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  1. Article ; Online: High-Throughput RT-PCR for small-molecule screening assays.

    Bittker, Joshua A

    Current protocols in chemical biology

    2011  Volume 4, Issue 1, Page(s) 49–63

    Abstract: Quantitative measurement of the levels of mRNA expression using real-time reverse transcription polymerase chain reaction (RT-PCR) has long been used for analyzing expression differences in tissue or cell lines of interest. This method has been used ... ...

    Abstract Quantitative measurement of the levels of mRNA expression using real-time reverse transcription polymerase chain reaction (RT-PCR) has long been used for analyzing expression differences in tissue or cell lines of interest. This method has been used somewhat less frequently to measure the changes in gene expression due to perturbagens such as small molecules or siRNA. The availability of new instrumentation for liquid handling and real-time PCR analysis as well as the commercial availability of start-to-finish kits for RT-PCR has enabled the use of this method for high-throughput small-molecule screening on a scale comparable to traditional high-throughput screening (HTS) assays. This protocol focuses on the special considerations necessary for using quantitative RT-PCR as a primary small-molecule screening assay, including the different methods available for mRNA isolation and analysis.
    Language English
    Publishing date 2011-10-04
    Publishing country United States
    Document type Journal Article
    ISSN 2160-4762
    ISSN (online) 2160-4762
    DOI 10.1002/9780470559277.ch110204
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  2. Article ; Online: The Use of Informer Sets in Screening: Perspectives on an Efficient Strategy to Identify New Probes.

    Clemons, Paul A / Bittker, Joshua A / Wagner, Florence F / Hands, Allison / Dančík, Vlado / Schreiber, Stuart L / Choudhary, Amit / Wagner, Bridget K

    SLAS discovery : advancing life sciences R & D

    2021  Volume 26, Issue 7, Page(s) 855–861

    Abstract: Small-molecule discovery typically involves large-scale screening campaigns, spanning multiple compound collections. However, such activities can be cost- or time-prohibitive, especially when using complex assay systems, limiting the number of compounds ... ...

    Abstract Small-molecule discovery typically involves large-scale screening campaigns, spanning multiple compound collections. However, such activities can be cost- or time-prohibitive, especially when using complex assay systems, limiting the number of compounds tested. Further, low hit rates can make the process inefficient. Sparse coverage of chemical structure or biological activity space can lead to limited success in a primary screen and represents a missed opportunity by virtue of selecting the "wrong" compounds to test. Thus, the choice of screening collections becomes of paramount importance. In this perspective, we discuss the utility of generating "informer sets" for small-molecule discovery, and how this strategy can be leveraged to prioritize probe candidates. While many researchers may assume that informer sets are focused on particular targets (e.g., kinases) or processes (e.g., autophagy), efforts to assemble informer sets based on historical bioactivity or successful human exposure (e.g., repurposing collections) have shown promise as well. Another method for generating informer sets is based on chemical structure, particularly when the compounds have unknown activities and targets. We describe our efforts to screen an informer set representing a collection of 100,000 small molecules synthesized through diversity-oriented synthesis (DOS). This process enables researchers to identify activity early and more extensively screen only a few chemical scaffolds, rather than the entire collection. This elegant and economic outcome is a goal of the informer set approach. Here, we aim not only to shed light on this process, but also to promote the use of informer sets more widely in small-molecule discovery projects.
    MeSH term(s) Drug Discovery/methods ; Drug Evaluation, Preclinical/methods ; Humans ; Small Molecule Libraries ; Structure-Activity Relationship
    Chemical Substances Small Molecule Libraries
    Language English
    Publishing date 2021-06-16
    Publishing country United States
    Document type Journal Article
    ZDB-ID 2885123-7
    ISSN 2472-5560 ; 2472-5552
    ISSN (online) 2472-5560
    ISSN 2472-5552
    DOI 10.1177/24725552211019410
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  3. Article ; Online: High-Throughput Screens To Identify Autophagy Inducers That Function by Disrupting Beclin 1/Bcl-2 Binding.

    Chiang, Wei-Chung / Wei, Yongjie / Kuo, Yi-Chun / Wei, Shuguang / Zhou, Anwu / Zou, Zhongju / Yehl, Jenna / Ranaghan, Matthew J / Skepner, Adam / Bittker, Joshua A / Perez, Jose R / Posner, Bruce A / Levine, Beth

    ACS chemical biology

    2018  Volume 13, Issue 8, Page(s) 2247–2260

    Abstract: Autophagy, a lysosomal degradation pathway, plays a crucial role in cellular homeostasis, development, immunity, tumor suppression, metabolism, prevention of neurodegeneration, and lifespan extension. Thus, pharmacological stimulation of autophagy may be ...

