TDP-43 is an RNA binding protein found to accumulate in the cytoplasm of brain and spinal cord from patients affected with amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). Nuclear TDP-43 protein regulates transcription through several mechanisms, and under stressed conditions it forms cytoplasmic aggregates that co-localize with stress granule (SG) proteins in cell culture. These granules are also found in the brain and spinal cord of patients affected with ALS and FTLD. The mechanism through which TDP-43 might contribute to neurodegenerative diseases is poorly understood. In order to investigate the pathophysiology of TDP-43 aggregation and to isolate potential therapeutic targets, we screened a chemical library of 75,000 compounds using high content analysis with PC12 cells that inducibly express human TDP-43 tagged with GFP. The screen identified 16 compounds that dose-dependently decreased the TDP-43 inclusions without significant cellular toxicity or changes in total TDP-43 expression levels. To validate the effect of the compounds, we tested compounds by Western Blot analysis and in a model that replicates some of the relevant disease phenotypes. The hits from this assay will be useful for elucidating regulation of TDP-43, stress granule response, and possible ALS therapeutics.
Amyotrophic lateral sclerosis; RNA granule; RNA binding protein; aggregation; high throughput screen; protein synthesis
Alternative splicing (AS) is an efficient mechanism that involves the generation of transcriptome and protein diversity from a single gene. Defects in pre-mRNA splicing are an important cause of numerous diseases, including cancer. AS of pre-mRNA as a target for cancer therapy has not been well studied. We have reported previously that a splicing factor, polypyrimidine tract-binding protein (PTB) is overexpressed in ovarian tumors, compared to matched normal controls, and knockdown (KD) of PTB expression by shRNA impairs ovarian tumor cell growth, colony formation and invasiveness. Given the complexity of PTB’s molecular functions, a chemical method for controlling PTB activity might provide a therapeutic and experimental tool. However, no commercially available PTB inhibitors have yet been described. To expand our ability to find novel inhibitors, we developed a robust, fluorometric, cell-based high throughput screening HTS assay in 96-well plates that reports on the splicing activity of PTB. In an attempt to use the cells for large-scale chemical screens to identify PTB modulators, we established cell lines stably expressing the reporter gene. Our results suggest that this high throughput assay could be used to identify small molecule modulators of PTB activity. Based on these findings and the role that upregulated PTB has on cell proliferation and malignant properties of tumors targeting PTB for inhibition with small molecules offers a promising strategy for cancer therapy.
PTB; ovarian cancer; fluorescence methods; cell-based HTS; alternative splicing
Wnt/β-catenin signaling has emerged as a central player in pathways implicated in the pathophysiology and treatment of neuropsychiatric disorders. To identify potential novel therapeutics for these disorders, high-throughput screening (HTS) assays reporting on Wnt/β-catenin signaling in disease relevant contexts are needed. The use of human patient-derived induced pluripotent stem cell (iPSC) models provides ideal disease relevant context if these stem cell cultures can be adapted for HTS-compatible formats. Here, we describe a sensitive, HTS-compatible Wnt/β-catenin signaling reporter system generated in homogeneous, expandable neural progenitor cells (NPCs) derived from human iPSCs. We validated this system by demonstrating dose responsive stimulation by several known Wnt/β-catenin signaling pathway modulators, including Wnt3a, a glycogen synthase kinase-3 (GSK3) inhibitor, and the bipolar disorder therapeutic lithium. These responses were robust and reproducible over time across many repeated assays. We then conducted a screen of ~1,500 compounds from a library of FDA-approved drugs and known bioactives, and confirmed HTS hits, revealing multiple chemical and biological classes of novel small molecule probes of Wnt/β-catenin signaling. Generating this type of pathway-selective, cell-based phenotypic assays in human iPSC-derived neural cells will advance the field of human experimental neurobiology toward the goal of identifying and validating targets for neuropsychiatric disorder therapeutics.
induced pluripotent stem cell (iPSC); neural progenitor cell (NPC); Wnt/β-catenin signaling; neuropsychiatric disorders; human neurons
von Hippel Lindau (VHL) disease is an autosomal dominant inherited disorder that results in multiple organ systems being affected. Treatment is mainly surgical, however, effective systemic therapies are needed. We developed and tested a cell-based screening tool to identify compounds that stabilize or upregulate full-length, point mutated VHL.
