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Journal of biomolecular screening [journal]
- Control-Plate Regression (CPR) Normalization for High-Throughput Screens with Many Active Features. [JOURNAL ARTICLE]
- J Biomol Screen 2013 Dec 18.
Systematic error is present in all high-throughput screens, lowering measurement accuracy. Because screening occurs at the early stages of research projects, measurement inaccuracy leads to following up inactive features and failing to follow up active features. Current normalization methods take advantage of the fact that most primary-screen features (e.g., compounds) within each plate are inactive, which permits robust estimates of row and column systematic-error effects. Screens that contain a majority of potentially active features pose a more difficult challenge because even the most robust normalization methods will remove at least some of the biological signal. Control plates that contain the same feature in all wells can provide a solution to this problem by providing well-by-well estimates of systematic error, which can then be removed from the treatment plates. We introduce the robust control-plate regression (CPR) method, which uses this approach. CPR's performance is compared to a high-performing primary-screen normalization method in four experiments. These data were also perturbed to simulate screens with large numbers of active features to further assess CPR's performance. CPR performs almost as well as the best performing normalization methods with primary screens and outperforms the Z-score and equivalent methods with screens containing a large proportion of active features.
- Analytical and preparative instrumentation. [Journal Article]
- J Biomol Screen 2014 Jan; 19(1):184-6.
- Identification of a Selective Agonist for Liver X Receptor α (LXRα) via Screening of a Synthetic Compound Library. [JOURNAL ARTICLE]
- J Biomol Screen 2013 Dec 13.
Liver X receptor α (LXRα) plays an important role in reverse cholesterol transport (RCT), and activation of LXRα could reduce atherosclerosis. In the present study, we developed a screening method to identify new potential LXRα agonists using an LXRα-GAL4 chimera reporter assay. A novel analogue of N,N-disubstituted 2,8-diazaspiro[4.5]decane, IMB-151, was identified as an LXRα agonist by using this method. IMB-151 showed a significant activation effect on LXRα, with an EC50 value of 1.47 µM. IMB-151 also increased the expression of ATP-binding cassette transporter A1 (ABCA1) and G1 (ABCG1) in RAW264.7 macrophages. The upregulating effects of IMB-151 on ABCA1 and ABCG1 markedly decreased when coincubated with geranylgeranyl pyrophosphate (GGPP) ammonium salt or LXRα small interfering RNA (siRNA). Our data indicated that the upregulation of ABCA1 and ABCG1 by IMB-151 depended on activation of LXRα. Moreover, IMB-151 significantly reduced cellular lipid accumulation and increased cholesterol efflux in RAW264.7 macrophages. Interestingly, IMB-151 slightly increased sterol response element binding protein 1c (SREBP-1c) protein expression levels in HepG2 cells compared with TO901317, and this indicated that IMB-151 might have less lipogenesis side effect in vivo. These results suggested that IMB-151 was identified as a selective agonist for LXRα by using a screening method and could be used as a potential antiatherosclerotic lead compound in the future.
- A Novel Microscopy-Based High-Throughput Screening Method to Identify Proteins That Regulate Global Histone Modification Levels. [JOURNAL ARTICLE]
- J Biomol Screen 2013 Dec 11.
Posttranslational modifications of histones play an important role in the regulation of gene expression and chromatin structure in eukaryotes. The balance between chromatin factors depositing (writers) and removing (erasers) histone marks regulates the steady-state levels of chromatin modifications. Here we describe a novel microscopy-based screening method to identify proteins that regulate histone modification levels in a high-throughput fashion. We named our method CROSS, for Chromatin Regulation Ontology SiRNA Screening. CROSS is based on an siRNA library targeting the expression of 529 proteins involved in chromatin regulation. As a proof of principle, we used CROSS to identify chromatin factors involved in histone H3 methylation on either lysine-4 or lysine-27. Furthermore, we show that CROSS can be used to identify chromatin factors that affect growth in cancer cell lines. Taken together, CROSS is a powerful method to identify the writers and erasers of novel and known chromatin marks and facilitates the identification of drugs targeting epigenetic modifications.
- Identification and In Vitro Characterization of Phage-Displayed VHHs Targeting VEGF. [JOURNAL ARTICLE]
- J Biomol Screen 2013 Dec 2.
Vascular endothelial growth factor (VEGF) is a potential target for cancer treatment because of its role in angiogenesis and its overexpression in most human cancers. Currently, anti-VEGF antibodies have been shown to be promising tools for therapeutic applications. However, large size, poor tumor penetration, immunogenicity, and production in cost- and labor-intensive conditions are major drawbacks of such agents. The antigen-binding regions of camelid single-chain antibodies (VHHs), due to their unique biophysical characteristics, offer an alternative to conventional antibodies for tumor-targeting purposes. The present study was undertaken to generate and characterize anti-VEGF VHHs from an immune VHH library using phage display. Four rounds of panning were performed, and selected VHHs were characterized using various immunological techniques. Assessment of the antigenic profile of VHHs was done using competition enzyme-linked immunosorbent assay (ELISA). Selected VHHs reacted strongly to VEGF in indirect ELISA and cross-reactivity ELISA tests. The binding affinity of three VHHs, ZFR-1, ZFR-2, and ZFR-5, ranged from 2.5 to 80 nM, and among them, ZFR-5, which was selected for proliferation assay, significantly inhibited the endothelial cell growth in a dose-dependent manner. Taken together, our results indicate that ZFR-5 and other VHHs may be promising tools in cancer research and treatment.
- Development and Validation of a High-Content Bimolecular Fluorescence Complementation Assay for Small-Molecule Inhibitors of HIV-1 Nef Dimerization. [JOURNAL ARTICLE]
- J Biomol Screen 2013 Nov 26.
