Genome-wide RNAi screening is a powerful, yet relatively immature technology that allows investigation into the role of individual genes in a process of choice. Most RNAi screens identify a large number of genes with a continuous gradient in the assessed phenotype. Screeners must then decide whether to examine just those genes with the most robust phenotype or to examine the full gradient of genes that cause an effect and how to identify the candidate genes to be validated. We have used RNAi in Drosophila cells to examine viability in a 384-well plate format and compare two screens, untreated control and treatment. We compare multiple normalization methods, which take advantage of different features within the data, including quantile normalization, background subtraction, scaling, cellHTS2 1, and interquartile range measurement. Considering the false-positive potential that arises from RNAi technology, a robust validation method was designed for the purpose of gene selection for future investigations. In a retrospective analysis, we describe the use of validation data to evaluate each normalization method. While no normalization method worked ideally, we found that a combination of two methods, background subtraction followed by quantile normalization and cellHTS2, at different thresholds, captures the most dependable and diverse candidate genes. Thresholds are suggested depending on whether a few candidate genes are desired or a more extensive systems level analysis is sought. In summary, our normalization approaches and experimental design to perform validation experiments are likely to apply to those high-throughput screening systems attempting to identify genes for systems level analysis.
RNAi; high-throughput screen; normalization; validation
Cyclic nucleotide phosphodiesterases (PDEs) comprise a superfamily of enzymes that serve as drug targets in many human diseases. There is a continuing need to identify high-specificity inhibitors that affect individual PDE families or even subtypes within a single family. We describe a fission yeast-based high throughput screen to detect inhibitors of heterologously-expressed cAMP PDEs. The utility of this system is demonstrated by the construction and characterization of strains that express mammalian PDE2A, PDE4A, PDE4B, and PDE8A and respond appropriately to known PDE2A and PDE4 inhibitors. High throughput screens of two bioactive compound libraries for PDE inhibitors using strains expressing PDE2A, PDE4A, PDE4B, and the yeast PDE Cgs2 identified known PDE inhibitors and members of compound classes associated with PDE inhibition. We verified that the furanocoumarin imperatorin is a PDE4 inhibitor based on its ability to produce a PDE4-specific elevation of cAMP levels. This platform can be used to identify PDE activators, as well as genes encoding PDE regulators, which could serve as targets for future drug screens.
Schizosaccharomyces pombe; cAMP; phosphodiesterase; high throughput; inhibitors
Development of drugs targeting lipid kinases has been delayed by the lack of robust screening assays. Methods are needed that can accommodate the presentation of different acceptor substrates in the optimal lipid environment. The Trancreener™ ADP Assay relies on homogenous immunodetection of ADP, using either fluorescence polarization (FP) or time-resolved fluorescence resonance energy transfer (TR-FRET) as a signal output. Detection of ADP - the invariant product of all kinase reactions - provides complete flexibility for varying lipid substrate parameters. We used this assay to optimize dispersal methods for C8 and C16 phosphatidylinositol 4,5 bisphosphate substrates and to assess the effects of chain length on the activity and inhibition of phosphoinositide-3-kinase (PI3K) isoforms. The non-physiological C8 substrate supported the highest activity. Known inhibitors were profiled using both the FP and TR-FRET based assays and there was excellent concordance (r2 = 0.93) in the IC50 values. The overall rank order of inhibitors was the same using the C8 and C16 substrates, except for minor deviations. ATP hydrolysis in the absence of substrate was detected with the PI3Kα isoform, and inhibitors affected PI3Kα intrinsic ATP hydrolysis activity similarly to lipid phosphorylation.
ADP detection; phosphoinositide 3-kinase; fluorescence polarization; time-resolved fluorescence resonance energy transfer; intrinsic ATPase activity
Haspin/Gsg2 is a kinase that phosphorylates Histone H3 at Thr-3 (H3T3ph) during mitosis. Its depletion by RNA interference results in failure of chromosome alignment and a block in mitosis. Haspin therefore is a novel target for development of anti-mitotic agents. We report the development of a high throughput time-resolved fluorescence resonance energy transfer (TR-FRET) kinase assay for Haspin. Histone H3 peptide was used as a substrate, and a Europium-labeled H3T3ph phosphospecific monoclonal antibody was used to detect phosphorylation. A library of 137632 small molecules was screened at Km concentrations of ATP and peptide to allow identification of diverse inhibitor types. Reconfirmation of hits and IC50 determinations were carried out with the TR-FRET assay and by a radiometric assay using recombinant Histone H3 as the substrate. A preliminary assessment of specificity was made by testing inhibition of two unrelated kinases. EC50 values in cells were determined using a cell-based ELISA assay of H3T3ph. Five compounds were selected as leads based on potency and chemical structure considerations. These leads form the basis for the development of specific inhibitors of Haspin that will have clear utility in basic research and possible use as starting points for development of anti-mitotic anticancer therapeutics.