The 786-0 cell line was infected with full-length W117A mutated VHL linked to a C-terminal Venus fluorescent protein. This VHL-W117A-Venus line was used to screen the Prestwick drug library and was tested against the known proteasome inhibitors MG132 and bortezomib. Western blot validation and evaluation of downstream functional readouts, including HIF and GLUT1 levels, were performed.
Bortezomib, MG132, and the Prestwick compounds 8-azaguanine, thiostrepton and thioguanosine were found to reliably upregulate VHL-W117A-Venus in 786-0 cells. 8-azaguanine was found to downregulate HIF2α levels, and was augmented by the presence of VHL W117A. VHL p30 band intensities varied as a function of compound used, suggesting alternate post-translational processing. In addition, nuclear-cytoplasmic localization of pVHL varied amongst the different compounds.
786-0 cells containing VHL-W117A-Venus can be successfully used to identify compounds that upregulate VHL levels, and that have a differential effect on pVHL intracellular localization and posttranslational processing. Further screening efforts will broaden the number of pharmacophores available to develop therapeutic agents that will upregulate and refunctionalize mutated VHL.
VHL upregulation; proteostasis; high-throughput screen; Prestwick
Eya proteins are essential co-activators of the Six family of homeobox transcription factors and also contain a unique protein tyrosine phosphatase activity, belonging to the haloacid dehalogenase family of phosphatases. The phosphatase activity of Eya is important for a subset of Six1-mediated transcription, making this a unique type of transcriptional control. It is also responsible for directing cells to the repair instead of apoptosis pathway upon DNA damage. Furthermore, the phosphatase activity of Eya is critical for transformation, migration, invasion, and metastasis of breast cancer cells. Thus, inhibitors of the Eya phosphatase activity may be anti-tumorigenic and anti-metastatic, as well as sensitize cancer cells to DNA damage inducing therapies. In this paper, we identified a previously unknown chemical series using high throughput screening that inhibits the Eya2 phosphatase activity with IC50s ranging from 1.8 to 79 μM. Compound activity was confirmed using an alternative malachite green assay and H2AX, a known Eya substrate. Importantly, these Eya2 phosphatase inhibitors show specificity and do not significantly inhibit several other cellular phosphatases. Our studies identify the first selective Eya2 phosphatase inhibitors that can potentially be developed into chemical probes for functional studies of Eya phosphatase or into anti-cancer drugs in the future.
Phosphatase; Eyes Absent 2; Eya2; Eya2 inhibitor; Six1
Chemotherapeutics tumor resistance is a principal reason for treatment failure and clinical and experimental data indicate that multidrug transporters such as ATP-binding Cassette (ABC) B1 and ABCG2 play a leading role by preventing cytotoxic intracellular drug concentrations. Functional efflux inhibition of existing chemotherapeutics by these pumps continues to present a promising approach for treatment. A contributing factor to the failure of existing inhibitors in clinical applications is limited understanding of specific substrate/inhibitor/pump interactions. We have identified selective efflux inhibitors by profiling multiple ABC transporters against a library of small molecules to find molecular probes to further explore such interactions. In our primary screening protocol using JC-1 as a dual-pump fluorescent reporter substrate we identified a piperazine substituted pyrazolo[1,5-a]pyrimidine substructure with promise for selective efflux inhibition. As a result of a focused SAR-driven chemistry effort we describe compound 1 (CID44640177), an efflux inhibitor with selectivity toward ABCG2 over ABCB1. Compound 1 is also shown to potentiate the activity of mitoxantrone in vitro as well as preliminarily in vivo in an ABCG2 over-expressing tumor model. At least two analogs significantly reduce tumor size in combination with the chemotherapeutic topotecan. To our knowledge, low nanomolar chemoreversal activity coupled with direct evidence of efflux inhibition for ABCG2 is unprecedented.
