In light of the current outbreak of Ebola virus disease, there is an urgent need to develop effective therapeutics to treat Ebola infection, and drug repurposing screening is a potentially rapid approach for identifying such therapeutics. We developed a biosafety level 2 (BSL-2) 1536-well plate assay to screen for entry inhibitors of Ebola virus-like particles (VLPs) containing the glycoprotein (GP) and the matrix VP40 protein fused to a beta-lactamase reporter protein and applied this assay for a rapid drug repurposing screen of Food and Drug Administration (FDA)-approved drugs. We report here the identification of 53 drugs with activity of blocking Ebola VLP entry into cells. These 53 active compounds can be divided into categories including microtubule inhibitors, estrogen receptor modulators, antihistamines, antipsychotics, pump/channel antagonists, and anticancer/antibiotics. Several of these compounds, including microtubule inhibitors and estrogen receptor modulators, had previously been reported to be active in BSL-4 infectious Ebola virus replication assays and in animal model studies. Our assay represents a robust, effective and rapid high-throughput screen for the identification of lead compounds in drug development for the treatment of Ebola virus infection.
Antipsychotics; drug repurposing screen; Ebola virus; Ebola virus glycoprotein; estrogen receptor modulator; microtubule inhibitor; virus entry; VP40
Since its establishment in 2008, the US Tox21 inter-agency collaboration has made great progress in developing and evaluating cellular models for the evaluation of environmental chemicals as a proof of principle. Currently, the program has entered its production phase (Tox21 Phase II) focusing initially on the areas of modulation of nuclear receptors and stress response pathways. During Tox21 Phase II, the set of chemicals to be tested has been expanded to nearly 10,000 (10K) compounds and a fully automated screening platform has been implemented. The Tox21 robotic system combined with informatics efforts is capable of screening and profiling the collection of 10K environmental chemicals in triplicate in a week. In this article, we describe the Tox21 screening process, compound library preparation, data processing, and robotic system validation.
10K compound library; in vitro assays; quantitative high-throughput screening; robotic platform; Tox21 collaboration; toxicity testing
The U.S. Tox21 program has screened a library of approximately 10,000 (10K) environmental chemicals and drugs in three independent runs for estrogen receptor alpha (ERα) agonist and antagonist activity using two types of ER reporter gene cell lines, one with an endogenous full length ERα (ER-luc; BG1 cell line) and the other with a transfected partial receptor consisting of the ligand binding domain (ER-bla; ERα β-lactamase cell line), in a quantitative high-throughput screening (qHTS) format. The ability of the two assays to correctly identify ERα agonists and antagonists was evaluated using a set of 39 reference compounds with known ERα activity. Although both assays demonstrated adequate (i.e. >80%) predictivity, the ER-luc assay was more sensitive and the ER-bla assay more specific. The qHTS assay results were compared with results from previously published ERα binding assay data and showed >80% consistency. Actives identified from both the ER-bla and ER-luc assays were analyzed for structure-activity relationships (SARs) revealing known and potentially novel ERα active structure classes. The results demonstrate the feasibility of qHTS to identify environmental chemicals with the potential to interact with the ERα signaling pathway and the two different assay formats improve the confidence in correctly identifying these chemicals.
Knowledge of oncogenic mutations can inspire therapeutic strategies that are synthetically lethal, affecting cancer cells while sparing normal cells. Lenalidomide is an active agent in the activated B-cell-like (ABC) subtype of diffuse large B cell lymphoma (DLBCL), but its mechanism of action is unknown. Lenalidomide kills ABC DLBCL cells by augmenting interferon β (IFNβ) production, owing to the oncogenic MYD88 mutations in these lymphomas. In a cereblon-dependent fashion, lenalidomide downregulates IRF4 and SPIB, transcription factors that together prevent IFNβ production by repressing IRF7 and also amplify pro-survival NF-κB signaling by transactivating CARD11. Blockade of B cell receptor (BCR) signaling using the BTK inhibitor ibrutinib also downregulates IRF4 and consequently synergizes with lenalidomide in killing ABC DLBCLs, suggesting attractive therapeutic strategies.