    Abstract Autophagy, a lysosomal degradation pathway, plays a crucial role in cellular homeostasis, development, immunity, tumor suppression, metabolism, prevention of neurodegeneration, and lifespan extension. Thus, pharmacological stimulation of autophagy may be an effective approach for preventing or treating certain human diseases and/or aging. We sought to establish a method for developing new chemical compounds that specifically induce autophagy. To do this, we developed two assays to identify compounds that target a key regulatory node of autophagy induction-specifically, the binding of Bcl-2 (a negative regulator of autophagy) to Beclin 1 (an allosteric modulator of the Beclin 1/VPS34 lipid kinase complex that functions in autophagy initiation). These assays use either a split-luciferase assay to measure Beclin 1/Bcl-2 binding in cells or an AlphaLISA assay to directly measure direct Beclin 1/Bcl-2 binding in vitro. We screened two different chemical compound libraries, comprising ∼300 K compounds, to identify small molecules that disrupt Beclin 1/Bcl-2 binding and induce autophagy. Three novel compounds were identified that directly inhibit Beclin 1/Bcl-2 interaction with an IC
    MeSH term(s) Autophagy/drug effects ; Beclin-1/metabolism ; Cell Survival/drug effects ; Drug Evaluation, Preclinical/methods ; HeLa Cells ; High-Throughput Screening Assays/methods ; Humans ; Protein Binding/drug effects ; Protein Interaction Maps/drug effects ; Proto-Oncogene Proteins c-bcl-2/metabolism
    Chemical Substances BCL2 protein, human ; Beclin-1 ; Proto-Oncogene Proteins c-bcl-2
    Language English
    Publishing date 2018-06-21
    Publishing country United States
    Document type Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't
    ISSN 1554-8937
    ISSN (online) 1554-8937
    DOI 10.1021/acschembio.8b00421
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  4. Article ; Online: High Throughput Screen Identifies Interferon γ-Dependent Inhibitors of Toxoplasma gondii Growth.

    Radke, Joshua B / Carey, Kimberly L / Shaw, Subrata / Metkar, Shailesh R / Mulrooney, Carol / Gale, Jennifer P / Bittker, Joshua A / Hilgraf, Robert / Comer, Eamon / Schreiber, Stuart L / Virgin, Herbert W / Perez, Jose R / Sibley, L David

    ACS infectious diseases

    2018  Volume 4, Issue 10, Page(s) 1499–1507

    Abstract: Toxoplasma gondii is an obligate intracellular parasite capable of causing severe disease due to congenital infection and in patients with compromised immune systems. Control of infection is dependent on a robust Th1 type immune response including ... ...

    Abstract Toxoplasma gondii is an obligate intracellular parasite capable of causing severe disease due to congenital infection and in patients with compromised immune systems. Control of infection is dependent on a robust Th1 type immune response including production of interferon gamma (IFN-γ), which is essential for control. IFN-γ activates a variety of antimicrobial mechanisms in host cells, which are then able to control intracellular parasites such as T. gondii. Despite the effectiveness of these pathways in controlling acute infection, the immune system is unable to eradicate chronic infections that can persist for life. Similarly, while antibiotic treatment can control acute infection, it is unable to eliminate chronic infection. To identify compounds that would act synergistically with IFN-γ, we performed a high-throughput screen of diverse small molecule libraries to identify inhibitors of T. gondii. We identified a number of compounds that inhibited parasite growth in vitro at low μM concentrations and that demonstrated enhanced potency in the presence of a low level of IFN-γ. A subset of these compounds act by enhancing the recruitment of light chain 3 (LC3) to the parasite-containing vacuole, suggesting they work by an autophagy-related process, while others were independent of this pathway. The pattern of IFN-γ dependence was shared among the majority of analogs from 6 priority scaffolds, and analysis of structure activity relationships for one such class revealed specific stereochemistry associated with this feature. Identification of these IFN-γ-dependent leads may lead to development of improved therapeutics due to their synergistic interactions with immune responses.
    MeSH term(s) Autophagy/physiology ; Green Fluorescent Proteins/metabolism ; Growth Inhibitors/analysis ; Growth Inhibitors/chemistry ; Growth Inhibitors/metabolism ; HeLa Cells ; High-Throughput Screening Assays/methods ; Humans ; Immunity, Innate ; Interferon-gamma/metabolism ; Linear Models ; Luciferases/analysis ; Microtubule-Associated Proteins/metabolism ; Protein Binding ; Small Molecule Libraries ; Stereoisomerism ; Th1 Cells/immunology ; Toxoplasma/growth & development ; Vacuoles/metabolism
    Chemical Substances Growth Inhibitors ; MAP1LC3A protein, human ; Microtubule-Associated Proteins ; Small Molecule Libraries ; Green Fluorescent Proteins (147336-22-9) ; Interferon-gamma (82115-62-6) ; Luciferases (EC 1.13.12.-)
    Language English
    Publishing date 2018-08-28
    Publishing country United States
    Document type Journal Article
    ISSN 2373-8227
    ISSN (online) 2373-8227
    DOI 10.1021/acsinfecdis.8b00135
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  5. Article ; Online: Automatically detecting workflows in PubChem.