Multi-drug resistance; ABC Transporter; ABCG2; ABCB1; Efflux inhibition
Using fluorescence resonance energy transfer (FRET), we performed a high-throughput screen (HTS) in a reconstituted membrane system, seeking compounds that reverse inhibition of sarco-/endoplasmic reticulum Ca-ATPase (SERCA) by its endogenous regulator, phospholamban (PLB). Such compounds have long been sought to correct aberrant Ca2+ regulation in heart failure. Donor-SERCA was reconstituted in phospholipid membranes with or without acceptor-PLB, and FRET was measured in a steady-state fluorescence microplate reader. A 20,000-compound library was tested in duplicate. Compounds that decreased FRET by more than three standard deviations were considered hits. From 43 primary hits (0.2%), 31 (72%) were found to be false positives upon more thorough testing. The remaining 12 hits were tested in assays of Ca-ATPase activity, and six of these activated SERCA significantly, by as much as 60%, and several also enhanced cardiomyocyte contractility. These compounds directly activated SERCA from heart and other tissues. These results validate our FRET approach and set the stage for medicinal chemistry and pre-clinical testing. We were concerned about the high rate of false positives, resulting from the low precision of steady-state fluorescence. Preliminary studies with a novel fluorescence lifetime plate reader show 20-fold higher precision. This instrument can dramatically increase the quality of future HT.
calcium pump; calcium transport; phospholamban; reconstituted membrane; fluorescence lifetime
Spinal muscular atrophy (SMA) is a neurodegenerative disorder that is characterized by progressive loss of motor neuron function. It is caused by the homozygous loss of the SMN1 (survival of motor neuron 1) gene and a decrease in full-length SMN protein. SMN2 is a nearly identical homolog of SMN1 that, due to alternative splicing, expresses predominantly truncated SMN protein. SMN2 represents an enticing therapeutic target. Increasing expression of full-length SMN from the SMN2 gene might represent a treatment for SMA. We describe a newly designed cell-based reporter assay that faithfully and reproducibly measures full-length SMN expression from the SMN2 gene. This reporter can detect increases of SMN protein by an array of compounds previously shown to regulate SMN2 expression and by the overexpression of proteins that modulate SMN2 splicing. It also can be used to evaluate changes at both the transcriptional and splicing level. This assay can be a valuable tool for the identification of novel compounds that increase SMN2 protein levels and the optimization of compounds already known to modulate SMN2 expression. We present here preliminary data from a high-throughput screen using this assay to identify novel compounds that increase expression of SMN2.
spinal muscular atrophy; survival of motor neuron; SMN1; SMN2; cell-based assay; high-content screening; HTS
The generation of an action potential is a complex process in excitable cells which involves the temporal opening and closing of several voltage-dependent ion channels in the cell membrane. The shape of an action potential can carry information concerning the state of the involved ion channels and their relationship to cellular processes. Alteration of these ion channels by the administration of toxins, drugs, and biochemicals can change the action potential’s shape in a specific way which can be characteristic for a given compound. Thus, action potential shape analysis could be a valuable tool to classify toxins and measure drug effects based on their mechanism of action. In an effort to begin classifying the effect of toxins on the shape of intracellularly recorded action potentials, patch clamp experiments were performed on NG108-15 hybrid cells in the presence of Veratridine, TEA, and quinine. To analyze the effect a computer model of the action potential mechanism was generated to determine to what extent each ion channel was affected during administration of these compounds based on the changes in the model parameters. Our work is a first step towards establishing a new assay system to detect and identify toxins by action potential shape analysis.
