[Show abstract][Hide abstract] ABSTRACT: Epigenetic regulation of gene expression is essential in many biological processes and its deregulation contributes to pathology including tumor formation. We used an image-based cell assay that measures the induction of a silenced GFP-estrogen receptor reporter to identify novel classes of small molecules involved in the regulation of gene expression. Using this Locus Derepression assay, we queried 283,122 compounds by quantitative high-throughput screening evaluating compounds at multiple concentrations. After confirmation and independent validation, the Locus Derepression assay identified 19 small molecules as new actives that induce the GFP message over two fold. Viability assays demonstrated 17 of these actives have anti-proliferative activity and two of them show selectivity for cancer vs. patient matched normal cells and cause unique changes in gene expression patterns in cancer cells. Hence, these compounds represent chemical tools for understanding the molecular mechanisms of epigenetic control of transcription and for modulating cell growth pathways.
ACS Chemical Biology 09/2014; 9(11). DOI:10.1021/cb500532x · 5.33 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Retinoic acid-related orphan receptor RORγt plays a pivotal role in the differentiation of TH17 cells. Antagonizing RORγt transcriptional activity is a potential means to treat TH17-related autoimmune diseases. Herein, we describe the identification of a series of diphenylpropanamides as novel and selective RORγ antagonists. Diphenylpropanamide 4n inhibited transcriptional activity of RORγt, but not RORα, in cells. In addition, it suppressed human TH17 cell differentiation at sub-micromolar concentrations.
[Show abstract][Hide abstract] ABSTRACT: Malaria is a deadly infectious disease in many tropical and subtropical countries. Previous efforts to eradicate malaria have failed, largely due to the emergence of drug-resistant parasites, insecticide-resistant mosquitoes and, in particular, the lack of drugs or vaccines to block parasite transmission. ATP-binding cassette (ABC) transporters are known to play a role in drug transport, metabolism, and resistance in many organisms, including malaria parasites. To investigate whether a Plasmodium falciparum ABC transporter (Pf14_0244 or PfABCG2) modulates parasite susceptibility to chemical compounds or plays a role in drug resistance, we disrupted the gene encoding PfABCG2, screened the recombinant and the wild-type 3D7 parasites against a library containing 2,816 drugs approved for human or animal use, and identified an antihistamine (ketotifen) that became less active against the PfABCG2-disrupted parasite in culture. In addition to some activity against asexual stages and gametocytes, ketotifen was highly potent in blocking oocyst development of P. falciparum and the rodent parasite Plasmodium yoelii in mosquitoes. Tests of structurally related tricyclic compounds identified additional compounds having similar activities in inhibiting transmission. Additionally, ketotifen appeared to have some activity against relapse of Plasmodium cynomolgi in rhesus monkeys. Further clinical evaluation of ketotifen and related compounds, including synthetic new derivatives, in blocking malaria transmission may provide new weapons for the current effort of malaria eradication.
[Show abstract][Hide abstract] ABSTRACT: High-throughput screening (HTS) provides a rapid and comprehensive approach to identifying compounds that target specific biological processes as well as genes that are essential to those processes. Here we describe a HTS assay for small molecules that induce either DNA re-replication or endoreduplication (i.e. excess DNA replication) selectively in cells derived from human cancers. Such molecules will be useful not only to investigate cell division and differentiation, but they may provide a novel approach to cancer chemotherapy. Since induction of DNA re-replication results in apoptosis, compounds that selectively induce DNA re-replication in cancer cells without doing so in normal cells could kill cancers in vivo without preventing normal cell proliferation. Furthermore, the same HTS assay can be adapted to screen siRNA molecules to identify genes whose products restrict genome duplication to once per cell division. Some of these genes might regulate the formation of terminally differentiated polyploid cells during normal human development, whereas others will prevent DNA re-replication during each cell division. Based on previous studies, we anticipate that one or more of the latter genes will prove to be essential for proliferation of cancer cells but not for normal cells, since many cancer cells are deficient in mechanisms that maintain genome stability.
[Show abstract][Hide abstract] ABSTRACT: Malaria remains a devastating disease largely because of widespread drug resistance. New drugs and a better understanding of the mechanisms of drug action and resistance are essential for fulfilling the promise of eradicating malaria. Using high-throughput chemical screening and genome-wide association analysis, we identified 32 highly active compounds and genetic loci associated with differential chemical phenotypes (DCPs), defined as greater than or equal to fivefold differences in half-maximum inhibitor concentration (IC(50)) between parasite lines. Chromosomal loci associated with 49 DCPs were confirmed by linkage analysis and tests of genetically modified parasites, including three genes that were linked to 96% of the DCPs. Drugs whose responses mapped to wild-type or mutant pfcrt alleles were tested in combination in vitro and in vivo, which yielded promising new leads for antimalarial treatments.