    Calhoun, Bradley T / Browning, Michael R / Chen, Brian R / Bittker, Joshua A / Swamidass, S Joshua

    Journal of biomolecular screening

    2012  Volume 17, Issue 8, Page(s) 1071–1079

    Abstract: Public databases that store the data from small-molecule screens are a rich and untapped resource of chemical and biological information. However, screening databases are unorganized, which makes interpreting their data difficult. We propose a method of ... ...

    Abstract Public databases that store the data from small-molecule screens are a rich and untapped resource of chemical and biological information. However, screening databases are unorganized, which makes interpreting their data difficult. We propose a method of inferring workflow graphs--which encode the relationships between assays in screening projects--directly from screening data and using these workflows to organize each project's data. On the basis of four heuristics regarding the organization of screening projects, we designed an algorithm that extracts a project's workflow graph from screening data. Where possible, the algorithm is evaluated by comparing each project's inferred workflow to its documentation. In the majority of cases, there are no discrepancies between the two. Most errors can be traced to points in the project where screeners chose additional molecules to test based on structural similarity to promising molecules, a case our algorithm is not yet capable of handling. Nonetheless, these workflows accurately organize most of the data and also provide a method of visualizing a screening project. This method is robust enough to build a workflow-oriented front-end to PubChem and is currently being used regularly by both our lab and our collaborators. A Python implementation of the algorithm is available online, and a searchable database of all PubChem workflows is available at http://swami.wustl.edu/flow.
    MeSH term(s) Algorithms ; Computational Biology ; Computer Graphics ; Data Mining/methods ; Database Management Systems ; Databases, Chemical ; Drug Evaluation, Preclinical/methods ; High-Throughput Screening Assays/methods ; Molecular Structure ; Small Molecule Libraries/pharmacology
    Chemical Substances Small Molecule Libraries
    Language English
    Publishing date 2012-09
    Publishing country United States
    Document type Journal Article ; Research Support, Non-U.S. Gov't
    ZDB-ID 1433680-7
    ISSN 1552-454X ; 1087-0571
    ISSN (online) 1552-454X
    ISSN 1087-0571
    DOI 10.1177/1087057112449054
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  6. Article ; Online: Enhancing the rate of scaffold discovery with diversity-oriented prioritization.

    Swamidass, S Joshua / Calhoun, Bradley T / Bittker, Joshua A / Bodycombe, Nicole E / Clemons, Paul A

    Bioinformatics (Oxford, England)

    2011  Volume 27, Issue 16, Page(s) 2271–2278

    Abstract: Motivation: In high-throughput screens (HTS) of small molecules for activity in an in vitro assay, it is common to search for active scaffolds, with at least one example successfully confirmed as an active. The number of active scaffolds better reflects ...

    Abstract Motivation: In high-throughput screens (HTS) of small molecules for activity in an in vitro assay, it is common to search for active scaffolds, with at least one example successfully confirmed as an active. The number of active scaffolds better reflects the success of the screen than the number of active molecules. Many existing algorithms for deciding which hits should be sent for confirmatory testing neglect this concern.
    Results: We derived a new extension of a recently proposed economic framework, diversity-oriented prioritization (DOP), that aims-by changing which hits are sent for confirmatory testing-to maximize the number of scaffolds with at least one confirmed active. In both retrospective and prospective experiments, DOP accurately predicted the number of scaffold discoveries in a batch of confirmatory experiments, improved the rate of scaffold discovery by 8-17%, and was surprisingly robust to the size of the confirmatory test batches. As an extension of our previously reported economic framework, DOP can be used to decide the optimal number of hits to send for confirmatory testing by iteratively computing the cost of discovering an additional scaffold, the marginal cost of discovery.
    Contact: swamidass@wustl.edu
    Supplementary information: Supplementary data are available at Bioinformatics online.
    MeSH term(s) Algorithms ; Cluster Analysis ; High-Throughput Screening Assays
    Language English
    Publishing date 2011-06-17
    Publishing country England
    Document type Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't ; Validation Study
    ZDB-ID 1422668-6
    ISSN 1367-4811 ; 1367-4803
    ISSN (online) 1367-4811
    ISSN 1367-4803
    DOI 10.1093/bioinformatics/btr369
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  7. Article ; Online: Utility-aware screening with clique-oriented prioritization.