Myotonic dystrophy (DM) is a multi-system neuromuscular disorder for which there is no treatment. We have developed a medium throughput phenotypic assay, based on the identification of nuclear foci in DM patient cell lines using in situ hybridization and high-content imaging to screen for potentially useful therapeutic compounds. A series of further assays based on molecular features of DM have also been employed. Two compounds that reduce and/or remove nuclear foci have been identified, Ro 31-8220 and chromomycin A3. Ro 31-8220 is a PKC inhibitor, previously shown to affect the hyperphosphorylation of CELF1 and ameliorate the cardiac phenotype in a DM1 mouse model. We show that the same compound eliminates nuclear foci, reduces MBNL1 protein in the nucleus, affects ATP2A1 alternative splicing and reduces steady-state levels of CELF1 protein. We demonstrate that this effect is independent of PKC activity and conclude that this compound may be acting on alternative kinase targets within DM pathophysiology. Understanding the activity profile for this compound is key for the development of targeted therapeutics in the treatment of DM.
A recent large outbreak of fungal infections by Exserohilum rostratum from contaminated compounding solutions has highlighted the need to rapidly screen available pharmaceuticals that could be useful in therapy. The present study utilized two newly-developed high throughput assays to screen approved drugs and pharmaceutically active compounds for identification of potential antifungal agents. Several known drugs were found that have potent effects against E. rostratum including the triazole antifungal posaconazole. Posaconazole is likely to be effective against infections involving septic joints and may provide an alternative for refractory central nervous system infections. The anti-E. rostratum activities of several other drugs including bithionol (an anti-parasitic drug), tacrolimus (an immunosuppressive agent) and floxuridine (an antimetabolite) were also identified from the drug repurposing screens. In addition, activities of other potential antifungal agents against E. rostratum were excluded, which may avoid unnecessary therapeutic trials and reveals the limited therapeutic alternatives for this outbreak. In summary, this study has demonstrated that drug repurposing screens can be quickly conducted within a useful time-frame. This would allow clinical implementation of identified alternative therapeutics and should be considered as part of the initial public health response to new outbreaks or rapidly-emerging microbial pathogens.
The human pathogen Giardia lamblia is an anaerobic protozoan parasite that causes giardiasis, one of the most common diarrheal diseases worldwide. Although several drugs are available for the treatment of giardisis, resistance to these drugs has been reported and is likely to increase. The Giardia carbamate kinase (glCK) plays an essential role in Giardia metabolism and has no homologs in humans, making it an attractive candidate for anti-Giardia drug development. We have developed a luminescent enzyme coupled assay to measure the activity of glCK by quantitating the amount of ATP produced by the enzyme. This assay is homogeneous and has been miniaturized into a 1536-well plate format. A pilot screen against 4,096 known compounds using this assay yielded a signal-to-basal ratio of 11.5 fold and Z’ factor of 0.8 with a hit rate of 0.9 % of inhibitors of glCK. Therefore, this Giardia lamblia carbamate kinase assay is useful for high throughput screening of large compound collection for identification of the inhibitors for drug development.
Carbamate kinase; Giardia; high throughput screening; assay development.
We describe how room temperature storage of a 1,120 member compound library prepared in either DMSO or in a hydrated DMSO/water (67/33) mixture affects the reproducibility of potency values as monitored using cytochrome P450 1A2 and 2D6 isozyme assays. The bioluminescent assays showed Z′-factors of 0.71 and 0.62, with 18% and 32% of the library found as active against the CYP 1A2 and 2D6 isozymes respectively. We tested the library using quantitative high-throughput screening to generate potency values for every library member which was measured at seven time intervals spanning 37 weeks. We calculated the minimum significant ratio (MSR) from these potency values at each time interval and we found that for the library stored in DMSO, the CYP 1A2 and 2D6 assay MSRs progressed from approximately 2.0 to 5.0. The hydrated conditions showed similar performance in both MSR progression and analytical QC results. Based on this study we recommend that DMSO samples be stored in 1,536-well plates for < 4 months at room temperature. Further, the study shows the magnitude of potency changes that can occur in a robust bioassay due to compound sample storage.
HTS; compound storage; DMSO; quantitative HTS
Small-molecule compounds approved for use as drugs may be “repurposed” for new indications and studied to determine the mechanisms of their beneficial and adverse effects. A comprehensive collection of all small-molecule drugs approved for human use would be invaluable for systematic repurposing across human diseases, particularly for rare and neglected diseases, for which the cost and time required for development of a new chemical entity are often prohibitive. Previous efforts to build such a comprehensive collection have been limited by the complexities, redundancies, and semantic inconsistencies of drug naming within and among regulatory agencies worldwide; a lack of clear conceptualization of what constitutes a drug; and a lack of access to physical samples. We report here the creation of a definitive, complete, and nonredundant list of all approved molecular entities as a freely available electronic resource and a physical collection of small molecules amenable to high-throughput screening.