[Show abstract][Hide abstract] ABSTRACT: In an effort to expand the stereochemical and structural complexity of chemical libraries used in drug discovery, the Center for Chemical Methodology and Library Development at Boston University has established an infrastructure to translate methodologies accessing diverse chemotypes into arrayed libraries for biological evaluation. In a collaborative effort, the NIH Chemical Genomics Center determined IC(50)'s for Plasmodium falciparum viability for each of 2,070 members of the CMLD-BU compound collection using quantitative high-throughput screening across five parasite lines of distinct geographic origin. Three compound classes displaying either differential or comprehensive antimalarial activity across the lines were identified, and the nascent structure activity relationships (SAR) from this experiment used to initiate optimization of these chemotypes for further development.
Proceedings of the National Academy of Sciences 04/2011; 108(17):6775-80. DOI:10.1073/pnas.1017666108 · 9.67 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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, the authors 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 6 concentrations up to 92 µM to generate titration-response curves and determine the potency and efficacy of all compounds. The qHTS data set 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 µM IC(50). This series represents a new class of thyroid hormone receptor-coactivator modulators.
[Show abstract][Hide abstract] ABSTRACT: Previous studies have shown DNA re-replication can be induced in cells derived from human cancers under conditions in which it is not possible for cells derived from normal tissues. Because 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 1,280 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, and 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.
Molecular Cancer Research 03/2011; 9(3):294-310. DOI:10.1158/1541-7786.MCR-10-0570 · 4.38 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Thyroid hormone receptors (TRs) are members of the nuclear hormone receptor (NR) superfamily and regulate development, growth, and metabolism. Upon binding thyroid hormone, TR undergoes a conformational change that allows the release of corepressors and the recruitment of coactivators, which in turn regulate target gene transcription. Although a number of TR antagonists have been developed, most are analogs of the endogenous hormone that inhibit ligand binding. In a screen for inhibitors that block the association of TRβ with steroid receptor coactivator 2 (SRC2), we identified a novel methylsulfonylnitrobenzoate (MSNB)-containing series that blocks this interaction at micromolar concentrations. Here we have studied a series of MSNB analogs and characterized their structure activity relationships. MSNB members do not displace thyroid hormone T3 but instead act by direct displacement of SRC2. MSNB series members are selective for the TR over the androgen, vitamin D, and PPARγ NR members, and they antagonize thyroid hormone-activated transcription action in cells. The methylsulfonylnitro group is essential for TRβ antagonism. Side-chain alkylamine substituents showed better inhibitory activity than arylamine substituents. Mass spectrum analysis suggested that MSNB inhibitors bind irreversibly to Cys-298 within the AF-2 cleft of TRβ to disrupt SRC2 association.
[Show abstract][Hide abstract] ABSTRACT: The microtubule-associated protein tau contributes directly or indirectly to key structural and regulatory cellular functions. Under pathological conditions, tau becomes sequestered into insoluble neurofibrillary tangles that promote axonal transport deficits, ultimately lead to synaptic dysfunction and neuronal loss. Beta-amyloid protein undergoes a similar type of aggregation as tau, and is implicated in the pathology of Alzheimer's disease. Recent reports describing screening of libraries of small molecules for their ability to inhibit tau fibrillization have shown that some classes of compounds, such as anthraquinones, phenothiazines, and porphyrins have the ability to inhibit tau fibril formation. While these hits may be unlikely to be developed into novel therapeutics for the treatment of tauopathies, the results suggest that in vitro assays used for these screenings are capable of detecting inhibitors of tau fibrillization. Thus, further qHTS for small molecule inhibitors of heparin-induced tau fibril formation promises to provide the identification novel and more tractable hits. HTS screening identified the probe ML103 (CID-9795907) as a member of a series of tau oligomerization/fibrillization inhibitors that can be used to study tau protein aggregation in vitro, as well as a starting point for drug development for the treatment of Alzheimer's Disease (AD) and other tauopathies.
Probe Reports from the NIH Molecular Libraries Program, 01/2010; National Center for Biotechnology Information (US).
