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Journal of biomolecular screening [journal]
- Multiparametric Analysis of Screening Data: Growing Beyond the Single Dimension to Infinity and Beyond. [JOURNAL ARTICLE]
- J Biomol Screen 2014 Mar 5.
Advances in instrumentation now allow the development of screening assays that are capable of monitoring multiple readouts such as transcript or protein levels, or even multiple parameters derived from images. Such advances in assay technologies highlight the complex nature of biology and disease. Harnessing this complexity requires integration of all the different parameters that can be measured rather than just monitoring a single dimension as is commonly used. Although some of the methods used to combine multiple measurements, such as principal component analysis, are commonly used for microarray analysis, biologists are not yet using many of the tools that have been developed in other fields to address such issues. Visualization of multiparametric data sets is one of the major challenges in this field, and a depiction of the results in a manner that can be readily interpreted is essential. This article describes a number of assay systems being used to generate such data sets en masse, and the methods being applied to their visualization and analysis. We also discuss some of the challenges of applying methods developed in other fields to biology.
- Enzymatic Characterization of ER Stress-Dependent Kinase, PERK, and Development of a High-Throughput Assay for Identification of PERK Inhibitors. [JOURNAL ARTICLE]
- J Biomol Screen 2014 Mar 5.
PERK is serine/threonine kinase localized to the endoplasmic reticulum (ER) membrane. PERK is activated and contributes to cell survival in response to a variety of physiological stresses that affect protein quality control in the ER, such as hypoxia, glucose depravation, increased lipid biosynthesis, and increased protein translation. Pro-survival functions of PERK are triggered by such stresses, suggesting that development of small-molecule inhibitors of PERK may be efficacious in a variety of disease scenarios. Hence, we have conducted a detailed enzymatic characterization of the PERK kinase to develop a high-throughput-screening assay (HTS) that will permit the identification of small-molecule PERK inhibitors. In addition to establishing the Km of PERK for both its primary substrate, eIF2α, and for adenosine triphosphate, further mechanistic studies revealed that PERK targets its substrate via either a random/steady-state ordered mechanism. For HTS, we developed a time-resolved fluorescence resonance energy transfer-based assay that yielded a robust Z' factor and percent coefficient of variation value, enabling the successful screening of 79,552 compounds. This approach yielded one compound that exhibited good in vitro and cellular activity. These results demonstrate the validity of this screen and represent starting points for drug discovery efforts.
- A Basic Post-SET Extension of NSDs Is Essential for Nucleosome Binding In Vitro. [JOURNAL ARTICLE]
- J Biomol Screen 2014 Mar 4.
The nuclear receptor SET domain-containing family of proteins (NSD1, NSD2, and NSD3) is known to mono- and dimethylate lysine 36 of histone H3 (H3K36). Overexpression and translocation of NSDs have been widely implicated in a variety of diseases including cancers. Although the substrate specificity of NSDs has been a subject of many valuable studies, the activity of these proteins has never been fully characterized in vitro. In this study, we present full kinetic characterization of NSD1, NSD2, and NSD3 and provide robust in vitro assays suitable for screening these proteins in a 384-well format using nucleosome as a substrate. Through monitoring the changes in substrate specificity of a series of NSD constructs and using molecular modeling, we show that a basic post-SET extension common to all three NSDs (corresponding to residues 1209 to 1226 of NSD2) is essential for proper positioning on nucleosome substrates.
- Combined Analysis of Phenotypic and Target-Based Screening in Assay Networks. [JOURNAL ARTICLE]
- J Biomol Screen 2014 Feb 21.
Small-molecule screens are an integral part of drug discovery. Public domain data in PubChem alone represent more than 158 million measurements, 1.2 million molecules, and 4300 assays. We conducted a global analysis of these data, building a network of assays and connecting the assays if they shared nonpromiscuous active molecules. This network spans both phenotypic and target-based screens, recapitulates known biology, and identifies new polypharmacology. Phenotypic screens are extremely important for drug discovery, contributing to the discovery of a large proportion of new drugs. Connections between phenotypic and biochemical, target-based screens can suggest strategies for repurposing both small-molecule and biologic drugs. For example, a screen for molecules that prevent cell death from a mutated version of superoxide-dismutase is linked with ALOX15. This connection suggests a therapeutic role for ALOX15 inhibitors in amyotrophic lateral sclerosis. An interactive version of the network is available online (http://swami.wustl.edu/flow/assay_network.html).
