at 2 nM. DMSO at 0.... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=3082031 Sat, 12 Dec 2009 00:18:48 +0100 3082031 http://www.medworm.com/index.php?rid=3082030&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F14%2F10%2F1257%3Frss%3D1 The PI3K/Akt signaling pathway plays a key role in cancer cell growth, survival, and tumor angiogenesis. 3-Phosphoinositide-dependent protein kinase 1 (PDK1) is a Ser/Thr protein kinase, which catalyzes the phosphorylation of a conserved residue in the activation loop of a number of AGC kinases, including proto-oncogenes Akt, p70S6K, and RSK kinases. To find new small-molecule inhibitors of this important regulator kinase, the authors have developed PDK1-specific high-throughput enzymatic assays in time-resolved fluorescence resonance energy transfer (TR-FRET) and AlphaScreen® formats, monitoring phosphorylation of a biotinylated peptide substrate derived from the activation loop of Akt. Development of homogeneous assays enabled screening of a focused kinase library of ~21,500 compound... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=3082030 Sat, 12 Dec 2009 00:18:48 +0100 3082030 http://www.medworm.com/index.php?rid=3082029&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F14%2F10%2F1251%3Frss%3D1 Bone morphogenetic protein II (BMP2), a member of the transforming growth factor—β (TGF-β) superfamily, is highly expressed in osteoblasts and is a crucial regulator of osteogenic differentiation. Many observations clearly indicate the high potency of BMP2 as an inducer of osteogenesis, and it may be a novel therapeutic target for diseases associated with bone loss, especially in menopausal and postmenopausal women. To discover new agents that enhance the expression of the mouse BMP2, the authors developed a high-throughput assay to screen a synthetic and natural compound library. The cell-based high-throughput screen was conducted in 96-well plates using the clonal murine calvarial MC3T3-E1 cells. These cells were stably transfected with mouse BMP2 promoter-luciferase and ... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=3082029 Sat, 12 Dec 2009 00:18:48 +0100 3082029 http://www.medworm.com/index.php?rid=3082028&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F14%2F10%2F1245%3Frss%3D1 To provide an experimental basis for a comprehensive molecular modeling evaluation study, 500 fragments from the Maybridge fragment library were soaked into crystals of bovine pancreatic trypsin and the structures determined by X-ray crystallography. The soaking experiments were performed in both single and pooled aliquots to determine if combination of fragments is an appropriate strategy. A further set of data was obtained from co-crystallizing the pooled fragments with the protein. X-ray diffraction data were collected on approximately 1000 crystals at the Australian Synchrotron, and these data were subsequently processed, and the preliminary analysis was performed with a custom software application (Jigsaw), which combines available software packages for structure solution and analysis... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=3082028 Sat, 12 Dec 2009 00:18:48 +0100 3082028 http://www.medworm.com/index.php?rid=3082027&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F14%2F10%2F1236%3Frss%3D1 Artificial neural networks (ANNs) are trained using high-throughput screening (HTS) data to recover active compounds from a large data set. Improved classification performance was obtained on combining predictions made by multiple ANNs. The HTS data, acquired from a methionine aminopeptidases inhibition study, consisted of a library of 43,347 compounds, and the ratio of active to nonactive compounds, R A/N, was 0.0321. Back-propagation ANNs were trained and validated using principal components derived from the physicochemical features of the compounds. On selecting the training parameters carefully, an ANN recovers one-third of all active compounds from the validation set with a 3-fold gain in R A/N value. Further gains in RA/N values were obtained upon combining the predictions made by a ... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=3082027 Sat, 12 Dec 2009 00:18:47 +0100 3082027 http://www.medworm.com/index.php?rid=3082026&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F14%2F10%2F1228%3Frss%3D1 The generation of an action potential (AP) is a complex process in excitable cells that involves the temporal opening and closing of several voltage-dependent ion channels within the cell membrane. The shape of an AP can carry information concerning the state of the involved ion channels as well as their relationship to cellular processes. Alteration of these ion channels by the administration of toxins, drugs, and biochemicals can change the AP’s shape in a specific way, which can be characteristic for a given compound. Thus, AP shape analysis could be a valuable tool for toxin classification and the measurement of drug effects based on their mechanism of action. In an effort to begin classifying the effect of toxins on the shape of intracellularly recorded APs, patch-clamp experime... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=3082026 Sat, 12 Dec 2009 00:18:47 +0100 3082026 http://www.medworm.com/index.php?rid=3082025&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F14%2F10%2F1216%3Frss%3D1 The National Toxicology Program is developing a high-throughput screening (HTS) program to set testing priorities for compounds of interest, to identify mechanisms of action, and potentially to develop predictive models for human toxicity. This program will generate extensive data on the activity of large numbers of chemicals in a wide variety of biochemical- and cell-based assays. The first step in relating patterns of response among batteries of HTS assays to in vivo toxicity is to distinguish between positive and negative compounds in individual assays. Here, the authors report on a statistical approach developed to identify compounds positive or negative in an HTS cytotoxicity assay based on data collected from screening 1353 compounds for concentration-response effects in 9 human and ... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=3082025 Sat, 12 Dec 2009 00:18:47 +0100 3082025 http://www.medworm.com/index.php?rid=3082024&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F14%2F10%2F1207%3Frss%3D1 One of the challenges in developing cell lines for high-throughput screening in drug discovery is the labor- and time-intensive process required to create stable clonal cell lines that express specific reporters or drug targets. The authors report here the generation of a site-specific retargeting platform in 3 different cell lines: adherent HEK293, suspension CHO-S, and a human embryonic cell line (BGO1V). These platform cell lines were generated by using a combination of 2 site-specific integrases to develop a system that allows one to efficiently target a gene of interest to a specific locus and generates rapid production of homogeneous cell pools that stably express the gene of interest. The phiC31 integrase was used to create a platform line by placing a target site for the R4 integra... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=3082024 Sat, 12 Dec 2009 00:18:47 +0100 3082024 http://www.medworm.com/index.php?rid=3082023&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F14%2F10%2F1195%3Frss%3D1 RGS proteins are critical modulators of G-protein-coupled receptor (GPCR) signaling given their ability to deactivate G subunits via GTPase-accelerating protein (GAP) activity. Their selectivity for specific GPCRs makes them attractive therapeutic targets. However, measuring GAP activity is complicated by slow guanosine diphosphate (GDP) release from G and lack of solution phase assays for detecting free GDP in the presence of excess guanosine triphosphate (GTP). To overcome these hurdles, the authors developed a Gi1 mutant with increased GDP dissociation and decreased GTP hydrolysis rates, enabling detection of GAP activity using steady-state GTP hydrolysis. Gi1(R178M/A326S) GTPase activity was stimulated 6- to 12-fold by RGS proteins known to act on Gi subunits and not affected by those ... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=3082023 Sat, 12 Dec 2009 00:18:47 +0100 3082023 http://www.medworm.com/index.php?rid=3082022&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F14%2F10%2F1185%3Frss%3D1 This study shows the adaptation of the standard cell lysis method to a screening assay with improved throughput and assay characteristics. The assay takes advantage of the 384-well format, standard laboratory automation, and cryopreserved CHO-K1 cells stably overexpressing human GlyT1a transporter (CHO-K1/hGlyT1a) that were validated and banked in advance of screening. The assay was evaluated for the time course of glycine uptake, Km, Vmax, Z' factor analysis, and IC50 value determination with reference GlyT1 inhibitors. Screening of 118,000 compounds at 10 µM identified 4556 compounds (3.9%) as inhibitors. Positive compounds (>" xbd="1937" xhg="1914" ybd="1135" yhg="1097"/>50% inhibition) were retested in the assay at 4 inhibitor concentrations. Compounds demonstrating greater... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=3082022 Sat, 12 Dec 2009 00:18:47 +0100 3082022 http://www.medworm.com/index.php?rid=3082021&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F14%2F10%2F1176%3Frss%3D1 Drug treatment for human lung cancers remains unsatisfactory, despite the identification of many potential therapeutic targets (such as mutant KRAS protein) and the approval of agents that inhibit the tyrosine kinase activity of mutant epidermal growth factor receptor (EGFR). To seek new therapeutic strategies against lung tumors, the authors have screened 189,290 small molecules for their ability to retard growth of human lung adenocarcinoma cell lines, which harbor mutations in EGFR or KRAS. Four candidates that are structurally different from common tyrosine kinase inhibitors were selected for further study. The authors describe one small molecule (designated lung cancer screen—1 [LCS-1]) in detail here. Identification of the targets of LCS-1 and other growth inhibitors found in t... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=3082021 Sat, 12 Dec 2009 00:18:47 +0100 3082021 http://www.medworm.com/index.php?rid=3082020&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F14%2F10%2F1165%3Frss%3D1 Cancer cells are known to experience a high level of stress and may require constant repair for survival and proliferation. Recent studies showed that inhibition of heat shock factor 1 (HSF1), the key regulator for the stress-activated transcription of heat shock protein (HSP), can reduce the tumorigenic potential of cancer cells. Such a "nononcogene addiction" phenomenon makes HSF1 an attractive cancer drug target. Here, the authors report an image-based high-content screening (HCS) assay for HSF1 functional inhibitors. A heat shock—based methodology was used to stimulate the stress response followed by quantitative measurement of HSF1/HSP70 granules for compound-induced inhibitory effects. The authors discovered a small molecule from a compound library that inhibits HSF1 granule fo... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=3082020 Sat, 12 Dec 2009 00:18:46 +0100 3082020 http://www.medworm.com/index.php?rid=3082019&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F14%2F10%2F1157%3Frss%3D1 High-throughput affinity-based technologies are rapidly growing in use as primary screening methods in drug discovery. In this review, their principles and applications are described and their impact on small-molecule drug discovery is evaluated. In general, these technologies can be divided into 2 groups: those that detect binding interactions by measuring changes to the protein target and those that detect bound compounds. Technologies detecting binding interactions by focusing on the protein have limited throughput but can reveal mechanistic information about the binding interaction; technologies detecting bound compounds have very high throughput, some even significantly higher than current high-throughput screening technologies, but offer limited information about the binding interact... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=3082019 Sat, 12 Dec 2009 00:18:46 +0100 3082019 http://www.medworm.com/index.php?rid=3082018&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Freprint%2F14%2F10%2F1155%3Frss%3D1 (Source: Journal of Biomolecular Screening) Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=3082018 Sat, 12 Dec 2009 00:18:46 +0100 3082018 http://www.medworm.com/index.php?rid=2988438&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Freprint%2F14%2F9%2F1149%3Frss%3D1 (Source: Journal of Biomolecular Screening) Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=2988438 Thu, 12 Nov 2009 16:41:25 +0100 2988438 http://www.medworm.com/index.php?rid=2988437&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Freprint%2F14%2F9%2F1142%3Frss%3D1 (Source: Journal of Biomolecular Screening) Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=2988437 Thu, 12 Nov 2009 16:41:25 +0100 2988437 http://www.medworm.com/index.php?rid=2988436&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F14%2F9%2F1134%3Frss%3D1 Cellular assay development for the endothelial differentiation gene (EDG) family of G-protein-coupled receptors (GPCRs) and related lysophospholipid (LP) receptors is complicated by endogenous receptor expression and divergent receptor signaling. Endogenously expressed LP receptors exist in most tissue culture cell lines. these LP receptors, along with other endogenously expressed GPCRs, contribute to off-target signaling that can complicate interpretation of second-messenger-based cellular assay results. these receptors also activate a diverse and divergent set of cellular signaling pathways, necessitating the use of a variety of assay formats with mismatched procedures and functional readouts. this complicates examination and comparison of these receptors across the entire family. the ta... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=2988436 Thu, 12 Nov 2009 16:41:25 +0100 2988436 http://www.medworm.com/index.php?rid=2988435&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F14%2F9%2F1129%3Frss%3D1 The authors describe a continuous protein methylation assay using the G9a protein lysine methyltransferase and its substrate protein WIZ (widely interspaced zinc finger motifs). The assay is based on the coupling of the biotinylated substrate protein to streptavidin-coated FlashPlates and the transfer of radioactive methyl groups from the S-adenosyl-L-methionine to the substrate. The reaction progress is monitored continuously by proximity scintillation counting. The assay is very accurate, convenient, well suited for automation, and highly reproducible with standard errors in the range of 5%. Because of few pipetting steps and continuous data readout, it is ideal for high-throughput applications such as screening of inhibitors, testing many enzyme variants, or analyzing differences in met... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=2988435 Thu, 12 Nov 2009 16:41:25 +0100 2988435 http://www.medworm.com/index.php?rid=2988434&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F14%2F9%2F1119%3Frss%3D1 In this study, the authors report the development of a novel membrane potential-sensitive dye (MPSD) assay for HCN channel modulators that has been miniaturized into 384-well fluorescent imaging plate reader (FLIPR) high-throughput screening (HTS) format. When optimized (by cell plating density, plate type, cell recovery from cryopreservation), the wellto-well signal variability was low, with a Z' = 0.73 and coefficient of variation = 6.4%, whereas the MPSD fluorescence signal amplitude was -23,700 ± 1500 FLIPR3 relative fluorescence units (a linear relationship was found between HCN1 MPSD fluorescence signal and the cell plating density) and was completely blocked by 30 µM ZD7288. The assay tolerated up to 1% DMSO, inclusion of which did not significantly change the signal ki... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=2988434 Thu, 12 Nov 2009 16:41:25 +0100 2988434 http://www.medworm.com/index.php?rid=2988433&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F14%2F9%2F1109%3Frss%3D1 -Cobratoxin (Cbtx), the neurotoxin isolated from the venom of the Thai cobra Naja kaouthia , causes paralysis by preventing acetylcholine (ACh) binding to nicotinic acetylcholine receptors (nAChRs). In the current study, the region of the Cbtx molecule that is directly involved in binding to nAChRs is used as the target for anticobratoxin drug design. The crystal structure (1YI5) of Cbtx in complex with the acetylcholine binding protein (AChBP), a soluble homolog of the extracellular binding domain of nAChRs, was selected to prepare an -cobratoxin active binding site for docking. The amino acid residues (Ser182-Tyr192) of the AChBP structure, the binding site of Cbtx, were used as the positive control to validate the prepared Cbtx active binding site (root mean square deviation < 1.2 &A... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=2988433 Thu, 12 Nov 2009 16:41:25 +0100 2988433 http://www.medworm.com/index.php?rid=2988431&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F14%2F9%2F1102%3Frss%3D1 The aim of this work was to perform a systematic study of the effect of nonionic detergents on the activity of the dengue virus NS2B-NS3 protease. To ensure a high activity of the protease, the assay procedures for the dengue virus and other flaviviral proteases published to date are performed in the presence of up to 35% glycerol, which does not represent the cellular physicochemical environment. In addition, the high viscosity of glycerol-containing solutions leads to various experimental problems in miniaturized assays. Using an internally quenched peptide substrate, the authors show that glycerol is not essential for enzymatic activity if certain nonionic detergents are added to the assay buffer. In addition, nonionic detergents may help to avoid false-positive screening results caused... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=2988431 Thu, 12 Nov 2009 16:41:25 +0100 2988431 http://www.medworm.com/index.php?rid=2988429&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F14%2F9%2F1092%3Frss%3D1 The phenomenon of antibiotic resistance has created a need for the development of novel antibiotic classes with nonclassical cellular targets. Unfortunately, target-based drug discovery against proteins considered essential for in vitro bacterial viability has yielded few new therapeutic classes of antibiotics. Targeting the large proportion of genes considered nonessential that have yet to be explored by high-throughput screening, for example, RecA, can complement these efforts. Recent evidence suggests that RecA-controlled processes are responsible for tolerance to antibiotic chemotherapy and are involved in pathways that ultimately lead to full-fledged antibiotic resistance. Therefore inhibitors of RecA may serve as therapeutic adjuvants in combination chemotherapy of bacterial infectio... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=2988429 Thu, 12 Nov 2009 16:41:25 +0100 2988429 http://www.medworm.com/index.php?rid=2988427&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F14%2F9%2F1076%3Frss%3D1 To study CXCR2 modulated arrestin redistribution, the authors employed arrestin as a fusion protein containing either the Aequorea victoria—derived enhanced green fluorescent protein (EGFP) or a recently developed mutant of eqFP611, a red fluorescent protein derived from Entacmaea quadricolor. This mutant, referred to as RFP611, had earlier been found to assume a dimeric quarternary structure. It was therefore employed in this work as a "tandem" (td) construct for pseudo monomeric fusion protein labeling. Both arrestin fusion proteins, containing either td RFP611 (Arr td RFP611) or enhanced green fluorescent protein (EGFP; Arr EGFP), were found to colocalize with internalized fluorescently labeled Gro a few minutes after Gro addition. Intriguingly, however, Arr td RFP611 and Arr EGFP... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=2988427 Thu, 12 Nov 2009 16:41:25 +0100 2988427 http://www.medworm.com/index.php?rid=2988425&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F14%2F9%2F1067%3Frss%3D1 C5L2 (or GPR77) is a high-affinity receptor for the complement fragment C5a and its desarginated product, C5a-desArg. Unlike the classical C5a receptor CD88, C5L2 does not couple to intracellular G-protein-signaling pathways but is thought to function as a decoy receptor. The authors show that stimulation of C5L2 with C5a and C5a-desArg induces redistribution of green fluorescent protein—labeled β-arrestin2 to cytoplasmic vesicles. C3a and C3a-desArg were inactive in the β-arrestin translocation assay. Direct interaction of ligand-stimulated C5L2 with β-arrestin was confirmed using a novel β-galactosidase fragment complementation assay. In this assay, C5L2 was labeled with a mutationally altered peptide fragment of β-galactosidase, whereas β-arrestin2 wa... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=2988425 Thu, 12 Nov 2009 16:41:25 +0100 2988425 http://www.medworm.com/index.php?rid=2988424&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F14%2F9%2F1054%3Frss%3D1 The complexity of human biology has made prediction of health effects as a consequence of exposure to environmental chemicals especially challenging. Complex cell systems, such as the Biologically Multiplexed Activity Profiling (BioMAP) primary, human, cell-based disease models, leverage cellular regulatory networks to detect and distinguish chemicals with a broad range of target mechanisms and biological processes relevant to human toxicity. Here the authors use the BioMAP human cell systems to characterize effects relevant to human tissue and inflammatory disease biology following exposure to the 320 environmental chemicals in the Environmental Protection Agency’s (EPA’s) ToxCast phase I library. The ToxCast chemicals were assayed at 4 concentrations in 8 BioMAP cell systems,... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=2988424 Thu, 12 Nov 2009 16:41:25 +0100 2988424 http://www.medworm.com/index.php?rid=2988423&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F14%2F9%2F1045%3Frss%3D1 All solid malignancies share characteristic traits, including unlimited cellular proliferation, evasion of immune regulation, and the propensity to metastasize. The authors have previously described that a subnuclear structure, the perinucleolar compartment (PNC), is associated with the metastatic phenotype in solid tumor cancer cells. The percentage of cancer cells that contain PNCs (PNC prevalence) is indicative of the malignancy of a tumor both in vitro and in vivo, and thus PNC prevalence is a marker that reflects metastatic capability in a population of tumor cells. Although the function of the PNC remains to be determined, the PNC is highly enriched with small RNAs and RNA binding proteins. The initial chemical biology studies using a set of anticancer drugs that disassemble PNCs rev... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=2988423 Thu, 12 Nov 2009 16:41:25 +0100 2988423 http://www.medworm.com/index.php?rid=2988422&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Freprint%2F14%2F9%2F1043%3Frss%3D1 (Source: Journal of Biomolecular Screening) Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=2988422 Thu, 12 Nov 2009 16:41:25 +0100 2988422 http://www.medworm.com/index.php?rid=2850928&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Freprint%2F14%2F8%2F1039%3Frss%3D1 (Source: Journal of Biomolecular Screening) Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=2850928 Thu, 01 Oct 2009 17:19:46 +0100 2850928 http://www.medworm.com/index.php?rid=2850926&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Freprint%2F14%2F8%2F1032%3Frss%3D1 (Source: Journal of Biomolecular Screening) Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=2850926 Thu, 01 Oct 2009 17:19:46 +0100 2850926 http://www.medworm.com/index.php?rid=2850925&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F14%2F8%2F1017%3Frss%3D1 Since the advent of high-throughput screening (HTS) in the early 1990s, parallel multichannel liquid handlers have become a mainstay in every drug discovery setting. Although several peer-reviewed publications have discussed methods and criteria for stamping multiwell copies, there is very little information about establishing a standard operating procedure (SOP) for standard (microliter-level) serial dilutions of compounds used in dose-response experiments. The authors discuss the 4 main criteria any serial dilution process must pass (accuracy, precision, fold dilution, and outliers) and the process for establishing thresholds for all of these values in a compound management or biological screening laboratory. The thresholds need to be both low enough to be acceptable from a biological po... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=2850925 Thu, 01 Oct 2009 17:19:46 +0100 2850925 http://www.medworm.com/index.php?rid=2850923&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F14%2F8%2F1008%3Frss%3D1 In high-throughput biochemical assays performed in multiwell plates, the effect of test samples on the activity of the biochemical system is usually measured by optical means such as absorbance, fluorescence, luminescence, or scintillation counting. The test sample often causes detection interference when it remains in the well during the measurement. Interference may be due to light absorption, fluorescence quenching, sample fluorescence, chemical interaction of the sample with a detection reagent, or depression of the meniscus. A simple method is described that corrects for such interference well by well. The interference is measured in a separate artifact assay plate. An appropriate arithmetic correction is then applied to the measurement in the corresponding well of the activity assay ... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=2850923 Thu, 01 Oct 2009 17:19:46 +0100 2850923 http://www.medworm.com/index.php?rid=2850922&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F14%2F8%2F999%3Frss%3D1 A drug discovery startup company or academic lab entering the screening arena faces numerous challenges as it tries to manage the large quantity of data generated by a typical drug discovery screening campaign. Although there are sophisticated off-the-shelf software solutions available, their use requires substantial forethought and attention to detail if the data they capture are to be of sufficient quality to serve the various purposes to which it will be put. For newcomers to the field of screening data management in particular, the problem is compounded by a lack of literature covering the practical aspects of managing screening data. The authors provide some practical advice based on their experience of using a commercially available software suite. They discuss issues ranging from th... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=2850922 Thu, 01 Oct 2009 17:19:46 +0100 2850922 http://www.medworm.com/index.php?rid=2850921&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F14%2F8%2F991%3Frss%3D1 The botulinum neurotoxins (BoNTs) produced by Clostridium botulinum are the most poisonous protein substances known. The neutralizing antibodies against botulinum neurotoxin can effectively prevent and cure the toxicosis. Using purified Hc fragments of botulinum neurotoxin serotype A (BoNT/A-Hc) as antigen, 2 specific neutralizing antibodies mapping different epitopes were selected from a fully synthetic human antibody library. The 2 antibodies can effectively inhibit the binding between BoNT/A-Hc and differentiated PC-12 cells in vitro, and the neutralization was evaluated in vivo. Although no single mAb completely protected mice from toxin, they both could prolong time to death when challenged with 20 LD 50s (50% lethal doses) of BoNT/A. When used together, the mAbs completely neutralize... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=2850921 Thu, 01 Oct 2009 17:19:46 +0100 2850921 http://www.medworm.com/index.php?rid=2850920&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F14%2F8%2F980%3Frss%3D1 The aim of the present study was to assess the feasibility of a 3D tumor cell culture model, that is, multicellular tumor spheroids (MCTSs) as an adequate model for micrometastases and therefore as a pharmacological model for efficacy testing of trifunctional therapeutic antibodies. Unlike conventional monolayer cultures, spheroids allow researchers to study parameters, such as 3D cell shape, 3D cell arrangement and microenvironment, and penetration efficiency of defense cells that may largely influence the efficacy of antibody treatment in vivo. The authors established a long-term coculture of human MCTSs with peripheral blood mononuclear cells (PBMCs) to test the anticancer effect of the trifunctional, bispecific antibody catumaxomab (anti-EpCAM x anti-CD3) or similar therapeutic molecul... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=2850920 Thu, 01 Oct 2009 17:19:46 +0100 2850920 http://www.medworm.com/index.php?rid=2850919&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F14%2F8%2F970%3Frss%3D1 Protein-protein interactions (PPIs) play key roles in all cellular processes and hence are useful as potential targets for new drug development. To facilitate the screening of PPI inhibitors as anticancer drugs, the authors have developed a high-throughput screening (HTS) system using an in vitro protein fragment complementation assay (PCA) with monomeric Kusabira-Green fluorescent protein (mKG). The in vitro PCA system was established by the topological formation of a functional complex between 2 split inactive mKG fragments fused to target proteins, which fluoresces when 2 target proteins interact to allow complementation of the mKG fragments. Using this assay system, the authors screened inhibitors for TCF7/β-catenin, PAC1/PAC2, and PAC3 homodimer PPIs from 123,599 samples in their... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=2850919 Thu, 01 Oct 2009 17:19:46 +0100 2850919 http://www.medworm.com/index.php?rid=2850918&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F14%2F8%2F956%3Frss%3D1 Caspases are central to the execution of programmed cell death, and their activation constitutes the biochemical hallmark of apoptosis. In this article, the authors report the successful adaptation of a high-content assay method using the DEVDNucView488TM fluorogenic substrate, and for the first time, they show caspase activation in live cells induced by either drugs or siRNA. The fluorogenic substrate was found to be nontoxic over an exposure period of several days, during which the authors demonstrate automated imaging and quantification of caspase activation of the same cell population as a function of time. Overexpression of the antiapoptotic protein Bcl-XL, alone or in combination with the inhibitor Z-VAD-FMK, attenuated caspase activation in HeLa cells exposed to doxorubicin, etoposi... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=2850918 Thu, 01 Oct 2009 17:19:46 +0100 2850918 http://www.medworm.com/index.php?rid=2850917&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F14%2F8%2F944%3Frss%3D1 In this article, the authors describe the image analysis software DetecTiff©, which allows fully automated object recognition and quantification from digital images. The core module of the LabView©-based routine is an algorithm for structure recognition that employs intensity thresholding and size-dependent particle filtering from microscopic images in an iterative manner. Detected structures are converted into templates, which are used for quantitative image analysis. DetecTiff © enables processing of multiple detection channels and provides functions for template organization and fast interpretation of acquired data. The authors demonstrate the applicability of DetecTiff© for automated analysis of cellular uptake of fluorescencelabeled low-density lipoproteins as well... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=2850917 Thu, 01 Oct 2009 17:19:46 +0100 2850917 http://www.medworm.com/index.php?rid=2850916&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F14%2F8%2F936%3Frss%3D1 In this article, a single-label separation-free fluorescence technique is presented as a potential screening method for cell-based receptor antagonists and agonists.The time-resolved fluorescence technique, quenching resonance energy transfer (QRET), relies on a single-labeled binding partner in combination with a soluble quencher. The quencher efficiently suppresses the luminescence of the unbound labeled ligand, whereas the luminescence of the bound fraction is not affected. This approach allows the development of cell-based screening assays in a simple and cost-effective manner. The authors have applied the technique to the screening of β2-adrenoreceptor (β2AR) antagonists and agonists in intact human embryonic kidney HEK293i cells overexpressing human β2-adrenergic recep... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=2850916 Thu, 01 Oct 2009 17:19:46 +0100 2850916 http://www.medworm.com/index.php?rid=2850915&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F14%2F8%2F924%3Frss%3D1 The expansion of kinase assay technologies over the past decade has mirrored the growing interest in kinases as drug targets. As a result, there is no shortage of convenient, fluorescence-based methods available to assay targets that span the kinome. The authors recently reported on the development of a non-activity-based assay to characterize kinase inhibitors that depended on displacement of an Alexa Fluor® 647 conjugate of staurosporine (a "tracer") from a particular kinase. Kinase inhibitors were characterized by a change in fluorescence lifetime of the tracer when it was bound to a kinase relative to when it was displaced by an inhibitor. Here, the authors report on improvements to this strategy by reconfiguring the assay in a time-resolved fluorescence resonance energy transfer (... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=2850915 Thu, 01 Oct 2009 17:19:46 +0100 2850915 http://www.medworm.com/index.php?rid=2850914&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F14%2F8%2F913%3Frss%3D1 The potential of enzyme inhibition of a drug is frequently quantified in terms of IC50 values. Although this is a suitable quantity for reversible inhibitors, concerns arise when dealing with irreversible or mechanism-based inhibitors (MBIs). IC50 values of MBIs are time dependent, causing serious problems when aiming at ranking different compounds with respect to their inhibitory potential. As a consequence, most studies and ranking schemes related to MBIs rely on the inhibition constant (KI) and the rate of enzyme inactivation (kinact) rather than on IC50 values. In this article, the authors derive a novel relation between potentially time-dependent IC 50 values and KI, kinact parameters for different types of inhibition. This allows for direct estimation of KI and kinact values from tim... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=2850914 Thu, 01 Oct 2009 17:19:45 +0100 2850914 http://www.medworm.com/index.php?rid=2850913&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Freprint%2F14%2F8%2F907%3Frss%3D1 (Source: Journal of Biomolecular Screening) Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=2850913 Thu, 01 Oct 2009 17:19:45 +0100 2850913 http://www.medworm.com/index.php?rid=2757109&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Freprint%2F14%2F7%2F870%3Frss%3D1 (Source: Journal of Biomolecular Screening) Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=2757109 Mon, 31 Aug 2009 23:00:00 +0100 2757109 http://www.medworm.com/index.php?rid=2757108&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Freprint%2F14%2F7%2F865%3Frss%3D1 (Source: Journal of Biomolecular Screening) Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=2757108 Mon, 31 Aug 2009 23:00:00 +0100 2757108 http://www.medworm.com/index.php?rid=2757107&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Freprint%2F14%2F7%2F860%3Frss%3D1 (Source: Journal of Biomolecular Screening) Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=2757107 Mon, 31 Aug 2009 23:00:00 +0100 2757107 http://www.medworm.com/index.php?rid=2757106&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F14%2F7%2F853%3Frss%3D1 Cellular senescence is a persistently growth-arrested phenotype in normal and transformed cells induced by noncytotoxic stress. Cytostasis as a method of cancer treatment has recently generated significant interest. Research into the induction of cellular senescence as cancer therapy has been hindered by a lack of compounds that efficiently induce this response. The authors describe a semiautomated high-throughput method to identify library compounds that induce senescence using prostate cancer cells cultured in 96-well plates. Primary hits are identified by low cell numbers after 3 days in culture, measured by Hoechst 33342 fluorescence. A secondary visual assessment of senescence-associated β-galactosidase staining and cellular morphology in the same wells distinguishes senescence f... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=2757106 Mon, 31 Aug 2009 23:00:00 +0100 2757106 http://www.medworm.com/index.php?rid=2757105&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F14%2F7%2F845%3Frss%3D1 Reporter assays are widely used in research and drug discovery for analysis of signaling pathways in a cell-based format. Traditionally, reporter gene assays are run in a single-parameter mode, interrogating only 1 pathway per sample. To enable more complex assay formats for pathway analysis, the authors developed a multiplexed reporter cell-based assay that combines optical encoding with a β-lactamase reporter gene readout. The optical encoding is achieved by peptide-mediated delivery of quantum dots into reporter cell lines. Using different quantum dots, the authors were able to simultaneously analyze multiple signaling pathways in the same sample using fluorescence microscopy or flow cytometry. They selected 3 β-lactamase reporter cell lines for the analysis of tumor necrosis ... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=2757105 Mon, 31 Aug 2009 23:00:00 +0100 2757105 http://www.medworm.com/index.php?rid=2757104&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F14%2F7%2F838%3Frss%3D1 Lipid kinases are central players in lipid signaling pathways involved in inflammation, tumorigenesis, and metabolic syndrome. A number of these kinase targets have proven difficult to investigate in higher throughput cell-free assay systems. This challenge is partially due to specific substrate interaction requirements for several of the lipid kinase family members and the resulting incompatibility of these substrates with most established, homogeneous assay formats. Traditional, cell-free in vitro investigational methods for members of the lipid kinase family typically involve substrate incorporation of [-32P] and resolution of signal by thin-layer chromatography (TLC) and autoradiograph densitometry. This approach, although highly sensitive, does not lend itself to high-throughput testi... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=2757104 Mon, 31 Aug 2009 23:00:00 +0100 2757104 http://www.medworm.com/index.php?rid=2757103&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F14%2F7%2F824%3Frss%3D1 This article provides an example of a success where the Roadmap Initiative collaborative model, sponsored by the National Institutes of Health, brought together a deep knowledge of target biology from a principal investigator’s laboratory, a well-designed compound collection from the MLSMR, and an industrial-level screening facility and staff at the MLSCN center to identify pharmacologically active compounds, with outstanding selectivity data from a panel of more than 200 publicly accessible assays, through a high-throughput screen. (Journal of Biomolecular Screening 2009:824-837) (Source: Journal of Biomolecular Screening) Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=2757103 Mon, 31 Aug 2009 23:00:00 +0100 2757103 http://www.medworm.com/index.php?rid=2757102&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F14%2F7%2F811%3Frss%3D1 Receptor redistribution and β-arrestin recruitment assays provide a G-protein-subtype-independent method to measure ligand-stimulated activation of G-protein-coupled receptors. In particular β-arrestin assays are becoming an increasingly popular tool for drug discovery. The authors have compared a high-content-imaging-based Redistribution® assay and 2 nonimaging-based β-arrestin recruitment assays, TangoTM and PathHunter TM, for the cannabinoid receptor 1. Inasmuch as all 3 assays use receptors that are modified at the C-terminus, the authors verified their pharmacology via detection of Gi coupling of the receptor in cAMP assays using reference ligands. The potencies and efficacies of the cannabinoid receptor agonists CP55,940 and WIN55,212-2 correlated well between the ... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=2757102 Mon, 31 Aug 2009 23:00:00 +0100 2757102 http://www.medworm.com/index.php?rid=2757101&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F14%2F7%2F798%3Frss%3D1 Seven-transmembrane (7TM) receptors play an essential role in the regulation of a wide variety of physiological processes, making them one of the top target classes for pharmaceuticals. 7TM receptor function is mediated and modulated through 2 primary processes: G-protein and beta-arrestin signaling. Classically, it has been recognized that these 2 processes can interact with one another during 7TM receptor desensitization, but it has more recently been recognized that these 2 processes can also act independently of one another and can activate parallel signaling pathways. As such, the methods used to interrogate 7TM receptor signaling, both from a biological and a pharmaceutical perspective, may need to be reevaluated and the question of whether functionally selective compounds (compounds... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=2757101 Mon, 31 Aug 2009 23:00:00 +0100 2757101 http://www.medworm.com/index.php?rid=2757100&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F14%2F7%2F789%3Frss%3D1 GPR139 is an orphan G-protein-coupled receptor (GPCR) that is expressed nearly exclusively in the central nervous system and may play a role in the control of locomotor activity. The signal transduction pathway and pharmacological function of GPR139, however, are still controversial due to the lack of natural or synthetic ligands. The authors report the characterization of human GPR139 signaling pathway and identification of surrogate agonists and antagonists. In both transient and stable transfections of HEK293F cells, overexpression of GPR139 increased basal intracellular cAMP concentrations compared to control cells. Furthermore, forskolin and isoproterenol-stimulated cAMP responses were enhanced in GPR139-expressing cells, suggesting that GPR139 is predominantly coupled to Gs. The auth... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=2757100 Mon, 31 Aug 2009 23:00:00 +0100 2757100 http://www.medworm.com/index.php?rid=2757099&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F14%2F7%2F781%3Frss%3D1 G-protein-coupled receptors (GPCRs) are important therapeutic targets for many areas of drug research and development. Although chimeric G16 proteins are valuable tools for detecting the activation of Gi/o-coupled receptors, the details of the activation process remain unclear. The authors introduce a series of chimeras that combine both G16 and G i/o (G16/o, G16/i2, and G 16/i3) into a well-established transient expression system to examine the ability of these chimeras to interact with D2 long-form (D 2L) dopamine and 5-HT1A serotonin receptors. The pEC 50 data obtained for known agonists were similar to results from previous studies that used other cell-based assays, thus indicating sufficient sensitivity for the assay. Moreover, quinpirole exhibited similar intrinsic activity to dopami... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=2757099 Mon, 31 Aug 2009 23:00:00 +0100 2757099 http://www.medworm.com/index.php?rid=2757098&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F14%2F7%2F769%3Frss%3D1 -Amino butyric acid (GABA)—activated Cl— channels are critical mediators of inhibitory postsynaptic potentials in the CNS. To date, rational design efforts to identify potent and selective GABAA subtype ligands have been hampered by the absence of suitable high-throughput screening approaches. The authors describe 384-well population patch-clamp (PPC) planar array electrophysiology methods for the study of GABAA receptor pharmacology. In HEK293 cells stably expressing human 1β32 GABAA channels, GABA evoked outward currents at 0 mV of 1.05 ± 0.08 nA, measured 8 s post GABA addition. The IGABA was linear and reversed close to the theoretical ECl (—56 mV). Concentration-response curve analysis yielded a mean pEC50 value of 5.4 and Hill slope of 1.5, and for a ser... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=2757098 Mon, 31 Aug 2009 23:00:00 +0100 2757098 http://www.medworm.com/index.php?rid=2757097&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F14%2F7%2F755%3Frss%3D1 Stem cells, irrespective of their origin, have emerged as valuable reagents or tools in human health in the past 2 decades. Initially, a research tool to study fundamental aspects of developmental biology is now the central focus of generating transgenic animals, drug discovery, and regenerative medicine to address degenerative diseases of multiple organ systems. This is because stem cells are pluripotent or multipotent cells that can recapitulate developmental paths to repair damaged tissues. However, it is becoming clear that stem cell therapy alone may not be adequate to reverse tissue and organ damage in degenerative diseases. Existing small-molecule drugs and biologicals may be needed as "molecular adjuvants" or enhancers of stem cells administered in therapy or adult stem cells in th... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=2757097 Mon, 31 Aug 2009 23:00:00 +0100 2757097 http://www.medworm.com/index.php?rid=2757096&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Freprint%2F14%2F7%2F743%3Frss%3D1 (Source: Journal of Biomolecular Screening) Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=2757096 Mon, 31 Aug 2009 23:00:00 +0100 2757096 http://www.medworm.com/index.php?rid=2613483&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Freprint%2F14%2F6%2F737%3Frss%3D1 (Source: Journal of Biomolecular Screening) Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=2613483 Thu, 16 Jul 2009 23:00:00 +0100 2613483 http://www.medworm.com/index.php?rid=2613482&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Freprint%2F14%2F6%2F732%3Frss%3D1 (Source: Journal of Biomolecular Screening) Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=2613482 Thu, 16 Jul 2009 23:00:00 +0100 2613482 http://www.medworm.com/index.php?rid=2613481&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F14%2F6%2F723%3Frss%3D1 The innate immune system of mammals is a key defense mechanism against invading foreign pathogens. Innate immune stimulants may have applications as vaccine adjuvants as well as in the treatment of cancer and some viral diseases, and clinical studies have been performed using agonists of Toll-like receptors (TLRs) 7, 8, and 9. The high-throughput screens for such agonists have typically relied on the overexpression of a single TLR gene in an immortalized cell line and are inherently artificial systems that are restricted to the identification of agonists for a single receptor. The authors describe 2 assays for the identification of immunostimulants that employ primary human leukocytes cocultured with hepatitis C virus (HCV) replicon-expressing cells. In these assays, stimulation of innate ... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=2613481 Thu, 16 Jul 2009 23:00:00 +0100 2613481 http://www.medworm.com/index.php?rid=2613480&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F14%2F6%2F716%3Frss%3D1 Construction and supply of cell-based reagents for in vitro plate-based screens are often highlighted as a bottleneck within drug discovery. Recent years have seen the successful application of both cryopreservation and automation to increase the capacity and flexibility of cell provision. However, routine cell culture remains a fixed experimental process that requires cells to be prepared and used at specific times. We have investigated the potential of reduced temperature incubation to be used as a simple methodology for stopping and starting cell growth and introduce further flexibility into cell provision. Our results show that incubation of CHOK1, HEK293, and 1321N1 cells at 23 °C arrested growth while maintaining cell viability. Recovery of these paused cells at 37 °C resulte... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=2613480 Thu, 16 Jul 2009 23:00:00 +0100 2613480 http://www.medworm.com/index.php?rid=2613479&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F14%2F6%2F708%3Frss%3D1 The common practice of preparing storage libraries of compounds in 100% DMSO solution well in advance of bioassay brings with it difficulties that affect the accuracy of the data obtained. This publication presents a series of studies done on a subset of compounds that are difficult to bioassay because they precipitate from DMSO solution. These compounds are members of a frequently used, diverse compound library of the sort commonly used in the high-throughput screening (HTS) environment. Experiments were performed to determine the concentration of drug in solution above the precipitate, observe the time course and effect of various mixtures of solvents upon precipitation, measure the viscosity of cosolvents to determine compatibility with HTS, determine water absorption rates for various ... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=2613479 Thu, 16 Jul 2009 23:00:00 +0100 2613479 http://www.medworm.com/index.php?rid=2613478&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F14%2F6%2F700%3Frss%3D1 In the past decade, thermal melt/thermal shift assays have become a common tool for identifying ligands and other factors that stabilize specific proteins. Increased stability is indicated by an increase in the protein's melting temperature (Tm). In optimizing the assays for subsequent screening of compound libraries, it is important to minimize the variability of Tm measurements so as to maximize the assay's ability to detect potential ligands. The authors present an investigation of Tm variability in recombinant proteins from Plasmodium parasites. Ligands of Plasmodium proteins are particularly interesting as potential starting points for drugs for malaria, and new drugs are urgently needed. A single standard buffer (100 mM HEPES [pH 7.5], 150 mM NaCl) permitted estimation of Tm for 58 o... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=2613478 Thu, 16 Jul 2009 23:00:00 +0100 2613478 http://www.medworm.com/index.php?rid=2613477&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F14%2F6%2F690%3Frss%3D1 Typically, screening collections of pharmaceutical companies contain more than a million compounds today. However, for certain high-throughput screening (HTS) campaigns, constraints posed by the assay throughput and/or the reagent costs make it impractical to screen the entire deck. Therefore, it is desirable to effectively screen subsets of the collection based on a hypothesis or a diversity selection. How to select compound subsets is a subject of ongoing debate. The authors present an approach based on extended connectivity fingerprints to carry out diversity selection on a per plate basis (instead of a per compound basis). HTS data from 35 Novartis screens spanning 5 target classes were investigated to assess the performance of this approach. The analysis shows that selecting a fingerp... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=2613477 Thu, 16 Jul 2009 23:00:00 +0100 2613477 http://www.medworm.com/index.php?rid=2613476&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F14%2F6%2F679%3Frss%3D1 In this study, the effect of such unwanted inhibitors on several different enzyme targets was assessed based on screening results for over a million compounds. In particular, the shift in potency on variation of enzyme concentration was used as a means to identify nonstoichiometric inhibitors among the screening hits. These potency shifts depended on both compound structure and target enzyme. The approach was confirmed by statistical analysis of thousands of dose-response curves, which showed that the potency of competitive and therefore clearly stoichiometric inhibitors was not affected by increasing enzyme concentration. Light-scattering measurements of thermal protein unfolding further verified that compounds that stabilize protein structure by stoichiometric binding show the same poten... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=2613476 Thu, 16 Jul 2009 23:00:00 +0100 2613476 http://www.medworm.com/index.php?rid=2613475&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F14%2F6%2F668%3Frss%3D1 Cytochrome P450 enzyme (CYP450s) assays are critical enzymes in early-stage lead discovery and optimization in drug development. Currently available fluorescence-based reaction assays provide a rapid and reliable method for monitoring CYP450 enzyme activity but are confined to medium-throughput well-plate systems. The authors present a high-throughput, integrated screening platform for CYP450 assays combining enzyme encapsulation techniques, microarraying methods, and wide-field imaging. Alginate-containing microarrays consisting of up to 1134 CYP450 reaction elements were fabricated on functionalized glass slides (reaction volumes 20 to 80 nL, total enzyme content in pg) and imaged to yield endpoint activity, stability, and kinetic data. A charge-coupled device imager acquired quantitativ... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=2613475 Thu, 16 Jul 2009 23:00:00 +0100 2613475 http://www.medworm.com/index.php?rid=2613474&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F14%2F6%2F662%3Frss%3D1 The precise temperature control of the ABI Prism® 7900HT Sequence Detection System designed for detection of fluorescence of a biological sample in real-time PCR assays (TaqMan assays) was used to activate Thermo-TRP ion channels, enabling a novel 384-/96-well plate-based assay. Functional pharmacology was verified against the temperature activation using intracellular calcium fluorescence as a measure of ion channel activity. The assay is applicable to both heterologous expression systems and dorsal root ganglia primary cells. This will benefit several analgesic drug discovery programs searching for new Thermo-TRP modulators. (Journal of Biomolecular Screening 2009:662-667) (Source: Journal of Biomolecular Screening) Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=2613474 Thu, 16 Jul 2009 23:00:00 +0100 2613474 http://www.medworm.com/index.php?rid=2613473&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F14%2F6%2F653%3Frss%3D1 Removal of apoptotic cells and cellular debris by phagocytosis is essential for development, tissue homeostasis, and resolution of inflammation. Eat-me signals control the initiation of phagocytosis, holding a key to the understanding of phagocyte biology. Because of a lack of functional cloning strategy, eat-me signals are conventionally identified and characterized on a case-by-case basis. The feasibility of functional cloning of eat-me signals by phage display is investigated by characterizing the biological behavior of T7 phages displaying 2 well-known eat-me signals: growth arrest—specific gene 6 (Gas6) and milk fat globule—EGF8 (MFG-E8). Gas6-phage binds to all 3 known Gas6 receptors: Mer, Axl, and Tyro3 receptor tyrosine kinases. Gas6-phage and MFG-E8-phage are capable o... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=2613473 Thu, 16 Jul 2009 23:00:00 +0100 2613473 http://www.medworm.com/index.php?rid=2613472&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F14%2F6%2F643%3Frss%3D1 Mycothiol ligase (MshC) is a key enzyme in the biosynthesis of mycothiol, a small molecular weight thiol that is unique to actinomycetes and whose primary role is to maintain intracellular redox balance and remove toxins. MshC catalyzes the adenosine triphosphate (ATP)—dependent condensation of cysteine and glucosamine-inositol (GI) to produce cysteine-glucosamine-inositol (CGI). MshC is essential to Mycobacterium tuberculosis and therefore represents an attractive target for chemotherapeutic intervention. A screening protocol was developed to identify MshC inhibitors based on quantification of residual ATP using a coupled luminescent assay. The protocol was used to screen a library of 3100 compounds in a 384-well plate format (Z' ≥ 0.65). Fifteen hits (0.48%) were identified from... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=2613472 Thu, 16 Jul 2009 23:00:00 +0100 2613472 http://www.medworm.com/index.php?rid=2613471&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F14%2F6%2F636%3Frss%3D1 Fatty acid synthase (FAS), an essential enzyme for de novo lipogenesis, has been implicated in a number of disease states, including obesity, dyslipidemia, and cancer. To identify small-molecule inhibitors of FAS, the authors developed a bead-based scintillation proximity assay (SPA) to detect the fatty acid products of FAS enzymatic activity. This homogeneous SPA assay discriminates between a radiolabeled hydrophilic substrate of FAS (acetyl-coenzyme A) and the labeled lipophilic products of FAS (fatty acids), generating signal only when labeled fatty acids are present. The assay requires a single addition of unmodified polystyrene imaging SPA beads and can be miniaturized to 384- or 1536-well density with appropriate assay statistics for high-throughput screening. High-potency FAS inhibi... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=2613471 Thu, 16 Jul 2009 23:00:00 +0100 2613471 http://www.medworm.com/index.php?rid=2613470&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F14%2F6%2F627%3Frss%3D1 The prolyl-4-hydroxylase proteins regulate the hypoxia-inducible transcription factors (HIFs) by hydroxylation of proline residues targeting HIF-1 for proteasomal degradation. Using the purified catalytic domain of prolyl hydroxylase 2 (PHD2181-417), an enzymatic assay has been developed to test inhibitors of the enzyme in vitro. Because PHD2 hydroxylates HIF-1, with succinic acid produced as an end product, radiolabeled [5-14C]-2-oxoglutaric acid was used and formation of [14C]-succinic acid was measured to quantify PHD2181-417 enzymatic activity. Comparison of the separation of 2-oxoglutaric acid and succinic acid by either ion exchange chromatography or precipitation with phenylhydrazine showed similar results, but the quantification and throughput were vastly increased using the latter... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=2613470 Thu, 16 Jul 2009 23:00:00 +0100 2613470 http://www.medworm.com/index.php?rid=2613469&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F14%2F6%2F620%3Frss%3D1 The interaction between the hepatitis C virus (HCV) envelope glycoprotein E2 and the human tetraspanin protein CD81 is one of the key events involved in HCV cell entry. Therefore, compounds that interfere with this interaction may be useful tools for basic research and potential drugs for the treatment of HCV infection. The authors describe a medium-throughput assay for ligands of the E2 binding site on the CD81 receptor. In the assay, human hepatoma cells are incubated with the test compounds and stained with a fluorescently labeled anti-CD81 antibody (JS81). Flow cytometry is used to detect the level of bound antibody, reflecting the inhibitory potencies of the compounds. Eighty percent of compounds active in the assay show efficacy in an infection assay using luciferase reporter genome ... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=2613469 Thu, 16 Jul 2009 23:00:00 +0100 2613469 http://www.medworm.com/index.php?rid=2613468&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F14%2F6%2F610%3Frss%3D1 This report focuses on 5 RGS proteins (4, 6, 7, 8, and 16). To increase the content of screens, the authors assessed high-throughput screening of these RGS proteins in multiplex, by establishing binding constants of each RGS with Go in isolation, and then in a multiplex format with 5 RGS proteins present. To use this methodology as a higher-content multiplex protein-protein interaction screen, they established Z-factor values for RGS proteins in multiplex of 0.73 to 0.92, indicating this method is suitable for screening using FCPIA. To increase throughput, they also compressed a set of 8000 compounds by combining 4 compounds in a single assay well. Subsequent deconvolution of the compounds mixtures verified the identification of active compounds at specific RGS targets in their mixtures us... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=2613468 Thu, 16 Jul 2009 23:00:00 +0100 2613468 http://www.medworm.com/index.php?rid=2613467&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F14%2F6%2F596%3Frss%3D1 Prostate cancer is a leading cause of death among men due to the limited number of treatment strategies available for advanced disease. Discovery of effective chemotherapeutics involves the identification of agents that inhibit cancer cell growth. Increases in intracellular granularity have been observed during physiological processes that include senescence, apoptosis, and autophagy, making this phenotypic change a useful marker for identifying small molecules that induce cellular growth arrest or death. In this regard, epithelial-derived cancer cell lines appear uniquely susceptible to increased intracellular granularity following exposure to chemotherapeutics. We have established a novel flow cytometry approach that detects increases in side light scatter in response to morphological ch... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=2613467 Thu, 16 Jul 2009 23:00:00 +0100 2613467 http://www.medworm.com/index.php?rid=2504353&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Freprint%2F14%2F5%2F580%3Frss%3D1 (Source: Journal of Biomolecular Screening) Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=2504353 Wed, 10 Jun 2009 23:00:00 +0100 2504353 http://www.medworm.com/index.php?rid=2504352&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Freprint%2F14%2F5%2F579%3Frss%3D1 (Source: Journal of Biomolecular Screening) Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=2504352 Wed, 10 Jun 2009 23:00:00 +0100 2504352 http://www.medworm.com/index.php?rid=2504351&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Freprint%2F14%2F5%2F574%3Frss%3D1 (Source: Journal of Biomolecular Screening) Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=2504351 Wed, 10 Jun 2009 23:00:00 +0100 2504351 http://www.medworm.com/index.php?rid=2504350&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F14%2F5%2F566%3Frss%3D1 Disposable plastic labware is ubiquitous in contemporary pharmaceutical research laboratories. Plastic labware is routinely used for chemical compound storage and during automated liquid-handling processes that support assay development, high-throughput screening, structure-activity determinations, and liability profiling. However, there is little information available in the literature on the contaminants released from plastic labware upon DMSO exposure and their resultant effects on specific biological assays. The authors report here the extraction, by simple DMSO washing, of a biologically active substance from one particular size of disposable plastic tips used in automated compound handling. The active contaminant was identified as erucamide ((Z)-docos-13-enamide), a long-chain mono-u... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=2504350 Wed, 10 Jun 2009 23:00:00 +0100 2504350 http://www.medworm.com/index.php?rid=2504349&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F14%2F5%2F557%3Frss%3D1 The technological evolution of the 1990s in both combinatorial chemistry and high-throughput screening created the demand for rapid access to the compound deck to support the screening process. The common strategy within the pharmaceutical industry is to store the screening library in DMSO solution. Several studies have shown that a percentage of these compounds decompose in solution, varying from a few percent of the total to a substantial part of the library. In the COMDECOM (COMpound DECOMposition) project, the compound stability of screening compounds in DMSO solution is monitored in an accelerated thermal, hydrolytic, and oxidative decomposition program. A large database with stability data is collected, and from this database, a predictive model is being developed. The aim of this pr... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=2504349 Wed, 10 Jun 2009 23:00:00 +0100 2504349 http://www.medworm.com/index.php?rid=2504348&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F14%2F5%2F547%3Frss%3D1 It is common knowledge in the pharmaceutical industry that the quality of a company's compound collection has a major influence on the success of biological screening in drug discovery programs. DMSO is the widely accepted solvent of choice for storage of compounds, despite the hygroscopic nature of the solvent, which can lead to stability issues. Other factors that can affect compound stability (e.g., degradation, precipitation) include concentration of compound, intrinsic compound stability, presence of reactive contaminants, storage format-related factors (vessel, sealing, etc.), storage conditions (temperature, humidity, freeze-thaw technique and cycles, etc.), and storage time. To define the best practice for the storage and handling of solution samples, GlaxoSmithKline has undertaken... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=2504348 Wed, 10 Jun 2009 23:00:00 +0100 2504348 http://www.medworm.com/index.php?rid=2504347&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F14%2F5%2F538%3Frss%3D1 The authors describe how room temperature storage of a 1120-member compound library prepared in either DMSO or in a hydrated-DMSO/water (67/33) mixture affects the reproducibility of potency values as monitored using cytochrome P450 1A2 and 2D6 isozyme assays. The bioluminescent assays showed Z' factors of 0.71 and 0.62, with 17% and 32% of the library found as active against the CYP 1A2 and 2D6 isozymes, respectively. The authors tested the library using quantitative high-throughput screening to generate potency values for every library member, which was measured at 7 time intervals spanning 37 weeks. They calculated the minimum significant ratio (MSR) from these potency values at each time interval and found that for the library stored in DMSO, the CYP 1A2 and 2D6 assay MSRs progressed f... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=2504347 Wed, 10 Jun 2009 23:00:00 +0100 2504347 http://www.medworm.com/index.php?rid=2504346&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F14%2F5%2F531%3Frss%3D1 Recent literature has described the exciting development of a new universal detection technology for high-performance liquid chromatography (HPLC), as well as some exploratory work on its application to quantitative measurement of solutes at millimolar concentrations. The new methodology, known as charged aerosol detection (CAD), has been recognized as a viable alternative to evaporative light-scattering detection and refractive index detection that, like CAD, respond to molecular structures independently of their absorbance, or lack thereof, in the ultraviolet region of the electromagnetic spectrum. In this article, the authors exemplify their use of CAD in-line with HPLC and mass spectrometry (MS) to provide both stand-alone and complementary information that aids decision making for sam... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=2504346 Wed, 10 Jun 2009 23:00:00 +0100 2504346 http://www.medworm.com/index.php?rid=2504345&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F14%2F5%2F523%3Frss%3D1 The introduction of lean thinking and Six Sigma methodologies into the drug discovery process has become an important approach for ensuring efficient workflows while containing costs. For the compound management department at Bristol-Myers Squibb, this has resulted in a partnership with the research community to evaluate and streamline processes to enable cost-disciplined science. The authors describe the results of Lean Six Sigma approaches in the automation and informatics environment that have been optimized to support parallel processing of compounds. This new platform facilitates the rapid and simultaneous data generation from structure activity and structure liability assays. As a result of these compound management improvements, reduction of timelines and quicker decision making has... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=2504345 Wed, 10 Jun 2009 23:00:00 +0100 2504345 http://www.medworm.com/index.php?rid=2504344&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F14%2F5%2F515%3Frss%3D1 Historically, sample management successfully focused on providing compound quality and tracking distribution within a diverse geographic. However, if a competitive advantage is to be delivered in a changing environment of outsourcing, efficiency and customer service must now improve or face reconstruction. The authors have used discrete event simulation to model the compound process from chemistry to assay and applied lean manufacturing techniques to analyze and improve these processes. In doing so, they identified a value-adding process time of just 11 min within a procedure that took days. Modeling also allowed the analysis of equipment and human resources necessary to complete the expected demand in an acceptable cycle time. Layout and location of sample management and screening departm... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=2504344 Wed, 10 Jun 2009 23:00:00 +0100 2504344 http://www.medworm.com/index.php?rid=2504343&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F14%2F5%2F509%3Frss%3D1 Four years ago, the first acoustic droplet ejectors (ADEs) were launched on the market, providing a new generation of high-throughput noncontact liquid handlers that outclassed traditional contact instruments in almost every respect. This introduction of noncontact dispensing has triggered radical changes to the screening/compound management interface. Higher quality is achieved through greater accuracy and precision, whereas lower sample volumes can be used, and 1536 plate formats have become a reliable reality. Prior to the ADE instrument launch, 1536 assay-ready plate preparation was a high-effort enterprise requiring users to spend time developing liquid-handling methods along with daily fine-tuning of instruments to reach the desired level of performance. By overcoming the nanoliter d... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=2504343 Wed, 10 Jun 2009 23:00:00 +0100 2504343 http://www.medworm.com/index.php?rid=2504342&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F14%2F5%2F499%3Frss%3D1 To meet the needs of the increasingly rapid and parallelized lead optimization process, a fully integrated local compound storage and liquid handling system was designed and implemented to automate the generation of assay-ready plates directly from newly submitted and cherry-picked compounds. A key feature of the system is the ability to create project- or assay-specific compound-handling methods, which provide flexibility for any combination of plate types, layouts, and plate bar-codes. Project-specific workflows can be created by linking methods for processing new and cherry-picked compounds and control additions to produce a complete compound set for both biological testing and local storage in one uninterrupted workflow. A flexible cherry-pick approach allows for multiple, user-defined... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=2504342 Wed, 10 Jun 2009 23:00:00 +0100 2504342 http://www.medworm.com/index.php?rid=2504341&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F14%2F5%2F492%3Frss%3D1 This report describes the features and the performance of a new and significantly improved 1536-well microplate design. The design allows for simple, automation-friendly, and cost-effective storage of compound solutions for high-throughput screening. The plate design is based on Society for Biomolecular Sciences standards for microplates and can be molded from polystyrene or cycloolefin copolymer, thus making the plate suitable for use with acoustic dispensing as well as other conventional liquid dispensing in the nanoliter range. For a 9:1 DMSO/water mix as solvent, the novel plate design has shown to perform over 4 months with only minor losses in solvent. Thus, this novel plate design creates the basis for further reductions in compound storage volumes and allows for an increase in the ... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=2504341 Wed, 10 Jun 2009 23:00:00 +0100 2504341 http://www.medworm.com/index.php?rid=2504340&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F14%2F5%2F485%3Frss%3D1 Codeveloping alongside chemistry and in vitro screening, compound management was one of the first areas in research recognizing the need for efficient processes and workflows. Material management groups have centralized, automated, miniaturized and, importantly, found out what not to do with compounds. While driving down cost and improving quality in storage and processing, researchers still face the challenge of interfacing optimally with changing business processes, in screening groups, and with external vendors and focusing on biologicals in many companies. Here we review our strategy to provide a seamless link between compound acquisition and screening operations and the impact of material management on quality of the downstream processes. Although this is driven in part by new technol... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=2504340 Wed, 10 Jun 2009 23:00:00 +0100 2504340 http://www.medworm.com/index.php?rid=2504339&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F14%2F5%2F476%3Frss%3D1 This article presents the results of these experiments and how they affect compound integrity and the accuracy of compound management processes. (Journal of Biomolecular Screening 2009:476-484) (Source: Journal of Biomolecular Screening) Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=2504339 Wed, 10 Jun 2009 23:00:00 +0100 2504339 http://www.medworm.com/index.php?rid=2504338&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F14%2F5%2F468%3Frss%3D1 Assay-ready compound plates (ARPs) are sealed assay plates that contain DMSO solutions of screening compounds predispensed for particular assays. Assays are started by adding assay buffer and reagents to the ARPs. This concept offers important logistical advantages for screening such as decoupling of the plate preparation from the screening process and exchange of assay plates between different geographical locations. Compound solutions can be accurately and precisely dispensed by acoustic droplet ejection technology in the small volumes required for screening in the 1536-well format. At such low volumes, however, potential effects such as solvent evaporation, compound degradation, precipitation, or adsorption are reasons for concern with regard to assay reproducibility, performance, and s... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=2504338 Wed, 10 Jun 2009 23:00:00 +0100 2504338 http://www.medworm.com/index.php?rid=2504337&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F14%2F5%2F460%3Frss%3D1 This article will go on to describe the areas where Adaptive Focused Acoustics adds value as well as areas in which it has shown no clear benefit. (Source: Journal of Biomolecular Screening) Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=2504337 Wed, 10 Jun 2009 23:00:00 +0100 2504337 http://www.medworm.com/index.php?rid=2504336&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F14%2F5%2F452%3Frss%3D1 This study examines the cause of this variability and evaluates the accuracy of screening data in these case studies. A number of liquid-handling options have been explored to address the issues and establish a generic compound-handling workflow to support cell-based screening across our screening functions. The authors discuss the validation of the Labcyte Echo reformatter as an effective noncontact solution for generic compound-handling applications against diverse compound classes using triple-quad liquid chromatography/mass spectrometry. The successful validation and implementation challenges of this technology for direct dosing onto cells in cell-based screening is discussed. (Journal of Biomolecular Screening 2009:452-459) (Source: Journal of Biomolecular Screening) Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=2504336 Wed, 10 Jun 2009 23:00:00 +0100 2504336 http://www.medworm.com/index.php?rid=2504335&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F14%2F5%2F444%3Frss%3D1 The process of drug discovery has evolved considerably since the advent of high-throughput screening (HTS) in the 1980s. Experts and opinion leaders today are agreeing that the current trend in the field is a focus on increasing overall quality (target, screening, and compounds), use of multiple screening approaches for lead discovery, and more flexibility in the process. The associated need for increased flexibility and quality control to support existing HTS paradigms as well as lower throughput approaches such as fragment screening, computational chemistry, focused library building, and centralized lead optimization support has required an evolution in compound management (CM, aka sample management or library management). Although there is much less published peer-reviewed data in CM, d... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=2504335 Wed, 10 Jun 2009 23:00:00 +0100 2504335 http://www.medworm.com/index.php?rid=2504334&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Freprint%2F14%2F5%2F443%3Frss%3D1 (Source: Journal of Biomolecular Screening) Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=2504334 Wed, 10 Jun 2009 23:00:00 +0100 2504334 http://www.medworm.com/index.php?rid=2504333&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Freprint%2F14%2F5%2F441%3Frss%3D1 (Source: Journal of Biomolecular Screening) Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=2504333 Wed, 10 Jun 2009 23:00:00 +0100 2504333 http://www.medworm.com/index.php?rid=2380156&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Freprint%2F14%2F4%2F434%3Frss%3D1 (Source: Journal of Biomolecular Screening) Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=2380156 Wed, 29 Apr 2009 04:00:00 +0100 2380156 http://www.medworm.com/index.php?rid=2380155&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Freprint%2F14%2F4%2F426%3Frss%3D1 (Source: Journal of Biomolecular Screening) Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=2380155 Wed, 29 Apr 2009 04:00:00 +0100 2380155 http://www.medworm.com/index.php?rid=2380154&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F14%2F4%2F419%3Frss%3D1 Cyclophilin is a ubiquitous peptidyl prolyl cis/trans isomerase that plays critical roles in many biological processes. A number of cyclophilin inhibitors have been designed based on the structure of the immunosuppressant cyclosporin A. To discover inhibitors that have other structures, the authors established the high-throughput screening (HTS) method using FDSS6000 real-time fluorescence detector. The inhibitors identified with this HTS showed significant correlation with direct interaction as measured by surface plasmon resonance. This high-throughput assay system is a powerful tool for the discovery of peptidylprolyl isomerase inhibitors. (Journal of Biomolecular Screening 2009:419-424) (Source: Journal of Biomolecular Screening) Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=2380154 Wed, 29 Apr 2009 04:00:00 +0100 2380154 http://www.medworm.com/index.php?rid=2380153&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F14%2F4%2F412%3Frss%3D1 A novel assay for monitoring the activity of the bacterial enzyme UDP-N-acetylmuramyl-L-alanine:D-glutamate ligase (MurD ligase) is presented. MurD, which belongs to an enzyme family of Mur ligases, is essential for the synthesis of bacterial peptidoglycan and therefore represents an attractive target for the discovery of novel antibacterial agents. The inhibition assay described in this article is amenable to high-throughput screening. It is based on the detection of the accumulation of adenosine 5'-diphosphate (ADP), a product of the reaction catalyzed by MurD ligase, by conversion to a fluorescent signal via a coupled enzyme system, using the ADP QuestTM assay kit from DiscoveRx. The novel assay has been validated by obtaining KM,app values for substrates D-Glu, UDP- N-acetylmuramyl-L-a... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=2380153 Wed, 29 Apr 2009 04:00:00 +0100 2380153 http://www.medworm.com/index.php?rid=2380152&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F14%2F4%2F404%3Frss%3D1 This report describes a novel assay to monitor c-Met phosphorylation in cells using Amplified Luminescent Proximity Homogeneous Assay (AlphaScreenTM) technology. Using AlphaScreenTM, the authors were able to detect both global and site-specific phosphorylation of c-Met in transformed cell lines. Data obtained from the AlphaScreenTM assay were compared to data obtained from a high-content imaging (HCI) method developed in parallel to monitor c-Met phosphorylation at the single cell level. The AlphaScreenTM assay was miniaturized to a 384-well format with acceptable signal-to-background ratio (S/B) and Z' statistics and was employed to measure c-Met kinase activity in situ after treatment with potent c-Met-specific kinase inhibitors. The authors discuss the utility of quantifying endogenous ... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=2380152 Wed, 29 Apr 2009 04:00:00 +0100 2380152 http://www.medworm.com/index.php?rid=2380151&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F14%2F4%2F395%3Frss%3D1 A lead optimization library consisting of 800 HIV-1 nonnucleoside reverse transcriptase inhibitors (NNRTIs) was screened in parallel against 4 clinically relevant variants of HIV-1 RT (Wt, L100I, Y181C, and K103N) using a surface plasmon resonance—based biosensor. The aim was to identify inhibitors suitable in specific topical microbicides efficient for preventing the transmission of a range of clinically significant strains of HIV-1. The authors hypothesized that such compounds should have high affinity and slow dissociation rates for multiple variants of the target. To efficiently analyze the large amount of real-time data (sensorgrams) that were generated in the screening, they initially used signals from 3 selected time points to identify compounds with high affinity and slow dis... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=2380151 Wed, 29 Apr 2009 04:00:00 +0100 2380151 http://www.medworm.com/index.php?rid=2380150&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F14%2F4%2F381%3Frss%3D1 Assays for high-throughput screening of G-protein-coupled receptors (GPCRs) have typically revolved around receptor binding, guanine nucleotide binding, and second-messenger assays measuring intracellular cAMP and calcium levels. New assay development has been directed toward G-protein-independent signaling pathways, including protein redistribution in response to activated receptors. β-arrestin recruitment to agonist-stimulated GPCRs is the basis for the Transfluor®, PathHunter®, and TangoTM GPCR screening platforms. In the TangoTM GPCR technology, receptor activation results in the recruitment of a TEV protease:β-arrestin fusion protein to the activated receptor where the TEV protease releases the GAL4-VP16 tethered to the target GPCR by a 7—amino acid TEV proteas... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=2380150 Wed, 29 Apr 2009 04:00:00 +0100 2380150 http://www.medworm.com/index.php?rid=2380149&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F14%2F4%2F371%3Frss%3D1 This study has been focused on establishing a β-arrestin 2—based screening assay for the CB1R using BRET2 technology. When the existing BRET2 screening platform was applied to the CB1R, the authors discovered that the receptor interacted weakly with β-arrestin 2, resulting in unsatisfactory assay performance. To enhance the β-arrestin binding capacity, they replaced the C-terminal tail of the CB1R with tails from either the V2 or BRS3 receptors, both of which interact strongly with β-arrestin 2. Using this chimeric approach, the authors screened a small compound library and identified 21 antagonist and inverse agonist hits with IC50 and EC50 values ranging from 0.3 nM to 7.5 µM. Both primary and secondary screening were performed with Z' > 0.5, suggesting... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=2380149 Wed, 29 Apr 2009 04:00:00 +0100 2380149 http://www.medworm.com/index.php?rid=2380148&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F14%2F4%2F360%3Frss%3D1 Novel fluorescent derivatives of serotonin have been synthesized and used as tracers for the development of a 5-HT2C fluorescence polarization assay. Serotonin analogs that feature a fluorescent probe attached through an ether linkage at the tryptamine 5-position have high affinity for the 5-HT2C receptor, and affinity is dependent on both linker length and pendent dye. These variables have been optimized to generate Cy3B derivative 5a, which has 10-fold higher 5-HT2C affinity relative to serotonin (Kd = 0.23 nM). In receptor activation experiments, 5a acts as a full agonist of 5-HT2C. Upon binding to 5-HT2C cell membranes, 5a shows a robust increase in fluorescence polarization (FP) signal. In an FP binding assay using 5a as a tracer ligand, Ki values for known 5-HT2C agonists and antagon... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=2380148 Wed, 29 Apr 2009 04:00:00 +0100 2380148 http://www.medworm.com/index.php?rid=2380147&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F14%2F4%2F350%3Frss%3D1 In conclusion, they developed a sensitive, easy-to-use, and versatile method for the study of the interaction between profilin and different ligands. (Journal of Biomolecular Screening 2009:350-359) (Source: Journal of Biomolecular Screening) Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=2380147 Wed, 29 Apr 2009 04:00:00 +0100 2380147 http://www.medworm.com/index.php?rid=2380146&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F14%2F4%2F337%3Frss%3D1 Surface plasmon resonance (SPR) technology has emerged as a new and powerful technique to investigate the interaction between low-molecular-weight molecules and target proteins. In the present work, the authors assemble from a large compound collection a library of 2226 molecules (fragments having low molecular weights between 100 and 300 Da) to screen them for binding to chymase, a serine protease. Both the active chymase and a zymogen-like form of the protein were used in parallel to distinguish between specific and unspecific binding. The relative ligand-binding activity of the immobilized protein was periodically measured with a reference compound. The screening experiments were performed at 25 °C at a fragment concentration of 200 µM in the presence of 2% DMSO. Applying the fil... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=2380146 Wed, 29 Apr 2009 04:00:00 +0100 2380146 http://www.medworm.com/index.php?rid=2380145&cid=s_32016_67_f&fid=32016&url=http%3A%2F%2Fjbx.sagepub.com%2Fcgi%2Fcontent%2Fabstract%2F14%2F4%2F330%3Frss%3D1 Although the pathophysiology and molecular basis of sickle cell disease (SCD) were described more than half a century ago, an effective and safe therapy is not yet available. This may be explained by the lack of a suitable high-throughput technique that allows rapid screening of thousands of compounds for their antisickling effect. The authors have thus developed a novel high-throughput screening (HTS) assay based on detecting the ability of red blood cells (RBC) to traverse a column of tightly packed Sephacryl chromatography beads. When deoxygenated, sickle RBC are rigid and remain on the top of the column. However, when deoxygenated and treated with an effective antisickling agent, erythrocytes move through the Sephacryl media and produce a red dot on the bottom of the assay tubes. This ... Journal of Biomolecular Screening journals http://www.medworm.com/rss/comments.php?id=2380145 Wed, 29 Apr 2009 04:00:00 +0100 2380145