anti-mitotics; chromatin; Haspin; kinase; time-resolved resonance energy transfer
Using a highly reproducible and robust cell-based HTS assay, the authors screened a 100,000 compound library at 14 and 114 μM compound concentration against influenza strain A/Udorn/72 (H3N2). The “hit” rates (>50% inhibition of the viral cytopathic effect) from the 14 and 114 μM screens were 0.022% and 0.38%, respectively. The hits were evaluated for their antiviral activity, cell toxicity and selectivity in dose response experiments. The screen at the lower concentration yielded three compounds, which displayed moderate activity (SI50 = 10-49). Intriguingly, the screen at the higher concentration revealed several additional hits. Two of these hits were highly active with an SI50 > 50. Time of addition experiments revealed one compound that inhibited early and four other compounds that inhibited late in the virus life cycle; suggesting they affect entry and replication, respectively. The active compounds represent several different classes of molecules such as carboxanilides, 1-benzoyl-3-arylthioureas, sulfonamides and benzothiazinones, which have not been previously identified as having anti-viral/anti-influenza activity.
influenza; HTS; high-throughput screening; antivirals
As natural peroxisome proliferator-activated receptor-α (PPARα) ligands, high levels of fatty acids and glucose could lead to hyperactivation of PPARα, like that seen in diabetes. Important diabetes research goals are to uncover new metabolic or signaling pathways involved in hyperglycemic cellular injury and to develop therapeutics for preventing or reversing this injury. Consequently, 1040 putative antidiabetic agents were screened for their ability to 1) affect PPARα lipid binding, 2) directly bind PPARα, and 3) alter PPARα transactivation in the presence of high glucose. A high-throughput fluorescent binding assay was developed to examine each compound’s ability to restore fatty acyl-CoA binding to PPARα in the presence of high glucose concentrations. Approximately 1% of the compounds restored acyl-CoA binding by 60% or more. These compounds directly interacted with PPAR α with high affinity (nM Kds), validating the primary screen. Furthermore, these compounds altered PPARα transactivation, and 1 strongly reversed the hyperactivation of PPARα found in the presence of clofibrate and high glucose levels.
nuclear transcription factors; peroxisome proliferator-activated receptors; fluorescent binding assays; glucose
Calpain activation is hypothesized to be an early occurrence in the sequence of events resulting in neurodegeneration, as well as in the signaling pathways linking extracellular accumulation of Aβ peptides and intracellular formation of neurofibrillary tangles. In an effort to identify small molecules that prevent neurodegeneration in Alzheimer’s disease by early intervention in the cell death cascade, a cell-based assay in differentiated Sh-SY5Y cells was developed utilizing calpain activity as a read-out for the early stages of death in cells exposed to extracellular Aβ. This assay was optimized for high-throughput screening, and a library of approximately 120,000 compounds tested. It was expected that the compounds identified as calpain inhibitors would include those that act directly on the enzyme and those that prevented calpain activation by blocking an upstream step in the pathway. In fact, of the compounds that inhibited calpain activation by Aβ with IC50 values < 10 μM and showed little or no toxicity at concentrations up to 30 μM, none inhibit the calpain enzyme directly. Studies to identify the targets of these compounds in the cell death pathway are ongoing.
Astrocytoma/glioblastoma is the most common malignant form of brain cancer and is often unresponsive to current pharmacological therapies and surgical interventions. Despite several potential therapeutic agents against astrocytoma and glioblastoma (1), there are currently no effective therapies for astrocytoma, creating a great need for the identification of effective anti-tumor agents. We have developed a novel dual-reporter system in Trp53/Nf1-null astrocytoma cells to simultaneously and rapidly assay cell viability and cell cycle progression as evidenced by activity of the human E2F1 promoter in vitro. The dual-reporter high-throughput assay was used to screen experimental therapeutics for activity in Trp53/Nf1-null astrocytoma. Several compounds were identified demonstrating selectivity for astrocytoma over primary astrocytes. The dual-reporter system described here may be a valuable tool for identifying potential anti-tumor treatments that specifically target astrocytoma.
