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
TAR DNA binding protein 43 (TDP-43) is a nucleic acid binding protein that is associated with the pathology of cystic fibrosis and neurodegenerative diseases such as amyotrophic lateral sclerosis and frontotemporal lobar dementia. We have developed a robust, quantitative, nonradiometric high-throughput assay measuring oligonucleotide binding to TDP-43 using AlphaScreen® technology. Biotinylated single-stranded TAR DNA (bt-TAR-32) and 6 TG repeats (bt-TG6) bound with high affinity to TDP-43, with KD values of 0.75 nM and 0.63 nM, respectively. Both oligonucleotides exhibited slow dissociation rates, with half-lives of 750 min for bt-TAR-32 and 150 min for bt-TG6. The affinities of unlabeled oligonucleotides, as determined by displacement of either bt-TAR-32 or bt-TG6, were consistent with previous reports of nucleic acid interactions with TDP-43, where increasing TG or UG repeats yield greater affinity. A diversity library of 7360 compounds was screened for inhibition of TDP-43 binding to bt-TAR-32, and a series of compounds was discovered with nascent SAR and IC50 values ranging from 100 nM to 10 μM. These compounds may prove to be useful biochemical tools to elucidate the function of TDP-43 and may lead to novel therapeutics for indications where the TDP-43 nucleic acid interaction is causal to the associated pathology.
TDP-43; AlphaScreen; TAR DNA; ALS; cystic fibrosis
Tissue-nonspecific alkaline phosphatase (TNAP) plays a major role in maintaining a ratio of phosphate to inorganic pyrophosphate (Pi/PPi) in biological fluids that is conducive to controlled skeletal mineralization while preventing inappropriate ectopic calcification. Medial calcification associated with Enpp1 or Ank deficiency or with end–stage renal disease is associated with an increase in TNAP activity in arteries that leads to reduced levels of PPi and increased vascular calcification. Here, we describe in detail a high-throughput screening (HTS) campaign to identify inhibitors of TNAP, performed within the Molecular Library Screening Center Network (MLSCN). A homogeneous luminescent TNAP assay was developed and optimized for identification of compounds with diverse mechanism of action (MOA). The MLSCN compound collection, containing 64,394 molecules at the time of screening, was tested in the assay. Several novel inhibitory scaffold classes were identified and demonstrated to have diverse selectivity and mode of inhibition (MOI) profiles. Representatives of the novel scaffolds exhibited nanomolar potency surpassing the inhibitors known to date.
This paper sets a successful example in which pharmacologically active compounds, with outstanding selectivity in a panel of more than 200 assays, are identified from high throughput screening. Integral to the success of the project were a well-designed compound collection, an industrial-level screening facility and a deep knowledge of target biology that were brought together through the NIH-sponsored Roadmap Initiative.
NIH Roadmap Initiatives; MLSCN; TNAP inhibitors; diverse MOA; compound selectivity
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.
thyroid receptor; small molecule; HTS; coactivator; protein-protein interaction
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.
P. aeruginosa; type II secretion; high throughput screening; inhibitors
A number of diabetogenic stimuli interact to influence insulin promoter activity, making it an attractive target for both mechanistic studies and therapeutic interventions. High-throughput screening (HTS) for insulin promoter modulators has the potential to reveal novel inputs into the control of that central element of the pancreatic β-cell. A cell line from human islets in which the expression of insulin and other β-cell-restricted genes are modulated by an inducible form of the bHLH transcription factor E47 was developed. This cell line, T6PNE, was adapted for HTS by transduction with a vector expressing green fluorescent protein under the control of the human insulin promoter. The resulting cell line was screened against a library of known drugs for those that increase insulin promoter activity. Members of the phenothiazine class of neuroleptics increased insulin gene expression upon short-term exposure. Chronic treatment, however, resulted in suppression of insulin promoter activity, consistent with the effect of phenothiazines observed clinically to induce diabetes in chronically treated patients. In addition to providing insights into previously unrecognized targets and mechanisms of action of phenothiazines, the novel cell line described here provides a broadly applicable platform for mining new molecular drug targets and central regulators of β-cell differentiated function.
diabetes; chlorpromazine; ethopropazine
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.