Background: The large and increasing number of chemicals released into the environment demands more efficient and cost-effective approaches for assessing environmental chemical toxicity. The U.S. Tox21 program has responded to this challenge by proposing alternative strategies for toxicity testing, among which the quantitative high-throughput screening (qHTS) paradigm has been adopted as the primary tool for generating data from screening large chemical libraries using a wide spectrum of assays.
Objectives: The goal of this study was to develop methods to evaluate the data generated from these assays to guide future assay selection and prioritization for the Tox21 program.
Methods: We examined the data from the Tox21 pilot-phase collection of approximately 3,000 environmental chemicals profiled in qHTS format against a panel of 10 human nuclear receptors (AR, ERα, FXR, GR, LXRβ, PPARγ, PPARδ, RXRα, TRβ, and VDR) for reproducibility, concordance of biological activity profiles with sequence homology of the receptor ligand binding domains, and structure–activity relationships.
Results: We determined the assays to be appropriate in terms of biological relevance. We found better concordance for replicate compounds for the agonist-mode than for the antagonist-mode assays, likely due to interference of cytotoxicity in the latter assays. This exercise also enabled us to formulate data-driven strategies for discriminating true signals from artifacts, and to prioritize assays based on data quality.
Conclusions: The results demonstrate the feasibility of qHTS to identify the potential for environmentally relevant chemicals to interact with key toxicity pathways related to human disease induction.
assay performance; chemical genomics; cytotoxicity; nuclear receptors; qHTS; Tox21
Activation of Gq protein-coupled receptors can be monitored by measuring the increase in intracellular calcium with fluorescent dyes. Recent advances in fluorescent kinetic plate readers and liquid-handling technology have made it possible to follow these transient changes in intracellular calcium in a 1,536-well plate format for high-throughput screening (HTS). Here, we have applied the latest generation of fluorescence kinetic plate readers to multiplex the agonist and antagonist screens of a G protein-coupled receptor (GPCR). This multiplexed assay format provides an efficient and cost-effective method for HTS of Gq-coupled GPCR targets.
Nuclear factor-kappa B (NF-κB) is a transcription factor that plays a critical role across many cellular processes including embryonic and neuronal development, cell proliferation, apoptosis, immune responses to infection, and inflammation. Dysregulation of NF-κB signaling is associated with inflammatory diseases and certain cancers. Constitutive activation of NF-κB signaling has been found in some types of tumors including breast, colon, prostate, skin and lymphoid, hence therapeutic blockade of NF-κB signaling in cancer cells provides an attractive strategy for the development of anticancer drugs. To identify small molecule inhibitors of NF-κB signaling, we screened approximately 2,800 clinically approved drugs and bioactive compounds from the NIH Chemical Genomics Center Pharmaceutical Collection (NPC) in a NF-κB mediated β-lactamase reporter gene assay. Each compound was tested at fifteen different concentrations in a quantitative high throughput screening format. We identified nineteen drugs that inhibited NF-κB signaling, with potencies as low as 20 nM. Many of these drugs, including emetine, fluorosalan, sunitinib malate, bithionol, narasin, tribromsalan, and lestaurtinib, inhibited NF-κB signaling via inhibition of IκBα phosphorylation. Others, such as ectinascidin 743, chromomycin A3 and bortezomib utilized other mechanisms. Furthermore, many of these drugs induced caspase 3/7 activity and had an inhibitory effect on cervical cancer cell growth. Our results indicate that many currently approved pharmaceuticals have previously unappreciated effects on NF-κB signaling, which may contribute to anticancer therapeutic effects. Comprehensive profiling of approved drugs provides insight into their molecular mechanisms, thus providing a basis for drug repurposing.
caspase 3/7; cervical cancer; IκBα phosphorylation; NCGC Pharmaceutical Collection; NF-κB signaling
A series of substituted 6-arylquinazolin-4-amines were prepared and analyzed as inhibitors of Clk4. Synthesis, structure activity-relationships and the selectivity of a potent analogue against a panel of 402 kinases are presented. Inhibition of Clk4 by these agents at varied concentrations of assay substrates (ATP and receptor peptide) highly suggests that this chemotype is an ATP competitive inhibitor. Molecular docking provides further evidence that inhibition is the result of binding at the kinase hinge region. Selected compounds represent novel tools capable of potent and selective inhibition of Clk1, Clk4 and Dyrk1A.