    Swamidass, S Joshua / Calhoun, Bradley T / Bittker, Joshua A / Bodycombe, Nicole E / Clemons, Paul A

    Journal of chemical information and modeling

    2011  Volume 52, Issue 1, Page(s) 29–37

    Abstract: Most methods of deciding which hits from a screen to send for confirmatory testing assume that all confirmed actives are equally valuable and aim only to maximize the number of confirmed hits. In contrast, "utility-aware" methods are informed by models ... ...

    Abstract Most methods of deciding which hits from a screen to send for confirmatory testing assume that all confirmed actives are equally valuable and aim only to maximize the number of confirmed hits. In contrast, "utility-aware" methods are informed by models of screeners' preferences and can increase the rate at which the useful information is discovered. Clique-oriented prioritization (COP) extends a recently proposed economic framework and aims--by changing which hits are sent for confirmatory testing--to maximize the number of scaffolds with at least two confirmed active examples. In both retrospective and prospective experiments, COP enables accurate predictions of the number of clique discoveries in a batch of confirmatory experiments and improves the rate of clique discovery by more than 3-fold. In contrast, other similarity-based methods like ontology-based pattern identification (OPI) and local hit-rate analysis (LHR) reduce the rate of scaffold discovery by about half. The utility-aware algorithm used to implement COP is general enough to implement several other important models of screener preferences.
    MeSH term(s) Algorithms ; Animals ; Caenorhabditis elegans ; Caenorhabditis elegans Proteins/antagonists & inhibitors ; Caenorhabditis elegans Proteins/chemistry ; Chromosomal Proteins, Non-Histone/antagonists & inhibitors ; Chromosomal Proteins, Non-Histone/chemistry ; Drug Discovery/methods ; High-Throughput Screening Assays ; Humans ; Jumonji Domain-Containing Histone Demethylases/antagonists & inhibitors ; Jumonji Domain-Containing Histone Demethylases/chemistry ; Models, Molecular ; Small Molecule Libraries
    Chemical Substances Caenorhabditis elegans Proteins ; Chromosomal Proteins, Non-Histone ; MEX-5 protein, C elegans ; Small Molecule Libraries ; Jumonji Domain-Containing Histone Demethylases (EC 1.14.11.-) ; KDM4A protein, human (EC 1.5.-)
    Language English
    Publishing date 2011-12-20
    Publishing country United States
    Document type Journal Article ; Research Support, N.I.H., Extramural
    ZDB-ID 190019-5
    ISSN 1549-960X ; 0095-2338
    ISSN (online) 1549-960X
    ISSN 0095-2338
    DOI 10.1021/ci2003285
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  8. Article ; Online: An economic framework to prioritize confirmatory tests after a high-throughput screen.

    Swamidass, S Joshua / Bittker, Joshua A / Bodycombe, Nicole E / Ryder, Sean P / Clemons, Paul A

    Journal of biomolecular screening

    2010  Volume 15, Issue 6, Page(s) 680–686

    Abstract: How many hits from a high-throughput screen should be sent for confirmatory experiments? Analytical answers to this question are derived from statistics alone and aim to fix, for example, the false discovery rate at a predetermined tolerance. These ... ...

    Abstract How many hits from a high-throughput screen should be sent for confirmatory experiments? Analytical answers to this question are derived from statistics alone and aim to fix, for example, the false discovery rate at a predetermined tolerance. These methods, however, neglect local economic context and consequently lead to irrational experimental strategies. In contrast, the authors argue that this question is essentially economic, not statistical, and is amenable to an economic analysis that admits an optimal solution. This solution, in turn, suggests a novel tool for deciding the number of hits to confirm and the marginal cost of discovery, which meaningfully quantifies the local economic trade-off between true and false positives, yielding an economically optimal experimental strategy. Validated with retrospective simulations and prospective experiments, this strategy identified 157 additional actives that had been erroneously labeled inactive in at least one real-world screening experiment.
    MeSH term(s) False Positive Reactions ; Feasibility Studies ; High-Throughput Screening Assays/economics ; High-Throughput Screening Assays/methods ; Humans ; Reproducibility of Results
    Language English
    Publishing date 2010-06-14
    Publishing country United States
    Document type Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't
    ZDB-ID 1433680-7
    ISSN 1552-454X ; 1087-0571
    ISSN (online) 1552-454X
    ISSN 1087-0571
    DOI 10.1177/1087057110372803
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  9. Article: Nucleic acid evolution and minimization by nonhomologous random recombination.