unfolded protein response; endoplasmic reticulum stress; cell-based assay; luciferase reporter; natural products
Protein degradation via the ubiquitin-proteasome pathway is important for a diverse number of cellular processes ranging from cell signaling to development. Disruption of the ubiquitin pathway occurs in a variety of human diseases, including several cancers and neurological disorders. Excessive proteolysis of tumor suppressor proteins, such as p27, occurs in numerous aggressive human tumors. To discover small-molecule inhibitors that potentially prevent p27 degradation, we developed a series of screening assays, including a cell-based screen of a small-molecule compound library and two novel nucleotide exchange assays. Several small-molecule inhibitors, including NSC624206, were identified and subsequently verified to prevent p27 ubiquitination in vitro. The mechanism of NSC624206 inhibition of p27 ubiquitination was further unraveled using the nucleotide exchange assays and shown to be due to antagonizing ubiquitin activating enzyme (E1). We determined that NSC624206 and PYR-41, a recently reported inhibitor of ubiquitin E1, specifically block ubiquitin-thioester formation but have no effect on ubiquitin adenylation. These studies reveal a novel E1 inhibitor that targets a specific step of the E1 activation reaction. NSC624206 could, therefore, be potentially useful for the control of excessive ubiquitin-mediated proteolysis in vivo.
ubiquitin E1; inhibitor; p27kip1; ubiquitin; proteolysis
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.
compound promiscuity; assay ontology; reporter gene assays; high-throughput screening data analysis; cheminformatics
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.
Assay development; Cytokine; JAK3 inhibitors; STAT5 reporter; high-throughput chemical screening
Latent infection with Epstein-Barr Virus (EBV) is a carcinogenic cofactor in several lymphoid and epithelial cell malignancies. At present, there are no small molecule inhibitors that specifically target EBV latent infection or latency-associated oncoproteins. EBNA1 is an EBV-encoded sequence-specific DNA-binding protein that is consistently expressed in EBV-associated tumors and required for stable maintenance of the viral genome in proliferating cells. EBNA1 is also thought to provide cell survival function in latently infected cells. In this work we describe the development of a biochemical high-throughput screening (HTS) method using a homogenous fluorescence polarization (FP) assay monitoring EBNA1 binding to its cognate DNA binding site. An FP-based counterscreen was developed using another EBV-encoded DNA binding protein, Zta, and its cognate DNA binding site. We demonstrate that EBNA1 binding to a fluorescent labeled DNA probe provides a robust assay with a Z-factor consistently greater than 0.6. A pilot screen of a small molecule library of ~14,000 compounds identified 3 structurally related molecules that selectively inhibit EBNA1, but not Zta. All three compounds had activity in a cell-based assay specific for the disruption of EBNA1 transcription repression function. One of the compounds was effective in reducing EBV genome copy number in Raji Burkitt lymphoma cells. These experiments provide a proof-of-concept that small molecule inhibitors of EBNA1 can be identified by biochemical high-throughput screening of compound libraries. Further screening in conjunction with medicinal chemistry optimization may provide a selective inhibitor of EBNA1 and EBV latent infection.
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.
fluorescence polarization; gyrase; assay development; high-throughput screen; anthracycline
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.
protein arginine methyltransferases; PRMT; scintillation proximity assay; SPA; high-throughput screening; HTS
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.
Hepatitis C virus (HCV); IRES; luciferase; high-throughput screen; rabbit reticulocyte lysate
Methylthioadenosine phosphorylase (MTAP), a key enzyme in the methionine salvage pathway, is inactivated in a variety of human cancers. Since all human tissues express MTAP, it would be of potential interest to identify compounds that selectively inhibit the growth of MTAP deficient cells. To determine if MTAP inactivation could be targeted, we have performed a differential chemical genetic screen in isogenic MTAP+ and MTAP− S. cerevisiae. A low molecular weight compound library containing 30,080 unique compounds was screened for those that selectively inhibit growth of MTAP− yeast using a differential growth assay. One compound, containing a 1,3,4-thiadiazine ring, repeatedly showed a differential dose response, with MTAP− cells exhibiting a four-fold shift in IC50 compared to MTAP+ cells. Several structurally related derivatives of this compound also showed enhanced growth inhibition in MTAP− yeast. These compounds were also examined for growth inhibition of isogenic MTAP+ and MTAP− HT1080 fibrosarcoma cells, and four of the five compounds exhibited evidence of modest, but significant, increased potency in MTAP− cells. In summary, these studies show the feasibility of differential growth screening technology and have identified a novel class of compounds that can preferentially inhibit growth of MTAP− cells.