- Development of a Kinetic Assay for Late Endosome Movement. [JOURNAL ARTICLE]
- J Biomol Screen 2014 Feb 20.
Automated imaging screens are performed mostly on fixed and stained samples to simplify the workflow and increase throughput. Some processes, such as the movement of cells and organelles or measuring membrane integrity and potential, can be measured only in living cells. Developing such assays to screen large compound or RNAi collections is challenging in many respects. Here, we develop a live-cell high-content assay for tracking endocytic organelles in medium throughput. We evaluate the added value of measuring kinetic parameters compared with measuring static parameters solely. We screened 2000 compounds in U-2 OS cells expressing Lamp1-GFP to label late endosomes. All hits have phenotypes in both static and kinetic parameters. However, we show that the kinetic parameters enable better discrimination of the mechanisms of action. Most of the compounds cause a decrease of motility of endosomes, but we identify several compounds that increase endosomal motility. In summary, we show that kinetic data help to better discriminate phenotypes and thereby obtain more subtle phenotypic clustering.
- SEC-TID: A Label-Free Method for Small-Molecule Target Identification. [JOURNAL ARTICLE]
- J Biomol Screen 2014 Feb 19.
Bioactive small molecules are an invaluable source of therapeutics and chemical probes for exploring biological pathways. Yet, significant hurdles in drug discovery often come from lacking a comprehensive view of the target(s) for both early tool molecules and even late-stage drugs. To address this challenge, a method is provided that allows for assessing the interactions of small molecules with thousands of targets without any need to modify the small molecule of interest or attach any component to a surface. We describe size-exclusion chromatography for target identification (SEC-TID), a method for accurately and reproducibly detecting ligand-macromolecular interactions for small molecules targeting nucleic acid and several protein classes. We report the use of SEC-TID, with a library consisting of approximately 1000 purified proteins derived from the protein databank (PDB), to identify the efficacy targets tankyrase 1 and 2 for the Wnt inhibitor XAV939. In addition, we report novel interactions for the tumor-vascular disrupting agent vadimezan/ASA404 (interacting with farnesyl pyrophosphate synthase) and the diuretic mefruside (interacting with carbonic anhydrase XIII). We believe this method can dramatically enhance our understanding of the mechanism of action and potential liabilities for small molecules in drug discovery pipelines through comprehensive profiling of candidate druggable targets.
- Virtual Screening of Some Active Human Macrophage Migration Inhibitory Factor Antagonists. [JOURNAL ARTICLE]
- J Biomol Screen 2014 Feb 19.
Macrophage migration inhibitory factor (MIF) is an autocrine- and paracrine-acting cytokine that is involved in several inflammatory, autoimmune, infectious, and oncogenic diseases. Clinical data have shown that inhibition of MIF, especially its tautomerase activity, with small compounds has been beneficial in some disease models. A virtual screening (VS) experiment is conducted for searching some active compounds to inhibit the tautomerase activity of MIF from the ZINC database. By using an x-ray-determined structure OXIM-11 as the query and an in-house developed two-dimensional scaffold comparing method designated as Sfilter, we have screened out some 1500 compounds for ranking by our previously published docking method ADDock. After further ranking by ADDock on 119 compounds screened, we have decided to choose 17 of them for measuring their inhibitory activity IC50 against the MIF tautomerase experimentally. The IC50's are measured using both human monocytic THP-1 cell lysate and purified recombinant human MIF protein. We have found that the IC50's measured for three searched compounds (namely, ZINC02693801, ZINC00141102, and ZINC12368346) are better than that determined for ISO-1, a known MIF tautomerase inhibitor and standard used throughout our VS experiment. Moreover, the scaffolds of most of our active compounds searched are also quite different from those searched and published by others previously.