astrocytoma; Nf1; p53; E2F1; luciferase
Paramyxoviruses are negative strand non-segmented RNA viruses. Several members of this family constitute major human pathogens that, collectively, are responsible for major morbidity and mortality worldwide. In an effort to ultimately develop novel therapeutics against measles virus (MV), a prominent member of the paramyxovirus family, we report a high-throughput screening protocol that allows hit identification using non-recombinant primary MV strains as targets. Implementation of the assay has yielded 60 hit candidates from a 137,500-entry library. Counterscreening and generation of dose-response curves narrows this pool to 35 compounds with active concentrations ≤15.3 μM against the MV-Alaska strain and specificity indices ranging from 36 to >500. Library mining for structural analogs of several confirmed hits combined with re-testing of identified candidates reveals a low false-negative rate and, thus, a high accuracy of primary hit identification. Eleven of the confirmed hits were found to interfere with the viral entry machinery, while the remaining 24 compounds target post-entry steps of the viral life cycle. Activity testing against selected members of the paramyxovirus family reveals three patterns of activity: 1) exclusively MV-specific blockers; 2) inhibitors of MV and related viruses of the same genus; 3) broader-range inhibitors with activity against a different paramyxovirinae genus. Representatives of the last class may open avenues for the development of broad-range paramyxovirus inhibitors through hit-to-lead chemistry.
high-throughput screening; drug discovery; paramyxovirus; anti-infective drugs
Inherited deficiency of galactose-1-phosphate uridyltransferase (GALT) can result in a potentially lethal disorder called classic galactosemia. Although the neonatal lethality associated with this disease can be prevented through early diagnosis and a galactose-restricted diet, the lack of effective therapy continues to have consequences: developmental delay, neurological disorders, and premature ovarian failure are common sequelae in childhood and adulthood. Several lines of evidence indicate that an elevated level of galactose-1-phosphate (gal-1-p), the product of galactokinase (GALK), is a major, if not sole, pathogenic mechanism in patients with classic galactosemia. The authors hypothesize that elimination of gal-1-p production by inhibiting GALK will relieve GALT-deficient cells from galactose toxicity. To test this hypothesis, they obtained human GALK using a bacterial expression system. They developed a robust, miniaturized, high-throughput GALK assay (Z′ factor =0.91) and used this assay to screen against libraries composed of 50,000 chemical compounds with diverse structural scaffolds. They selected 150 compounds that, at an average concentration of 33.3 μM, inhibited GALK activity in vitro more than 86.5% and with a reproducibility score of at least 0.7 for a confirmatory screen under identical experimental conditions. Of these 150 compounds, 34 were chosen for further characterization. Preliminary results indicated that these 34 compounds have potential to serve as leads to the development of more effective therapy of classic galactosemia.
classic galactosemia; galactose-1-phosphate uridyltransferase (GALT); galactokinase (GALK); galactose-1-phosphate; GHMP small-molecule kinase; high-throughput screening; small-molecule inhibitors
The cyclic nucleotide phosphodiesterases (PDEs) are intracellular enzymes that catalyze the hydrolysis of 3', 5'-cyclic nucleotides, such as cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP), to their corresponding 5'-nucleotide monophosphates. These enzymes play an important role in controlling cellular concentrations of cyclic nucleotides and thus regulate a variety of cellular signaling events. PDEs are emerging as drug targets for several diseases including asthma, cardiovascular disease, ADHD, Parkinson’s disease, and Alzheimer’s disease. Though biochemical assays with purified recombinant PDE enzymes and cAMP or cGMP substrate are commonly used for compound screening, cell-based assays would provide a better assessment of compound activity in a more physiological context. Here we report the development and validation of a new cell-based PDE4 assay using a constitutively active GPCR as a driving force for cAMP production and a cyclic nucleotide gated (CNG) cation channel as a biosensor in 1536-well plates.
phosphodiesterase; PDE IV; cyclic nucleotide gated ion channels; cell-based assay; 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
Although proteases represent an estimated 5% to 10% of potential drug targets, inhibitors for metalloproteases (MPs) account for only a small proportion of all approved drugs, failures of which have typically been associated with lack of selectivity. In this study, the authors describe a novel and universal binding assay based on an actinonin derivative and show its binding activities for several MPs and its lack of activity toward all the non-MPs tested. This newly developed assay would allow for the rapid screening for inhibitors of a given MP and for the selectivity profiling of the resulting hits. The assay has successfully enabled for the first time simultaneous profiling of 8 well-known inhibitors against a panel of selected MPs. Previously published activities for these inhibitors were confirmed, and the authors have also discovered new molecular targets for some of them. The authors conclude that their profiling platform provides a generic assay solution for the identification of novel metalloprotease inhibitors as well as their selectivity profiling using a simple and homogeneous assay.
metalloprotease; inhibitor; profiling; fluorescence polarization; HTS