Methionine Salvage Pathway; Drug screening; Yeast; Genetic-chemical interaction
Members of the heat shock protein 70 (Hsp70) family of molecular chaperones are emerging as potential therapeutic targets. Their ATPase activity has classically been measured using colorimetric phosphate-detection reagents, such as quinaldine red (QR). While such assays are suitable for 96-well plate formats, they typically lose sensitivity when attempted in lower volume due to path length and meniscus effects. These limitations and Hsp70’s weak enzymatic activity have combined to create significant challenges in high throughput screening. To overcome these difficulties, we have adopted an energy transfer strategy that was originally reported by Zuck et al. (Anal. Biochem. 2005, 342:254–259). Briefly, white 384-well plates emit fluorescence when irradiated at 430 nm. In turn, this intrinsic fluorescence can be quenched by energy transfer with the QR-based chromophore. Using this more sensitive approach, we tested 55,400 compounds against DnaK, a prokaryotic member of the Hsp70 family. The assay performance was good (Z′ ~ 0.6, CV ~8%) and at least one promising new inhibitor was identified. In secondary assays, this compound specifically blocked stimulation of DnaK by its co-chaperone, DnaJ. Thus, this simple and inexpensive adaptation of a colorimetric method might be suitable for screening against Hsp70-family members.
phosphate; malachite green; ATPase; molecular chaperone; fluorescence assay
Cyclophilin A (CypA) is an overexpressed protein in lung cancer tumors and as a result is a potential therapeutic and diagnostic target. Here we utilize an H/D exchange- and MALDI mass spectrometry-based assay, termed single-point SUPREX (Stability of Unpurified Proteins from Rates of H/D Exchange), to screen two chemical libraries, including the 1280-compound LOPAC library and the 9600 compound DIVERSet library, for binding to CypA. This work represents the first application of single-point SUPREX using a pooled ligand approach, which we demonstrate is capable of screening rates as fast as six seconds/ligand. The false positive and false negative rates determined in the current work using a set of control samples were 0% and 9%, respectively. A false positive rate of 20% was found in screening the actual libraries. Eight novel ligands to CypA were discovered including: 2-(α-naphthoyl)ethyltrimethyl-ammonium iodide, (E)-3-(4-t-Butylphenylsulfonyl)-2-propenenitrile, 3-(N-benzyl-N-isopropyl)amino-1-(naphthalen-2-yl)propan-1-one, cis-diammineplatinum (II) chloride, 1-(3,5-dichlorophenyl)-1H-pyrrole-2,5-dione, N-(3-chloro-1,4-dioxo-1,4-dihydro-2-naphthalenyl)-N-cyclohexylacetamide, 1-[2-(3,4-dimethoxyphenyl)ethyl]-1H-pyrrole-2,5-dione, and 4-(2-methoxy-4-nitrophenyl)-1-methyl-10-oxa-4-azatricyclo[188.8.131.52~2,6~]dec-8-ene-3,5-dione. These compounds, which had moderate binding affinities to CypA (i.e., Kd values in the low micromolar range), provide new molecular scaffolds that might be useful in the development of CypA targeted diagnostic imaging or therapeutic agents for lung cancer.
Cyclophilin A; Matrix-Assisted Laser Desorption/Ionization; amide H/D exchange; high-throughput screening
α-Cobratoxin (Cbtx), the neurotoxin isolated from the venom of the Thai cobra Naja kaouthia, causes paralysis by preventing acetylcholine (ACh) binding to nicotinic acetylcholine receptors (nAChRs). In the current study, the region of the Cbtx molecule that is directly involved in binding to nAChRs is used as the target for anticobratoxin drug design. The crystal structure (1YI5) of Cbtx in complex with the acetylcholine binding protein (AChBP), a soluble homolog of the extracellular binding domain of nAChRs, was selected to prepare an α-cobratoxin active binding site for docking. The amino acid residues (Ser182-Tyr192) of the AChBP structure, the binding site of Cbtx, were used as the positive control to validate the prepared Cbtx active binding site (root mean square deviation < 1.2 Å). Virtual screening of the National Cancer Institute diversity set, a library of 1990 compounds with nonredundant pharmacophore profiles, using AutoDock against the Cbtx active site, revealed 39 potential inhibitor candidates. The adapted in vitro radioligand competition assays using [3H]epibatidine and [125I]bungarotoxin against the AChBPs from the marine species, Aplysia californica (Ac), and from the freshwater snails, Lymnaea stagnalis (Ls) and Bolinus truncates (Bt), revealed 4 compounds from the list of inhibitor candidates that had micromolar to nanomolar interferences for the toxin binding to AChBPs. Three hits (NSC42258, NSC121865, and NSC134754) can prolong the survival time of the mice if administered 30 min before injection with Cbtx, but only NSC121865 and NSC134754 can prolong the survival time if injected immediately after injection with Cbtx. These inhibitors serve as novel templates/scaffolds for the development of more potent and specific anticobratoxin.