[Show abstract][Hide abstract] ABSTRACT: Thyroid hormone receptors (TRs) are members of the nuclear hormone receptor superfamily and regulate many homeostatic processes, including basal metabolism, cardiovascular function, body weight, and lipid trafficking. Upon binding of the ligand triiodothyronine (T3), TR undergoes a conformational change that releases corepressors and recruits coactivators, such as Steroid Receptor Coactivator 2 (SRC2); in turn, these modulate the expression of target genes. In this report, we used a TRβ-SRC2 fluorescence polarization assay to screen the Molecular Libraries Small Molecule Repository (MLSMR) and identify a novel methylsulfonylnitrobenzoate (MSNB)-containing series that blocks the association of TRβ with a SRC2 peptide. This inhibitor probe molecule, ML151 (CID 5184800), blocked TRβ-SRC2 interaction with a potency of 1.8μM. Mechanistic studies revealed that ML151 (CID 5184800) is a covalent inhibitor and binds irreversibly to Cys298 within the AF-2 cleft of TRβ. This series will be useful for in vitro mechanistic studies of TR-SRC2 interactions, as well as other nuclear hormone receptor-coactivator interactions.
Probe Reports from the NIH Molecular Libraries Program, 01/2010; National Center for Biotechnology Information (US).
[Show abstract][Hide abstract] ABSTRACT: Studies of gene function and molecular mechanisms in Plasmodium falciparum are hampered by difficulties in characterizing and measuring phenotypic differences between individual parasites. We screened seven parasite lines for differences in responses to 1,279 bioactive chemicals. Hundreds of compounds were active in inhibiting parasite growth; 607 differential chemical phenotypes, defined as pairwise IC(50) differences of fivefold or more between parasite lines, were cataloged. We mapped major determinants for three differential chemical phenotypes between the parents of a genetic cross, and we identified target genes by fine mapping and testing the responses of parasites in which candidate genes were genetically replaced with mutant alleles. Differential sensitivity to dihydroergotamine methanesulfonate (1), a serotonin receptor antagonist, was mapped to a gene encoding the homolog of human P-glycoprotein (PfPgh-1). This study identifies new leads for antimalarial drugs and demonstrates the utility of a high-throughput chemical genomic strategy for studying malaria traits.
Nature Chemical Biology 10/2009; 5(10):765-71. DOI:10.1038/nchembio.215 · 13.00 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The NIH Chemical Genomics Center (NCGC) was the inaugural center of the Molecular Libraries and Screening Center Network (MLSCN). Along with the nine other research centers of the MLSCN, the NCGC was established with a primary goal of bringing industrial technology and experience to empower the scientific community with small molecule compounds for use in their research. We intend this review to serve as 1) an introduction to the NCGC standard operating procedures, 2) an overview of several of the lessons learned during the pilot phase and 3) a review of several of the innovative discoveries reported during the pilot phase of the MLSCN.
Current topics in medicinal chemistry 10/2009; 9(13):1181-93. DOI:10.2174/156802609789753644 · 3.40 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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 microM scored 35 and 71% of qHTS actives, respectively, as inactive and were therefore false negatives. Following counter screens to identify fluorescent and non-selective 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.
[Show abstract][Hide abstract] ABSTRACT: 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 approximately 292000 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 thioflavin T fluorescence and fluorescence polarization methods. Previously described classes of inhibitors as well as new scaffolds were identified, including novel aminothienopyridazines (ATPZs). A number of ATPZ analogues 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 Abeta(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.
[Show abstract][Hide abstract] ABSTRACT: Epigenetic regulation of gene expression is essential in embryonic development and contributes to cancer pathology. We used a cell-based imaging assay that measures derepression of a silenced green fluorescent protein (GFP) reporter to identify novel classes of compounds involved in epigenetic regulation. This locus derepression (LDR) assay was screened against a 69,137-member chemical library using quantitative high-throughput screening (qHTS), a titration-response method that assays compounds at multiple concentrations. From structure-activity relationships of the 411 actives recovered from the qHTS, 6 distinct chemical series were chosen for further study. A total of 48 qHTS actives and analogs were counterscreened using the parental line of the LDR cells, which lack the GFP reporter. Three series-8-hydroxy quinoline, quinoline-8-thiol, and 1,3,5-thiadiazinane-2-thione-were not fluorescent and reconfirmed activity in the LDR cells. The three active series did not inhibit histone deacetylase activity in nuclear extracts or reactivate the expression of the densely methylated p16 gene in cancer cells. However, one series induced expression of the methylated CDH13 gene and inhibited the viability of several lung cancer lines at submicromolar concentrations. These results suggest that the identified small molecules act on epigenetic or transcriptional components and validate our approach of using a cell-based imaging assay in conjunction with qHTS.