high-throughput; toxicity; cell models; electrophysiology; mechanisms
Removal of apoptotic cells and cellular debris by phagocytosis is essential for development, tissue homeostasis and resolution of inflammation. Eat-me signals control the initiation of phagocytosis, holding a key to our understanding of phagocyte biology. Due to lack of functional cloning strategy, eat-me signals are conventionally identified and characterized on a case-by-case basis. To investigate the feasibility of functionally cloning eat-me signals by phage display, we characterize the biological behavior of T7 phages displaying two well-known eat-me signals: growth arrest-specific gene 6 (Gas6) and milk fat globule-EGF8 (MFG-E8). Gas6-phage binds to all three known Gas6 receptors, Mer, Axl and Tyro3 receptor tyrosine kinases. Gas6-phage and MFG-E8-phage are capable of binding to phagocytes and non-phagocytes. However, both phages stimulate phage uptake only in phagocytes, including macrophages, microglia and retinal pigment epithelium (RPE) cells, but not in non-phagocytes. Furthermore, functional phage selection by phagocytosis in phagocytes enriches both Gas6-phage and MFG-E8-phage, suggesting that phage display can be used as a tool to functionally identify unknown eat-me signals from phage display cDNA library.
Eat-me signals; phagocytosis; phage display; Gas6; MFG-E8
Rift Valley fever virus (RVFV) is an emerging infectious pathogen that causes severe disease in humans and livestock and has the potential for global spread. Currently, there is no proven effective treatment for RVFV infection and there is no licensed vaccine. Inhibition of RNA binding to the essential viral nucleocapsid (N) protein represents a potential anti-viral therapeutic strategy because all of the functions performed by N during infection involve RNA binding. To target this interaction, we developed a fluorescence polarization-based high-throughput drug screening assay and tested 26,424 chemical compounds for their ability to disrupt an N-RNA complex. From libraries of FDA approved drugs, drug-like molecules and natural products extracts we identified several lead compounds that are promising candidates for medicinal chemistry.
nucleocapsid; RNA; Rift Valley fever virus; high-throughput screen; fluorescence polarization
MicroRNAs (miRNAs) are endogenous, single-stranded, noncoding RNAs of 21 to 23 nucleotides that regulate gene expression, typically by binding the 3′ untranslated regions of target messenger RNAs. It is estimated that miRNAs are involved in the regulation of 30% of all genes and almost every genetic pathway. Recently, the misregulation of miRNAs has been linked to various human diseases including cancer and viral infections, identifying miRNAs as potential targets for drug discovery. Thus, small-molecule modifiers of miRNAs could serve as lead structures for the development of new therapeutic agents and be useful tools in the elucidation of detailed mechanisms of miRNA function. As a result, we have developed a high-throughput screen for potential small-molecule regulators of the liver-specific microRNA miR-122, which is involved in hepatocellular carcinoma development and hepatitis C virus infection. Our small-molecule screen employs a Huh7 human hepatoma cell line stably transfected with a Renilla luciferase sensor for endogenous miR-122. The assay was optimized and validated using an miR-122 antisense agent and a previously identified small-molecule miR-122 inhibitor. The described reporter assay will enable the high-throughput screening of small-molecule miR-122 inhibitors and can be readily extended to other miRNAs.
high-throughput assay; cell-based assay; luciferase; microRNA; small-molecule inhibitor
Fragment-based screening has typically relied on X-ray or NMR methods to identify low affinity ligands that bind to therapeutic targets. These techniques are expensive in terms of material and time, so it useful to have a higher-throughput method to reliably pre-screen a fragment library to identify a subset of compounds for structural analysis. Calorimetry provides a label-free method to assay binding and enzymatic activity that is unaffected by the spectroscopic properties of the sample. Conventional microcalorimetry is hampered by requiring large quantities of reagents and long measurement times. Nanocalorimeters can overcome these limitations of conventional ITC. Here we have used enthalpy arrays, which are arrays of nanocalorimeters, to perform an enzyme activity based fragment screen for competitive inhibitors of phosphodiesterase 4A (PDE4A). Several inhibitors with KI<2 mM were identified and moved to X-ray crystallization trials. Although the co-crystals did not yield high-resolution data, evidence of binding was observed and the chemical structures of the hits were consistent with motifs of known PDE4 inhibitors. This study shows how array calorimetry can be used as a pre-screening method for fragment-based lead discovery with enzyme targets, and it provides a list of candidate fragments for inhibition of PDE4A.