α-cobratoxin; virtual screening; docking; neurotoxin; nicotinic acetylcholine receptor
The tyrosine kinase Wee1 is part of a key cellular sensing mechanism that signals completion of DNA replication, ensuring proper timing of entry into mitosis. Wee1 acts as an inhibitor of mitotic entry by phosphorylating cyclin-dependent kinase CDK1. Wee1 activity is mainly regulated at the protein level through its phosphorylation and subsequent degradation by the ubiquitin proteasome pathway. To facilitate identification of small molecules preventing Wee1 degradation, a homogeneous cell-based assay was developed using HeLa cells transiently transfected with a Wee1-Luciferase fusion protein. To insure uHTS compatibility, the assay was scaled to 1,536-well plate format and cells were transfected in bulk and cryopreserved. This miniaturized homogenous assay demonstrated robust performance, with a calculated Z′ factor of 0.65±0.05. The assay was screened against a publicly available library of ~218,000 compounds in order to identify Wee1 stabilizers. Nonselective, cytotoxic and promiscuous compounds were rapidly triaged through the use of a similarly formatted counterscreen that measured stabilization of a N-cyclin B-Luciferase fusion protein, as well as execution of viability assessment in the parental HeLa cell line. This screening campaign led to the discovery of four unrelated cell-permeable small molecules that showed selective Wee1-Luciferase stabilization with micromolar potency. One of these compounds, SID4243143, was shown to inhibit cell cycle progression, underscoring the importance of Wee1 degradation to the cell cycle. Our results suggest that this uHTS approach is suitable for identifying selective chemical probes that prevent Wee1 degradation, and generally applicable to discovering inhibitors of the ubiquitin proteasome pathway.
Wee1; degradation; stabilizer; reporter assay; transient transfection; cryopreserved cells; ubiquitin; proteasome
RNA interference-based screening is a powerful new genomic technology which addresses gene function en masse. To evaluate factors influencing hit list composition and reproducibility, we performed two identically designed small interfering RNA (siRNA)-based, whole genome screens for host factors supporting yellow fever virus infection. These screens represent two separate experiments completed five months apart and allow the direct assessment of the reproducibility of a given siRNA technology when performed in the same environment. Candidate hit lists generated by sum rank, median absolute deviation, z-score, and strictly standardized mean difference were compared within and between whole genome screens. Application of these analysis methodologies within a single screening dataset using a fixed threshold equivalent to a p-value ≤ 0.001 resulted in hit lists ranging from 82 to 1,140 members and highlighted the tremendous impact analysis methodology has on hit list composition. Intra- and inter-screen reproducibility was significantly influenced by the analysis methodology and ranged from 32% to 99%. This study also highlighted the power of testing at least two independent siRNAs for each gene product in primary screens. To facilitate validation we conclude by suggesting methods to reduce false discovery at the primary screening stage.
In this study we present the first comprehensive comparison of multiple analysis strategies, and demonstrate the impact of the analysis methodology on the composition of the “hit list”. Therefore, we propose that the entire dataset derived from functional genome-scale screens, especially if publicly funded, should be made available as is done with data derived from gene expression and genome-wide association studies.