    Bittker, Joshua A / Le, Brian V / Liu, David R

    Nature biotechnology

    2002  Volume 20, Issue 10, Page(s) 1024–1029

    Abstract: We have developed a simple method for exploring nucleic acid sequence space by nonhomologous random recombination (NRR) that enables DNA fragments to randomly recombine in a length-controlled manner without the need for sequence homology. We compared the ...

    Abstract We have developed a simple method for exploring nucleic acid sequence space by nonhomologous random recombination (NRR) that enables DNA fragments to randomly recombine in a length-controlled manner without the need for sequence homology. We compared the results of using NRR and error-prone PCR to evolve DNA aptamers that bind streptavidin. Starting with two parental sequences of modest avidin affinity, evolution using NRR resulted in aptamers with 15- to 20-fold higher affinity than the highest-affinity aptamers evolved using error-prone PCR, and 27- or 46-fold higher affinities than parental sequences derived using systematic evolution of ligands by exponential enrichment (SELEX). NRR also facilitates the identification of functional regions within evolved sequences. Inspection of a small number of NRR-evolved clones identified a 40-base DNA sequence, present in multiple copies in each clone, that binds streptavidin. Our findings suggest that NRR may enhance the effectiveness of nucleic acid evolution and the ease of identifying structure-activity relationships among evolved sequences.
    MeSH term(s) Base Sequence ; Cloning, Molecular ; DNA/genetics ; DNA Primers ; Directed Molecular Evolution/methods ; Evolution, Molecular ; Molecular Sequence Data ; Nucleic Acid Amplification Techniques/methods ; Polymerase Chain Reaction ; Random Amplified Polymorphic DNA Technique/methods ; Recombination, Genetic ; Reproducibility of Results ; Sensitivity and Specificity ; Sequence Analysis, DNA/methods ; Sequence Homology, Nucleic Acid ; Streptavidin/genetics
    Chemical Substances DNA Primers ; DNA (9007-49-2) ; Streptavidin (9013-20-1)
    Language English
    Publishing date 2002-09-09
    Publishing country United States
    Document type Comparative Study ; Evaluation Study ; Journal Article ; Research Support, Non-U.S. Gov't ; Research Support, U.S. Gov't, Non-P.H.S.
    ZDB-ID 1311932-1
    ISSN 1546-1696 ; 1087-0156
    ISSN (online) 1546-1696
    ISSN 1087-0156
    DOI 10.1038/nbt736
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  10. Article: Recent advances in the in vitro evolution of nucleic acids.

    Bittker, Joshua A / Phillips, Kevin J / Liu, David R

    Current opinion in chemical biology

    2002  Volume 6, Issue 3, Page(s) 367–374

    Abstract: Molecular evolution allows chemists and biologists to generate nucleic acids with tailor-made binding or catalytic activities. Recent examples of nucleic acid evolution in vitro provide insights into natural ribozyme evolution and also demonstrate ... ...

    Abstract Molecular evolution allows chemists and biologists to generate nucleic acids with tailor-made binding or catalytic activities. Recent examples of nucleic acid evolution in vitro provide insights into natural ribozyme evolution and also demonstrate potential applications of evolved DNA and RNA molecules. Efforts to expand the scope of nucleic acid evolution are also underway, including the development of novel methods for exploring nucleic acid sequence-space and the incorporation of non-natural chemical functionality into nucleic acid libraries.
    MeSH term(s) Base Sequence ; Catalysis ; DNA/chemistry ; DNA/genetics ; DNA/metabolism ; Evolution, Molecular ; RNA/chemistry ; RNA/genetics ; RNA/metabolism ; RNA, Catalytic
    Chemical Substances RNA, Catalytic ; RNA (63231-63-0) ; DNA (9007-49-2)
    Language English
    Publishing date 2002-05-21
    Publishing country England
    Document type Journal Article ; Research Support, Non-U.S. Gov't ; Research Support, U.S. Gov't, Non-P.H.S. ; Review
    ZDB-ID 1439176-4
    ISSN 1879-0402 ; 1367-5931
    ISSN (online) 1879-0402
    ISSN 1367-5931
    DOI 10.1016/s1367-5931(02)00321-6
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