kinase inhibition; pre-mRNA splicing; Clk; Dyrk1A
The Deepwater Horizon oil spill has led to the use of >1 M gallons of oil spill dispersants, which are mixtures of surfactants and solvents. Because of this large scale use there is a critical need to understand the potential for toxicity of the currently used dispersant and potential alternatives, especially given the limited toxicity testing information that is available. In particular, some dispersants contain nonylphenol ethoxylates (NPEs), which can degrade to nonylphenol (NP), a known endocrine disruptor. Given the urgent need to generate toxicity data, we carried out a series of in vitro high-throughput assays on eight commercial dispersants. These assays focused on the estrogen and androgen receptors (ER and AR), but also included a larger battery of assays probing other biological pathways. Cytotoxicity in mammalian cells was also quantified. No activity was seen in any AR assay. Two dispersants showed a weak ER signal in one assay (EC50 of 16 ppm for Nokomis 3-F4 and 25 ppm for ZI-400). NPs and NPEs also had a weak signal in this same ER assay. Note that Corexit 9500, the currently used product, does not contain NPEs and did not show any ER activity. Cytotoxicity values for six of the dispersants were statistically indistinguishable, with median LC50 values ∼100 ppm. Two dispersants, JD 2000, SAF-RON GOLD, were significantly less cytotoxic than the others with LC50 values approaching or exceeding 1000 ppm.
Activation of Gq protein-coupled receptors can be monitored by measuring the increase in intracellular calcium with fluorescent dyes. Recent advances in fluorescent kinetic plate readers and liquid-handling technology have made it possible to follow these transient changes in intracellular calcium in a 1536-well plate format for high-throughput screening. Here, we have applied the latest generation of fluorescence kinetic plate readers to multiplex the agonist and antagonist screens of a GPCR. This multiplexed assay format provides an efficient and cost-effective method for high-throughput screening of Gq-coupled GPCR targets.
GPCR; calcium assay; FDSS; multiplex; high-throughput screening
The thyroid stimulating hormone receptor (TSHR) belongs to the glycoprotein hormone receptor subfamily of seven-transmembrane spanning receptors. TSHR is expressed in thyroid follicular cells and is activated by TSH, which regulates growth and function of these cells. Recombinant TSH is used in diagnostic screens for thyroid cancer, especially in patients after thyroid cancer surgery. Currently, no selective small molecule agonist of the TSHR is available. To screen for novel TSHR agonists, we miniaturized a cell-based cAMP assay into 1536-well plate format. This assay uses a HEK293 cell line stably expressing the TSHR and a cyclic nucleotide gated ion channel (CNG), which functions as a biosensor. From a quantitative high-throughput screen of 73,180 compounds in parallel with a parental cell line (without the TSHR), 276 primary active compounds were identified. The activities of the selected active compounds were further confirmed in an orthogonal HTRF cAMP-based assay. 49 compounds in several structural classes have been confirmed as small molecule TSHR agonists that will serve as starting compounds for chemical optimization and studies of thyroid physiology in health and disease.
Thyroid-stimulating hormone TSH; TSHR; TSHR agonist; quantitative high throughput screening; qHTS; HTS; probe identification; CNG; PubChem
An efficient and versatile Compound Management operation is essential for the success of all downstream processes in high-throughput screening (HTS) and small molecule lead development. Staff, equipment, and processes need to be not only reliable, but remain flexible and prepared to incorporate paradigm changes. In the present report, we describe a system and associated processes which enable handling of compounds for both screening and follow-up purposes at the NIH Chemical Genomics Center (NCGC), a recently-established HTS and probe development center within the Molecular Libraries Initiative of the NIH Roadmap. Our screening process, termed quantitative HTS (qHTS), involves assaying the complete compound library, currently containing >200,000 members, at a series of dilutions to construct a full concentration-response profile. As such, Compound Management at the NCGC has been uniquely tasked to prepare, store, register, and track a vertically-developed plate dilution series (i.e., inter-plate titrations) in the 384-well format. These are compressed into a series of 1,536-well plates and are registered to track all subsequent plate storage. Here, we present details on the selection of equipment to enable automated, reliable and parallel compound manipulation in 384- and 1,536-well formats, protocols for preparation of inter-plate dilution series for qHTS, as well as qHTS-specific processes and issues.