RNA interference; analysis; RNAi screen analysis; siRNA; RNAi; siRNA screening; sum rank; median absolute deviation; strictly standardized mean difference; genome-wide; whole-genome; comparison; overlap; hit list
Drug treatment for human lung cancers remains unsatisfactory, despite the identification of many potential therapeutic targets (such as mutant KRAS protein) and the approval of agents that inhibit the tyrosine kinase activity of mutant epidermal growth factor receptor (EGFR). To seek new therapeutic strategies against lung tumors, we have screened 189, 290 small molecules for their ability to retard growth of human lung adenocarcinoma cell lines, which harbor mutations in EGFR or KRAS. Four candidates that are structurally different from common tyrosine kinase inhibitors were selected for further study. We describe one small molecule (designated lung cancer screen-1, LCS-1) in detail here. Identification of the targets of LCS-1 and other growth inhibitors found in this screen may help to develop new agents for treatment of lung adenocarcinomas, including those driven by mutant EGFR and KRAS.
high throughput drug screen; lung cancer; EGFR; KRAS
Fas-Associated protein with Death Domain (FADD) was originally reported as a pro-apoptotic adaptor molecule that mediates receptor induced apoptosis. Recent studies have revealed a potential role of FADD in NF-κB activation, embryogenesis, and cell cycle regulation and proliferation. Over-expression of FADD and its phosphorylation have been associated with the transformed phenotype in many cancers and is therefore a potential target for therapeutic intervention. In an effort to delineate signaling events that lead to FADD phosphorylation and to identify novel compounds that impinge on this pathway, we developed a cell based reporter for FADD kinase activity. The reporter assay, optimized for a high throughput screen (HTS), measures bioluminescence in response to modulation of FADD kinase activity in live cells. In addition, the potential use of the reporter cell line in the rapid evaluation of pharmacologic properties of HTS hits in mouse models has been demonstrated.
FADD; phosphorylation; non-invasive molecular imaging; bioluminescence; kinase activity
The leukocyte-specific integrin CD11b/CD18 plays a key role in the biological function of these cells and represents a validated therapeutic target for inflammatory diseases. Currently, the low affinity interaction between CD11b/CD18 integrin and its respective ligand poses a challenge in the development of cell-based adhesion assays for the high-throughput screening (HTS) environment. Here the authors describe a simple cell-based adhesion assay that can be readily used for HTS for the discovery of functional regulators of CD11b/CD18. The assay consistently produces acceptable Z′ values (> 0.5) for HTS. After testing the assay using 2 established blocking antibodies as reference biologicals, the authors performed a proof-of-concept primary screen using a library of 6612 compounds and identified both agonist and antagonist hits.
integrin; no-wash assay; cell adhesion assay; primary screen
Studies of the phosphodiesterase PDE7 family are impeded by there being only one commercially-available PDE7 inhibitor, BRL50481. We have employed a high throughput screen of commercial chemical libraries, using a fission yeast-based assay, to identify PDE7 inhibitors that include steroids, podocarpanes, and an unusual heterocyclic compound, BC30. In vitro enzyme assays measuring the potency of BC30 and two podocarpanes, in comparison with BRL50481, produce data consistent with those from yeast-based assays. In other enzyme assays, BC30 stimulates the PDE4D catalytic domain, but not full-length PDE4D2, suggesting an allosteric site of action. BC30 significantly enhances the anti-inflammatory effect of the PDE4 inhibitor rolipram as measured by release of TNFα from activated monocytes. These studies introduce several new PDE7 inhibitors that may be excellent candidates for medicinal chemistry due to the requirements for drug-like characteristics placed on them by the nature of the yeast-based screen.
Schizosaccharomyces pombe; cAMP; phosphodiesterase; high throughput; inhibitors; PDE7
Shigella flexneri is a human enteropathogen that infects ca. 165 million people and claims more than 1 million lives per year worldwide. Although shigellosis has been considered a disease of the “Third World,” like many other contagious diseases, it does occur in developed countries. The emergence of drug and multi-drug-resistant strains of Shigella emphasize the need for novel antibiotic development. VirF, an AraC-type transcriptional regulator, is responsible for the expression of all downstream virulence factors that control intracellular invasion and cell-to-cell spread of Shigella. Gene knockout studies have validated that inhibition of VirF expression is sufficient to block the normal life cycle of Shigella in the host and thereby increase susceptibility to the host immune system. The authors have developed a high-throughput, cell-based assay to monitor inhibition of VirF using β-galactosidase as a reporter protein. Using an avirulent strain of Shigella, they have screened libraries containing ~42,000 small molecules. Following confirmation and dose-response analysis, they have identified 25 compounds that demonstrate VirF inhibition in vivo ≥55% in comparison to the controls and little general antibacterial activity (measured by cell growth, OD600). The authors are in the process of confirming these “hits” in several secondary assays to assess the mechanism of action.
VirF; Shigella flexneri; AraC family; HTS; transcriptional activators