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1.  A Quantitative High Throughput Screen Identifies Novel Inhibitors of the Interaction of Thyroid Receptor β with a Peptide of Steroid Receptor Coactivator 2 
Journal of biomolecular screening  2011;16(6):618-627.
The thyroid hormone receptors (TR) are members of the nuclear hormone receptor (NHR) superfamily that regulate development, growth, and metabolism. Upon ligand binding, TR releases bound corepressors and recruits coactivators to modulate target gene expression. Steroid Receptor Coactivator 2 (SRC2) is an important coregulator that interacts with TRβ to activate gene transcription. To identify novel inhibitors of the TRβ and SRC2 interaction, we performed a quantitative high throughput screen (qHTS) of a TRβ-SRC2 fluorescence polarization assay against more than 290,000 small molecules. The qHTS assayed compounds at six concentrations up to 92 uM to generate titration-response curves and determine the potency and efficacy of all compounds. The qHTS dataset enabled the characterization of actives for structure-activity relationships as well as for potential artifacts such as fluorescence interference. Selected qHTS actives were tested in the screening assay using fluoroprobes labeled with Texas Red or fluorescein. The retest identified 19 series and 4 singletons as active in both assays with 40% or greater efficacy, free of compound interference and not toxic to mammalian cells. Selected compounds were tested as independent samples and a methylsulfonylnitrobenzoate series inhibited the TRβ-SRC2 interaction with 5 uM IC50. This series represents a new class of thyroid hormone receptor-coactivator modulators.
PMCID: PMC3162318  PMID: 21482722
thyroid receptor; small molecule; HTS; coactivator; protein-protein interaction
2.  An Image Based, High-Throughput, Screening Assay For Molecules That Induce Excess DNA Replication In Human Cancer Cells 
Molecular cancer research : MCR  2011;9(3):294-310.
Previous studies have shown DNA re-replication can be induced in cells derived from human cancers under conditions in which cells derived from normal tissues are not. Since DNA re-replication induces cell death, this strategy could be applied to the discovery of potential anticancer therapeutics. Therefore, an imaging assay amenable to high-throughput screening was developed that measures DNA replication in excess of four genomic equivalents in the nuclei of intact cells and indexes cell proliferation. This assay was validated by screening a library of 1280 bioactive molecules on both normal and tumor-derived cells where it proved more sensitive than current methods for detecting excess DNA replication. This screen identified known inducers of excess DNA replication such as inhibitors of microtubule dynamics, as well as novel compounds that induced excess DNA replication in both normal and cancer cells. In addition, two compounds were identified that induced excess DNA replication selectively in cancer cells, and one that induced endocycles selectively in cancer cells. Thus, this assay provides a new approach to the discovery of compounds useful for investigating the regulation of genome duplication and for the treatment of cancer.
PMCID: PMC3060295  PMID: 21257818
3.  The Identification of Aminothienopyridazine Inhibitors of Tau Assembly by Quantitative High-Throughput Screening† 
Biochemistry  2009;48(32):7732-7745.
Inclusions comprised of fibrils of the microtubule (MT)-associated protein tau are found in the brains of those with Alzheimer’s disease (AD) and other neurodegenerative tauopathies. The pathology that is observed in these diseases is believed to result from the formation of toxic tau oligomers or fibrils, and/or from the loss of normal tau function due to its sequestration into insoluble deposits. Hence, small molecules that prevent tau oligomerization and/or fibrillization might have therapeutic value. Indeed, examples of such compounds have been published but nearly all have properties that render them unsuitable as drug candidates. For these reasons, we conducted quantitative high-throughput screening (qHTS) of ~292,000 compounds to identify drug-like inhibitors of tau assembly. The fibrillization of a truncated tau fragment that contains four MT-binding domains was monitored in an assay that employed complementary thioflavine T fluorescence and fluorescence polarization methods. Previously described classes of inhibitors as well as new scaffolds were identified, including novel aminothienopyridazines (ATPZ’s). A number of ATPZ analogs were synthesized and structure-activity relationships were defined. Further characterization of representative ATPZ compounds showed they do not interfere with tau-mediated MT assembly, and they are significantly more effective at preventing the fibrillization of tau than the Aβ(1–42) peptide which forms AD senile plaques. Thus, the ATPZ molecules described here represent a novel class of tau assembly inhibitors that merit further development for testing in animal models of AD-like tau pathology.
PMCID: PMC2773749  PMID: 19580328
4.  A Quantitative High-Throughput Screen for Modulators of IL-6 Signaling: A Model for Interrogating Biological Networks using Chemical Libraries 
Molecular bioSystems  2009;5(9):1039-1050.
Small molecule modulators are critical for dissecting and understanding signaling pathways at the molecular level. Interleukin 6 (IL-6) is a cytokine that signals via the JAK/STAT pathway and is implicated in cancer and inflammation. To identify modulators of this pathway, we screened a chemical collection against an IL-6 responsive cell line stably expressing a beta-lactamase reporter gene fused to a sis-inducible element (SIE-bla cells). This assay was optimized for a 1536-well microplate format and screened against 11,693 small molecules using quantitative high-throughput screening (qHTS), a method that assays a chemical library at multiple concentrations to generate titration-response profiles for each compound. The qHTS recovered 564 actives with well-fit curves that clustered into 32 distinct chemical series of 13 activators and 19 inhibitors. A retrospective analysis of the qHTS data indicated that single concentration data at 1.5 and 7.7 uM scored 35 and 71% of qHTS actives, respectively, as inactive and were therefore false negatives. Following counter screens to identify fluorescent and nonselective series, we found four activator and one inhibitor series that modulated SIE-bla cells but did not show similar activity in reporter gene assays induced by EGF and hypoxia. Small molecules within these series will make useful tool compounds to investigate IL-6 signaling mediated by JAK/STAT activation.
PMCID: PMC2747079  PMID: 19668870
IL-6; small molecule; HTS; STAT; assay

Results 1-5 (5)