nanocalorimetry; enzyme assay; label-free assay; fragment-based lead discovery; X-ray crystallography
The authors conducted a high-throughput screening campaign for inhibitors of SV40 large T antigen ATPase activity to identify candidate antivirals that target the replication of polyomaviruses. The primary assay was adapted to 1536-well microplates and used to screen the National Institutes of Health Molecular Libraries Probe Centers Network library of 306 015 compounds. The primary screen had an Z value of ~0.68, signal/background = 3, and a high (5%) DMSO tolerance. Two counterscreens and two secondary assays were used to prioritize hits by EC50, cytotoxicity, target specificity, and off-target effects. Hits that inhibited ATPase activity by >44% in the primary screen were tested in dose–response efficacy and eukaryotic cytotoxicity assays. After evaluation of hit cytotoxicity, drug likeness, promiscuity, and target specificity, three compounds were chosen for chemical optimization. Chemical optimization identified a class of bisphenols as the most effective biochemical inhibitors. Bisphenol A inhibited SV40 large T antigen ATPase activity with an IC50 of 41 μM in the primary assay and 6.2 μM in a cytoprotection assay. This compound class is suitable as probes for biochemical investigation of large T antigen ATPase activity, but because of their cytotoxicity, further optimization is necessary for their use in studying polyomavirus replication in vivo.
SV40; chemistry; large T antigen; HTS; antivirals
One of the objectives of the National Institutes of Allergy and Infectious Diseases (NIAID) Biodefense Program is to identify or develop broad-spectrum antimicrobials for use against bioterrorism pathogens and emerging infectious agents. As a part of that program, our institution has screened the 10 000-compound MyriaScreen Diversity Collection of high-purity druglike compounds against three NIAID category A and one category B priority pathogens in an effort to identify potential compound classes for further drug development. The effective use of a Clinical and Laboratory Standards Institute–based high-throughput screening (HTS) 96-well–based format allowed for the identification of 49 compounds that had in vitro activity against all four pathogens with minimum inhibitory concentration values of ≤16 μg/mL. Adaptation of the HTS process was necessary to conduct the work in higher-level containment, in this case, biosafety level 3. Examination of chemical scaffolds shared by some of the 49 compounds and assessment of available chemical databases indicates that several may represent broad-spectrum antimicrobials whose activity is based on novel mechanisms of action.
anti-infective drugs; automation; cell-based assays; compound repositories; high-content screening
Methylation is a ubiquitous covalent modification used to control the function of diverse biomolecules including hormones, neurotransmitters, xenobiotics, proteins, nucleic acids and lipids. Histone methyltransferases (HMTs) are currently of high interest as drug targets because of their role in epigenetic regulation, however most HMT assay methods are either not amenable to an HTS environment or are applicable to a limited number of enzymes. We developed a generic methyltransferase assay method using fluorescent immunodetection of AMP, which is formed from the MT reaction product S-adenosylhomocysteine in a dual enzyme coupling step. The detection range of the assay, its suitability for HTS, including stability of reagents following dispensing and after addition to reactions as well as the potential for interference from drug like molecules was investigated. In addition, the use of the assay for measuring inhibitor potencies with peptide or intact protein substrates was examined through pilot screening with selected reference enzymes including HMT G9a. By combining a novel enzymatic coupling step with the well characterized Transcreener® AMP/GMP assay, we have developed a robust HTS assay for HMTs which should be broadly applicable to other types of methyltransferases as well.
epigenetics; histone methyltransferase; DNA methyltransferase; methyltransferase assay; high throughput screening
Testing small molecules for their ability to modify cysteine residues of proteins in the early stages of drug discovery is expected to accelerate our ability to develop more selective drugs with lesser side effects. In addition, this approach also enables the rapid evaluation of the mode of binding of new drug candidates in respect to thiol-reactivity and metabolism by glutathione. Herein, we describe the development of a fluorescence-based high throughput assay that allows the identification of thiol-reactive compounds. A thiol-containing fluorescent probe MSTI was synthesized and used to evaluate small molecules from the LOPAC collection of bioactive molecules. LOPAC compounds that are known to react with sulfur nucleophiles were identified with this assay, for example, irreversible protease inhibitors, nitric oxide releasing compounds, and proton-pump inhibitors. The results confirm that both electrophilic and redox reactive compounds can be quickly identified in a high throughput manner enabling the assessment of screening libraries in respect to thiol-reactive compounds.
