Automated microscopes have enabled the unprecedented collection of images at a rate that precludes visual inspection. Automated image analysis is required to identify interesting samples and extract quantitative information for high content screening (HCS). However, researchers are impeded by the lack of metrics and software tools to identify image-based aberrations that pollute data, limiting an experiment's quality. We have developed and validated approaches to identify those image acquisition artifacts that prevent optimal extraction of knowledge from high-throughput microscopy experiments. We have implemented these as a versatile, open-source toolbox of algorithms and metrics readily usable by biologists to improve data quality in a wide variety of biological experiments.

Aberrant protein-protein interactions are attractive drug targets in a variety of neurodegenerative diseases due to the common pathology of accumulation of protein aggregates. In amyotrophic lateral sclerosis, mutations in SOD1 cause the formation of aggregates and inclusions that may sequester other proteins and disrupt cellular processes. It has been demonstrated that mutant SOD1, but not wild-type SOD1, interacts with the axonal transport motor dynein and that this interaction contributes to motor neuron cell death, suggesting that disrupting this interaction may be a potential therapeutic target. However, it can be challenging to configure a high-throughput screening (HTS)–compatible assay to detect inhibitors of a protein-protein interaction. Here we describe the development and challenges of an HTS for small-molecule inhibitors of the mutant SOD1-dynein interaction. We demonstrate that the interaction can be formed by coexpressing the A4V mutant SOD1 and dynein intermediate complex in cells and that this interaction can be disrupted by compounds added to the cell lysates. Finally, we show that some of the compounds identified from a pilot screen to inhibit the protein-protein interaction with this method specifically disrupt the interaction between the dynein complex and mtSOD1 but not the dynein complex itself when applied to live cells.

Class O forkhead box (FOXO) transcription factors are downstream targets of the PI3K/AKT signaling pathway, which is upregulated in many tumors. AKT phosphorylates and inactivates FOXO1 by relocating it to the cytoplasm. Because FOXO1 functions as a tumor suppressor by negatively regulating cell-cycle progression and cell survival, compounds that promote FOXO1 localization to the nucleus might have therapeutic value in oncology. Here we describe the identification of such compounds by using an image-based, high content screen. Compounds that were active in retaining FOXO1 in the nucleus were tested to determine their pathway-specificity and isoform-specificity by using high content assays for Rev and FOXO3, respectively.

There are no effective antivirals currently available for the treatment of flavivirus infection in humans. As such, the identification and characterization of novel drug target sites are critical to developing new classes of antiviral drugs. The flavivirus NS5 N-terminal capping enzyme (CE) is vital for the formation of the viral RNA cap structure, which directs viral polyprotein translation and stabilizes the 5′ end of the viral genome. The structure of the flavivirus CE has been solved, and a detailed understanding of the CE–guanosine triphosphate (GTP) and CE–RNA cap interactions is available. Because of the essential nature of the interaction for viral replication, disrupting CE–GTP binding is an attractive approach for drug development. The authors have previously developed a robust assay for monitoring CE–GTP binding in real time. They adapted this assay for high-throughput screening and performed a pilot screen of 46 323 commercially available compounds. A number of small-molecule inhibitors capable of displacing a fluorescently labeled GTP in vitro were identified, and a second functional assay was developed to identify false positives. The results presented indicate that the flavivirus CE cap-binding site is a valuable new target site for antiviral drug discovery and should be further exploited for broad-spectrum anti-flaviviral drug development.

The molecular pathology of many protein misfolding, toxic gain-of-function diseases, such as amyotrophic lateral sclerosis (ALS), is not well understood. Although protein misfolding and aggregation are common themes in these diseases, efforts to identify cellular factors that regulate this process in an unbiased fashion and on a global scale have been lacking. Using an adapted version of an extant β-gal-based protein solubility assay, an expression screen for cellular modulators of solubility of an ALS-causing mutant SOD1 was carried out in mammalian cells. Following fluorescence-activated cell sorting enrichment of a mouse spinal cord cDNA library for gene products that increased SOD1 solubility, high-throughput screening of the cDNA pools from this enriched fraction was employed to identify pools containing relevant modulators. Positive pools, containing approximately 10 cDNA clones each, were diluted and rescreened iteratively until individual clones that improved SOD1 folding/solubility were identified. Genes with profound effects in the solubility assay were selected for validation by independent biochemical assays. Six of 10 validated genes had a significant effect on SOD1 solubility and folding in a SOD1 promoter-driven β-gal assay, indicating that global screening of cellular targets using such protein solubility/folding assay is viable and can be adapted for other misfolding diseases.