[Show abstract][Hide abstract] ABSTRACT: To aid in the interpretation of high-throughput screening (HTS) results derived from luciferase-based assays, we used quantitative HTS, an approach that defines the concentration-response behavior of each library sample, to profile the ATP-dependent luciferase from Photinus pyralis against more than 70,000 samples. We found that approximately 3% of the library was active, containing only compounds with inhibitory concentration-responses, of which 681 (0.9%) exhibited IC 50 < 10 microM. Representative compounds were shown to inhibit purified P. pyralis as well as several commercial luciferase-based detection reagents but were found to be largely inactive against Renilla reniformis luciferase. Light attenuation by the samples was also examined and found to be more prominent in the blue-shifted bioluminescence produced by R. reniformis luciferase than in the bioluminescence produced by P. pyralis luciferase. We describe the structure-activity relationship of the luciferase inhibitors and discuss the use of this data in the interpretation of HTS results and configuration of luciferase-based assays.
[Show abstract][Hide abstract] ABSTRACT: Alzheimer disease is diagnosed postmortem by the density and spatial distribution of beta-amyloid plaques and tau-bearing neurofibrillary tangles. The major protein component of each lesion adopts cross-beta-sheet conformation capable of binding small molecules with submicromolar affinity. In many cases, however, Alzheimer pathology overlaps with Lewy body disease, characterized by the accumulation of a third cross-beta-sheet forming protein, alpha-synuclein. To determine the feasibility of distinguishing tau aggregates from beta-amyloid and alpha-synuclein aggregates with small molecule probes, a library containing 72,455 small molecules was screened for antagonists of tau-aggregate-mediated changes in Thioflavin S fluorescence, followed by secondary screens to distinguish the relative affinity for each substrate protein. Results showed that >10-fold binding selectivity among substrates could be achieved, with molecules selective for tau aggregates containing at least three aromatic or rigid moieties connected by two rotatable bonds.
Neurobiology of Disease 01/2008; 28(3):251-60. DOI:10.1016/j.nbd.2007.07.018 · 5.08 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: High-throughput screening (HTS) assays enable the testing of large numbers of chemical substances for activity in diverse areas of biology. The biological responses measured in HTS assays span isolated biochemical systems containing purified receptors or enzymes to signal transduction pathways and complex networks functioning in cellular environments. This Review addresses factors that need to be considered when implementing assays for HTS and is aimed particularly at investigators new to this field. We discuss assay design strategies, the major detection technologies and examples of HTS assays for common target classes, cellular pathways and simple cellular phenotypes. We conclude with special considerations for configuring sensitive, robust, informative and economically feasible HTS assays.
Nature Chemical Biology 09/2007; 3(8):466-79. DOI:10.1038/nchembio.2007.17 · 13.00 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: High-throughput screening (HTS) of chemical compounds to identify modulators of molecular targets is a mainstay of pharmaceutical development. Increasingly, HTS is being used to identify chemical probes of gene, pathway, and cell functions, with the ultimate goal of comprehensively delineating relationships between chemical structures and biological activities. Achieving this goal will require methodologies that efficiently generate pharmacological data from the primary screen and reliably profile the range of biological activities associated with large chemical libraries. Traditional HTS, which tests compounds at a single concentration, is not suited to this task, because HTS is burdened by frequent false positives and false negatives and requires extensive follow-up testing. We have developed a paradigm, quantitative HTS (qHTS), tested with the enzyme pyruvate kinase, to generate concentration-response curves for >60,000 compounds in a single experiment. We show that this method is precise, refractory to variations in sample preparation, and identifies compounds with a wide range of activities. Concentration-response curves were classified to rapidly identify pyruvate kinase activators and inhibitors with a variety of potencies and efficacies and elucidate structure-activity relationships directly from the primary screen. Comparison of qHTS with traditional single-concentration HTS revealed a high prevalence of false negatives in the single-point screen. This study demonstrates the feasibility of qHTS for accurately profiling every compound in large chemical libraries (>10(5) compounds). qHTS produces rich data sets that can be immediately mined for reliable biological activities, thereby providing a platform for chemical genomics and accelerating the identification of leads for drug discovery.
Proceedings of the National Academy of Sciences 09/2006; 103(31):11473-8. DOI:10.1073/pnas.0604348103 · 9.67 Impact Factor