Promiscuous inhibitors; glutathione; fluorescence; high throughput screening; thiol-reactive or electrophilic compound
Misregulation of the Wnt pathway has been shown to be responsible for a variety of human diseases, most notably cancers. Screens for inhibitors of this pathway have been performed almost exclusively using cultured mammalian cells or with purified proteins. We have previously developed a biochemical assay using Xenopus egg extracts to recapitulate key cytoplasmic events in the Wnt pathway. Using this biochemical system, we show that a recombinant form of the Wnt coreceptor, LRP6, regulates the stability of two key components of the Wnt pathway (β-catenin and Axin) in opposing fashion. We have now fused β-catenin and Axin to firefly and Renilla luciferase, respectively, and demonstrate that the fusion proteins behave similarly as their wild-type counterparts. Using this dual luciferase readout, we adapted the Xenopus extracts system for high-throughput screening. Results from these screens demonstrate signal distribution curves that reflect the complexity of the library screened. Of several compounds identified as cytoplasmic modulators of the Wnt pathway, one was further validated as a bona fide inhibitor of the Wnt pathway in cultured mammalian cells and Xenopus embryos. We show that other embryonic pathways may be amendable to screening for inhibitors/modulators in Xenopus egg extracts.
Wnt signaling; Xenopus extracts; β-catenin; Axin; LRP6; flavonoids
Small ubiquitin-like modifier (SUMO1-3) is a small group of proteins that are ligated to lysine residues in target proteins. SUMO conjugation is a highly dynamic process, as SUMOylated proteins are rapidly deconjugated by SUMO proteases. SUMO conjugation/deconjugation plays pivotal roles in major cellular pathways, and is associated with a number of pathological conditions. It is therefore of significant clinical interest to develop new strategies to screen for compounds to specifically interfere with SUMO conjugation/deconjugation. Here, we describe a novel high throughput screening-compatible assay to identify inhibitors of SUMO proteases. The assay is based on AlphaScreen technology and uses His-tagged SUMO2 conjugated to Strep-tagged SUMO3 as a SUMO protease substrate. A bacterial SUMOylation system was used to generate this substrate. A three-step purification strategy was employed to yield substrate of high quality. Our data indicated that this unique substrate can be readily detected in the AlphaScreen assays in a dose-dependent manner. Cleavage reactions by SUMO protease with or without inhibitor were monitored based on AlphaScreen signals. Furthermore, the assay was adapted to a 384-well format, and the interplate and interday variability was evaluated in eight 384-well plates. The average Z’ factor was 0.83±0.04, confirming the suitability for high throughput screening applications.
AlphaScreen technology; assay development; inhibitor; SUMO proteases; SUMO
Prostate cancer is a leading cause of death among men due to the limited number of treatment strategies available for advanced disease. Discovery of effective chemotherapeutics involves the identification of agents that inhibit cancer cell growth. Increases in intracellular granularity have been observed during physiological processes that include senescence, apoptosis, and autophagy, making this phenotypic change a useful marker for identifying small molecules that induce cellular growth arrest or death. In this regard, epithelial-derived cancer cell lines appear uniquely susceptible to increased intracellular granularity following exposure to chemotherapeutics. We have established a novel flow cytometry approach that detects increases in side light scatter in response to morphological changes associated with intracellular granularity in the androgen-sensitive LNCaP and androgen-independent PC3 human prostate cancer cell lines. A cell-based assay was developed to screen for small molecule inducers of intracellular granularity using the HyperCyt® high-throughput flow cytometry platform. Validation was performed using the Prestwick Chemical Library, where known modulators of LNCaP intracellular granularity, such as testosterone, were identified. Nonandrogenic inducers of granularity were also detected. A further screen of ~25,000 small molecules led to the identification of a class of aryl-oxazoles that increased intracellular granularity in both cell lines, often leading to cell death. The most potent agents exhibited submicromolar efficacy in LNCaP and PC3 cells.