Nef is a human immunodeficiency virus 1 (HIV-1) accessory factor essential for viral pathogenesis and AIDS progression. Many Nef functions require dimerization, and small molecules that block Nef dimerization may represent antiretroviral drug leads. Here we describe a cell-based assay for Nef dimerization inhibitors based on bimolecular fluorescence complementation (BiFC). Nef was fused to nonfluorescent, complementary fragments of yellow fluorescent protein (YFP) and coexpressed in the same cell population. Dimerization of Nef resulted in juxtaposition of the YFP fragments and reconstitution of the fluorophore. For automation, the Nef-YFP fusion proteins plus a monomeric red fluorescent protein (mRFP) reporter were expressed from a single vector, separated by picornavirus "2A" linker peptides to permit equivalent translation of all three proteins. Validation studies revealed a critical role for gating on the mRFP-positive subpopulation of transfected cells, as well as use of the mRFP signal to normalize the Nef-BiFC signal. Nef-BiFC/mRFP ratios resulting from cells expressing wild-type versus dimerization-defective Nef were very clearly separated, with Z factors consistently in the 0.6 to 0.7 range. A fully automated pilot screen of the National Cancer Institute Diversity Set III identified several hit compounds that reproducibly blocked Nef dimerization in the low micromolar range. This BiFC-based assay has the potential to identify cell-active small molecules that directly interfere with Nef dimerization and function.
- Application of Titration-Based Screening for the Rapid Pilot Testing of High-Throughput Assays. [JOURNAL ARTICLE]
- J Biomol Screen 2013 Nov 18.
Pilot testing of an assay intended for high-throughput screening (HTS) with small compound sets is a necessary but often time-consuming step in the validation of an assay protocol. When the initial testing concentration is less than optimal, this can involve iterative testing at different concentrations to further evaluate the pilot outcome, which can be even more time-consuming. Quantitative HTS (qHTS) enables flexible and rapid collection of assay performance statistics, hits at different concentrations, and concentration-response curves in a single experiment. Here we describe the qHTS process for pilot testing in which eight-point concentration-response curves are produced using an interplate asymmetric dilution protocol in which the first four concentrations are used to represent the range of typical HTS screening concentrations and the last four concentrations are added for robust curve fitting to determine potency/efficacy values. We also describe how these data can be analyzed to predict the frequency of false-positives, false-negatives, hit rates, and confirmation rates for the HTS process as a function of screening concentration. By taking into account the compound pharmacology, this pilot-testing paradigm enables rapid assessment of the assay performance and choosing the optimal concentration for the large-scale HTS in one experiment.
- Overcoming the Challenges of Drug Discovery for Neglected Tropical Diseases: The A·WOL Experience. [JOURNAL ARTICLE]
- J Biomol Screen 2013 Nov 15.
Neglected tropical diseases (NTDs) are a group of 17 diseases that typically affect poor people in tropical countries. Each has been neglected for decades in terms of funding, research, and policy, but the recent grouping of them into one unit, which can be targeted using integrated control measures, together with increased advocacy has helped to place them on the global health agenda. The World Health Organization has set ambitious goals to control or eliminate 10 NTDs by 2020 and launched a roadmap in January 2012 to guide this global plan. The result of the launch meeting, which brought together representatives from the pharmaceutical industry, donors, and politicians, was the London Declaration: a series of commitments to provide more drugs, research, and funds to achieve the 2020 goals. Drug discovery and development for these diseases are extremely challenging, and this article highlights these challenges in the context of the London Declaration, before focusing on an example of a drug discovery and development program for the NTDs onchocerciasis and lymphatic filariasis (the anti-Wolbachia consortium, A·WOL).
- Droplet-Based Microfluidics: Enabling Impact on Drug Discovery. [JOURNAL ARTICLE]
- J Biomol Screen 2013 Nov 15.
Over the past two decades, the application of microengineered systems in the chemical and biological sciences has transformed the way in which high-throughput experimentation is performed. The ability to fabricate complex microfluidic architectures has allowed scientists to create new experimental formats for processing ultra-small analytical volumes in short periods and with high efficiency. The development of such microfluidic systems has been driven by a range of fundamental features that accompany miniaturization. These include the ability to handle small sample volumes, ultra-low fabrication costs, reduced analysis times, enhanced operational flexibility, facile automation, and the ability to integrate functional components within complex analytical schemes. Herein we discuss the impact of microfluidics in the area of high-throughput screening and drug discovery and highlight some of the most pertinent studies in the recent literature.
- Identification of Small Molecules That Selectively Inhibit Diacylglycerol Lipase-α Activity. [JOURNAL ARTICLE]
- J Biomol Screen 2013 Nov 15.
Recent genetic evidence suggests that the diacylglycerol lipase (DAGL-α) isoform is the major biosynthetic enzyme for the most abundant endocannabinoid, 2-arachidonoyl-glycerol (2-AG), in the central nervous system. Revelation of its essential role in regulating retrograde synaptic plasticity and adult neurogenesis has made it an attractive therapeutic target. Therefore, it has become apparent that selective inhibition of DAGL-α enzyme activity with a small molecule could be a strategy for the development of novel therapies for the treatment of disease indications such as depression, anxiety, pain, and cognition. In this report, the authors present the identification of small-molecule inhibitor chemotypes of DAGL-α, which were selective (≥10-fold) against two other lipases, pancreatic lipase and monoacylglycerol lipase, via high-throughput screening of a diverse compound collection. Seven chemotypes of interest from a list of 185 structural clusters, which included 132 singletons, were initially selected for evaluation and characterization. Selection was based on potency, selectivity, and chemical tractability. One of the chemotypes, the glycine sulfonamide series, was prioritized as an initial lead for further medicinal chemistry optimization.