Inhibitors of human dimethylarginine dimethylaminohydrolase-1 (DDAH-1) are of therapeutic interest for controlling pathological nitric oxide production. Only a limited number of biologically useful inhibitors have been identified, so structurally diverse lead compounds are desired. In contrast with previous assays that do not possesses adequate sensitivity for optimal screening, herein is reported a high-throughput assay that uses an alternative thiol-releasing substrate, S-methyl-L-thiocitrulline, and a thiol-reactive fluorophore, 7-diethylamino-3-(4′-maleimidylphenyl)-4-methylcoumarin, to enable continuous detection of product formation by DDAH-1. The assay is applied to query two commercial libraries totaling 4,446 compounds and two representative hits are described, including a known DDAH-1 inhibitor. This is the most sensitive DDAH-1 assay reported to date, and enables screening of compound libraries using [S] =KM conditions, while displaying Z′ factors from 0.6 – 0.8. Therefore, this strategy now makes possible high-throughput screening for human DDAH-1 inhibitors in pursuit of molecular probes and drugs to control excessive nitric oxide production.

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.

The type II secretion (T2S) system in Gram-negative bacteria is comprised of the Sec and Tat pathways for translocating proteins into the periplasm and an outer membrane secretin for transporting proteins into the extracellular space. To discover Sec/Tat/T2S pathway inhibitors as potential new therapeutics, we used a Pseudomonas aeruginosa bioluminescent reporter strain responsive to SecA depletion and inhibition to screen compound libraries and characterize the hits. The reporter strain placed a luxCDABE operon under regulation of a SecA depletion-responsive up-regulated promoter in a secA deletion background complemented with an ectopic lac-regulated secA copy. Bioluminescence was indirectly proportional to the IPTG concentration and stimulated by azide, a known SecA ATPase inhibitor. A total of 96 compounds (0.1% of 73,000) were detected as primary hits due to stimulation of luminescence with a z-score ≥5. Direct secretion assays of the 9 most potent hits, representing 5 chemical scaffolds, revealed that they do not inhibit SecA-mediated secretion of β-lactamase into the periplasm, but do inhibit T2S-mediated extracellular secretion of elastase with IC50 values from 5 – 25 μM. In addition, 7 of the 9 compounds also inhibited the T2S-mediated extracellular secretion of phospholipases C by P. aeruginosa and of protease activity by Burkholderia pseudomallei.

Despite advances toward understanding the prevention and treatment of many cancers, patients who suffer from oral squamous cell carcinoma (OSCC) confront a survival rate that has remained unimproved for more than two decades indicating our ability to treat them pharmacologically has reached a plateau. In an ongoing effort to improve the clinical outlook for this disease, we previously reported that an essential component of the mechanism by which the proteasome inhibitor bortezomib (PS-341, Velcade) induced apoptosis in OSCC required the activation of a terminal unfolded protein response (UPR). Predicated on these studies, we hypothesized that high throughput screening (HTS) of large diverse chemical libraries might identify more potent or selective small molecule activators of the apoptotic arm of the UPR to control or kill OSCC. We have developed complementary cell-based assays using stably transfected CHO-K1 cell lines that individually assess the PERK/eIF2α/CHOP (apoptotic) or the IRE1/XBP1 (adaptive) UPR sub-pathways. A ~66K compound collection was screened at the University of Michigan Center for Chemical Genomics that included a unique library of pre-fractionated natural product extracts. The mycotoxin methoxycitrinin was isolated from a natural extract and found to selectively activate the CHOP-luciferase reporter at 80μM. A series of citrinin derivatives were isolated from these extracts, including a unique congener that has not been previously described. In an effort to identify more potent compounds we examined the ability of citrinin and the structurally related mycotoxins ochratoxin A and patulin to activate the UPR. Strikingly, we found that patulin at 2.5 – 10μM induced a terminal UPR in a panel of OSCC cells that was characterized by an increase in CHOP, GADD34 and ATF3 gene expression and XBP1 splicing. A luminescent caspase assay and the induction of several BH3-only genes indicated that patulin could induce apoptosis in OSCC cells. These data support the use of this complementary HTS strategy to identify novel modulators of UPR signaling and tumor cell death.

High-throughput screening data repositories, such as PubChem, represent valuable resources for the development of small molecule chemical probes and can serve as entry points for drug discovery programs. While the loose data format offered by PubChem allows for great flexibility, important annotations, such as the assay format and technologies employed, are not explicitly indexed. We have previously developed a BioAssay Ontology (BAO) and curated over 350 assays with standardized BAO terms. Here we describe the use of BAO annotations to analyze a large set of assays that employ luciferase- and β-lactamase-based technologies. We identified promiscuous chemotypes pertaining to different sub-categories of assays and specific mechanisms by which these chemotypes interfere in reporter gene assays. Our results show that the data in PubChem can be used to identify promiscuous compounds that interfere non-specifically with particular technologies. Furthermore, we show that BAO is a valuable toolset for the identification of related assays and for the systematic generation of insights that are beyond the scope of individual assays or screening campaigns.