screening; qHTS; inter-plate titrations; serial dilution; concentration-response curve; dose-response curve; compound registration; liquid handling; automation; cherry-picking; follow-up
Retroviruses differ in their preferences for sites for viral DNA integration in the chromosomes of infected cells. Human immunodeficiency virus (HIV) integrates preferentially within active transcription units, whereas murine leukemia virus (MLV) integrates preferentially near transcription start sites and CpG islands. We investigated the viral determinants of integration-site selection using HIV chimeras with MLV genes substituted for their HIV counterparts. We found that transferring the MLV integrase (IN) coding region into HIV (to make HIVmIN) caused the hybrid to integrate with a specificity close to that of MLV. Addition of MLV gag (to make HIVmGagmIN) further increased the similarity of target-site selection to that of MLV. A chimeric virus with MLV Gag only (HIVmGag) displayed targeting preferences different from that of both HIV and MLV, further implicating Gag proteins in targeting as well as IN. We also report a genome-wide analysis indicating that MLV, but not HIV, favors integration near DNase I–hypersensitive sites (i.e., +/− 1 kb), and that HIVmIN and HIVmGagmIN also favored integration near these features. These findings reveal that IN is the principal viral determinant of integration specificity; they also reveal a new role for Gag-derived proteins, and strengthen models for integration targeting based on tethering of viral IN proteins to host proteins.
A required step in the replication cycle of retroviruses is the integration of a DNA copy of the viral genome into a host cell chromosome. Recent studies have shown that human immunodeficiency virus (HIV) and murine leukemia virus (MLV) favor integration near different chromosomal features. HIV preferentially targets active genes, while MLV prefers integration near start sites of gene transcription. The authors investigated integration-target site–selection by HIV derivatives substituted with segments of MLV to determine which viral proteins are responsible for integration-targeting preferences. They found that the viral integrase protein is the dominant determinant of integration-site selection, probably through its tethering to cellular proteins bound near preferred genomic regions. In addition, components of the viral structural polyprotein, Gag, appear to be involved in targeting. These findings provide a functional map of the viral proteins involved in directing integration-site selection.
We have analyzed the placement of sites of integration of avian sarcoma-leukosis virus (ASLV) and human immunodeficiency virus (HIV) DNA in the draft chicken genome sequence, with the goals of assessing species-specific effects on integration and allowing comparison to the distribution of chicken endogenous retroviruses (ERVs). We infected chicken embryo fibroblasts (CEF) with ASLV or HIV and sequenced 863 junctions between host and viral DNA. The relationship with cellular gene activity was analyzed by transcriptional profiling of uninfected or ASLV-infected CEF cells. ASLV weakly favored integration in active transcription units (TUs), and HIV strongly favored active TUs, trends seen previously for integration in human cells. The ERVs, in contrast, accumulated mostly outside TUs, including ERVs related to ASLV. The minority of ERVs present within TUs were mainly in the antisense orientation; consequently, the viral splicing and polyadenylation signals would not disrupt cellular mRNA synthesis. In contrast, de novo ASLV integration sites within TUs showed no orientation bias. Comparing the distribution of de novo ASLV integration sites to ERVs indicated that purifying selection against gene disruption, and not initial integration targeting, probably determined the ERV distribution. Further analysis indicated that ERVs in humans, mice, and rats showed similar distributions, suggesting purifying selection dictated their distributions as well.
The completion of the human genome sequence has made possible genome-wide studies of retroviral DNA integration. Here we report an analysis of 3,127 integration site sequences from human cells. We compared retroviral vectors derived from human immunodeficiency virus (HIV), avian sarcoma-leukosis virus (ASLV), and murine leukemia virus (MLV). Effects of gene activity on integration targeting were assessed by transcriptional profiling of infected cells. Integration by HIV vectors, analyzed in two primary cell types and several cell lines, strongly favored active genes. An analysis of the effects of tissue-specific transcription showed that it resulted in tissue-specific integration targeting by HIV, though the effect was quantitatively modest. Chromosomal regions rich in expressed genes were favored for HIV integration, but these regions were found to be interleaved with unfavorable regions at CpG islands. MLV vectors showed a strong bias in favor of integration near transcription start sites, as reported previously. ASLV vectors showed only a weak preference for active genes and no preference for transcription start regions. Thus, each of the three retroviruses studied showed unique integration site preferences, suggesting that virus-specific binding of integration complexes to chromatin features likely guides site selection.
Retroviruses have potential for gene therapy only if they do not activate endogenous genes. Of three tested retroviral vectors, ASLV showed no preference for integration into human transcription start regions