- Adaptive Focused Acoustics (AFA) Improves the Performance of Microtiter Plate ELISAs. [JOURNAL ARTICLE]
- J Biomol Screen 2014 Feb 19.
We investigated the use of Adaptive Focused Acoustics (AFA) technology to improve the performance of microtiter plate enzyme-linked immunosorbent assays (ELISAs). Experiments were performed with commercially available AFA instrumentation and off-the-shelf 96-well microtiter plate sandwich ELISAs. AFA was applied over a range of acoustic energies, temperatures, and durations to the antigen/antibody binding step of an ELISA for measuring HIV-1 p24 in tissue culture samples. AFA-mediated antigen/antibody binding was enhanced up to 2-fold over passive binding at comparable temperatures and was superior or comparable at low temperature (8-10 °C) to passive binding at 37 °C. Lower nonspecific binding (NSB), lower inter- and intra-assay coefficients of variation (CVs), higher Z' factors, and lower limits of detection (LODs) were measured in AFA-mediated assays compared with conventional passive binding. In a more limited study, AFA enhancement of antigen/antibody binding and lower NSB was measured in an ELISA for measuring IGFBP-3 in human plasma. We conclude from this study that application of AFA to antigen/antibody binding steps in microtiter plate ELISAs can enhance key assay performance parameters, particularly Z' factors and LODs. These features render AFA-mediated binding assays potentially more useful in applications such as high-throughput screening and in vitro diagnostics than assays processed with conventional passive antigen/antibody binding steps.
- Overview of Recent Progress in Protein-Expression Technologies for Small-Molecule Screening. [JOURNAL ARTICLE]
- J Biomol Screen 2014 Feb 13.
Production of novel soluble and membrane-localized protein targets for functional and affinity-based screening has often been limited by the inability of traditional protein-expression systems to generate recombinant proteins that have properties similar to those of their endogenous counterparts. Such targets have often been labeled as challenging. Although biological validation of these challenging targets for specific disease areas may be strong, discovery of small-molecule modulators can be greatly delayed or completely halted due to target-expression issues. In this article, the limitations of traditional protein-expression systems will be discussed along with new systems designed to overcome these challenges. Recent work in this field has focused on two major areas for both soluble and membrane targets: construct-design strategies to improve expression levels and new hosts that can carry out the posttranslational modifications necessary for proper target folding and function. Another area of active research has been on the reconstitution of solubilized membrane targets for both structural analysis and screening. Finally, the potential impact of these new systems on the output of small-molecule screening campaigns will be discussed.
- A Duplexed High-Throughput Screen to Identify Allosteric Modulators of the Glucagon-Like Peptide 1 and Glucagon Receptors. [JOURNAL ARTICLE]
- J Biomol Screen 2014 Feb 13.
Injectable, degradation-resistant peptide agonists for the glucagon-like peptide 1 (GLP-1) receptor (GLP-1R), such as exenatide and liraglutide, activate the GLP-1R via a complex orthosteric-binding site and are effective therapeutics for glycemic control in type 2 diabetes. Orally bioavailable orthosteric small-molecule agonists are unlikely to be developed, whereas positive allosteric modulators (PAMs) may offer an improved therapeutic profile. We hypothesize that allosteric modulators of the GLP-1R would increase the potency and efficacy of native GLP-1 in a spatial and temporally preserved manner and/or may improve efficacy or side effects of injectable analogs. We report the design, optimization, and initial results of a duplexed high-throughput screen in which cell lines overexpressing either the GLP-1R or the glucagon receptor were coplated, loaded with a calcium-sensitive dye, and probed in a three-phase assay to identify agonists, antagonists, and potentiators of GLP-1, and potentiators of glucagon. 175,000 compounds were initially screened, and progression through secondary assays yielded 98 compounds with a variety of activities at the GLP-1R. Here, we describe five compounds possessing different patterns of modulation of the GLP-1R. These data uncover PAMs that may offer a drug-development pathway to enhancing in vivo efficacy of both endogenous GLP-1 and peptide analogs.