JAK3 has become an ideal target for the therapeutic treatment of immune-related diseases, as well as for the prevention of organ allograft rejection. A number of JAK3 inhibitors have been identified by in vitro biochemical enzymatic assays, but the majority display significant off-target effects on JAK2. Therefore, there is an urgent need to develop new experimental approaches to identify compounds that specifically inhibit JAK3. Here, we showed that in 32D/IL-2Rβ cells, STAT5 becomes phosphorylated by IL-3/JAK2- or IL-2/JAK3-dependent pathway. Importantly, the selective JAK3 inhibitor CP-690,550 blocked the phosphorylation as well as the nuclear translocation of STAT5 following treatment of cells with IL-2, but not with IL-3. In an attempt to use the cells for large-scale chemical screens to identify JAK3 inhibitors, we established a cell line 32D/IL-2Rβ/6×STAT5 stably expressing a well-characterized STAT5 reporter gene. Treatment of this cell line with IL-2 or IL-3 dramatically increased the reporter activity in a high-throughput format. As expected, JAK3 inhibitors, CP-690,550 and JAK3 inhibitor VI, selectively inhibited the activity of the 6×STAT5 reporter following treatment with IL-2. By contrast, the pan-JAK inhibitor Curcumin non-selectively inhibited the activity of this reporter following treatment with either IL-2 or IL-3. Thus, this study indicates that our STAT5 reporter cell line can be used as an efficacious cellular model for chemical screens to identify low-molecular-weight inhibitors specific for JAK3.

DNA gyrase, a type II topoisomerase that introduces negative supercoils into DNA, is a validated antibacterial drug target. The holoenzyme is composed of 2 subunits, gyrase A (GyrA) and gyrase B (GyrB), which form a functional A2B2 heterotetramer required for bacterial viability. A novel fluorescence polarization (FP) assay has been developed and optimized to detect inhibitors that bind to the adenosine triphosphate (ATP) binding domain of GyrB. Guided by the crystal structure of the natural product novobiocin bound to GyrB, a novel novobiocin–Texas Red probe (Novo-TRX) was designed and synthesized for use in a high-throughput FP assay. The binding kinetics of the interaction of Novo-TRX with GyrB from Francisella tularensis has been characterized, as well as the effect of common buffer additives on the interaction. The assay was developed into a 21-μL, 384-well assay format and has been validated for use in high-throughput screening against a collection of Food and Drug Administration–approved compounds. The assay performed with an average Z′ factor of 0.80 and was able to identify GyrB inhibitors from a screening library.

Methylation of arginine residues, catalyzed by protein arginine methyltransferases (PRMTs), is one important protein post-translational modification involved in epigenetic regulation of gene expression. A fast and effective assay for PRMT can provide valuable information for dissecting the biological functions of PRMTs, as well as for screening small-molecule inhibitors of arginine methylation. Currently, among the methods used for PRMT activity measurement, many contain laborious separation procedures, which restrict the applications of these assays for high-throughput screening (HTS) in drug discovery. The authors report here a mix-and-measure method to measure PRMT activity based on the principle of scintillation proximity assay (SPA). In this assay, 3H-AdoMet was used as methyl donor, and biotin-modified histone H4 peptide served as a methylation substrate. Following the methylation reaction catalyzed by PRMTs, streptavidin-coated SPA beads were added to the reaction solution, and SPA signals were detected by a MicroBeta scintillation counter. No separation step is needed, which simplifies the assay procedure and greatly enhances the assay speed. Particularly, the miniaturization and robustness suggest that this method is suited for HTS of PRMT inhibitors.

Hepatitis C virus (HCV) is a considerable global health problem for which new classes of therapeutics are needed. We developed a high-throughput assay to identify compounds that selectively block translation initiation from the HCV internal ribosome entry site (HCV IRES). Rabbit reticulocyte lysate conditions were optimized to faithfully report on authentic HCV IRES-dependent translation relative to a 5′ capped mRNA control. We screened a library of ~430,000 small molecules for IRES inhibition, leading to ~1,700 initial hits. After secondary counter screening the vast majority of hits proved to be luciferase and general translation inhibitors. Despite well-optimized in vitro translation conditions, in the end we found no selective HCV IRES inhibitors but did discover a new scaffold of general translation inhibitor. The analysis of these molecules, and the finding that a large fraction of false positives resulted from off-target effects, highlights the challenges inherent in screens for RNA-specific inhibitors.