HyperCyt® high-throughput flow cytometry; small molecule screening; intracellular granularity; prostate cancer
Early success of kinase inhibitors has validated their use as drugs. However, discovery efforts have also suffered from high attrition rates; due to lack of cellular activity. We reasoned that screening for such candidates in live cells would identify novel cell permeable modulators for development. For this purpose, we have used our recently optimized EGFR biosensor (EGFRB) assay to screen for modulators of EGFR activity. Here, we report on its validation under HTS conditions displaying a S/N ratio of 21 and a Z’ value of 0.56; attributes of a robust cell based assay. We performed a pilot screen against a library of 6,912 compounds demonstrating good reproducibility and identifying 82 inhibitors and 66 activators with initial hit rates of 1.2% and 0.95 %, respectively. Follow up dose response studies revealed that 12 out of the 13 known EGFR inhibitors in the library confirmed as hits. ZM-306416, a VEGFR antagonist, was identified as a potent inhibitor of EGFR function. Flurandrenolide, beclomethasone and ebastine were confirmed as activators of EGFR function. Taken together, our results validate this novel approach and demonstrate its utility in the discovery of novel kinase modulators with potential use in the clinic.
EGFR; domain-based biosensor; high content analysis; live cell imaging
Activation of the antioxidant response element (ARE) up-regulates enzymes involved in detoxification of electrophiles and reactive oxygen species. The induction of ARE genes is regulated by the interaction between redox sensor protein, Keap1, and the transcription factor, Nrf2. Fluorescently labeled Nrf2 peptides containing the ETGE motif were synthesized and optimized as tracers in the development of a fluorescence polarization (FP) assay to identify small molecule inhibitors of Keap1-Nrf2 interaction. The tracers were optimized to increase the dynamic range of the assay and their binding affinities to the Keap1 Kelch domain. The binding affinities of Nrf2 peptide inhibitors obtained in our FP assay using FITC-9mer Nrf2 peptide amide as the probe were in good agreement with those obtained previously by a surface plasmon resonance (SPR) assay. The FP assay exhibits considerable tolerance towards DMSO and produced a Z'-factor greater than 0.6 in a 384-well format. Further optimization of the probe led to cyanine-labeled 9mer Nrf2 peptide amide, which can be used along with the FITC-9mer Nrf2 peptide amide in a high throughput screening (HTS) assay to discover small molecule inhibitors of Keap1-Nrf2 interaction.
Nrf2; Keap1; ARE; fluorescence polarization; high throughput screening; oxidative response
Recent advances in stem cell technology have enabled large scale production of human cells such as cardiomyocytes, hepatocytes and neurons for evaluation of pharmacological effect and toxicity of drug candidates. The assessment of compound efficacy and toxicity using human cells should lower the high clinical attrition rates of drug candidates by reducing the impact of species differences on drug efficacy and toxicity from animal studies. Methyl-β-cyclodextrin (MBCD) has shown to reduce lysosomal cholesterol accumulation in skin fibroblasts derived from patients with Niemann Pick type C disease and in the NPC1−/− mouse model. However, the compound has never been tested in human differentiated neurons. We have determined the cholesterol reduction effect of MBCD in neurons differentiated from human neural stem cells and commercially available astrocytes. The use of NSCs for producing differentiated neurons in large quantities can significantly reduce the production time and enhance the reproducibility of screening results. The EC50 values of MBCD on cholesterol reduction in human neurons and astrocytes were 66.9 and 110.7 µM, respectively. The results indicate that human neurons differentiated from the NSCs and human astrocytes are useful tools for evaluating pharmacological activity and toxicity of drug candidates to predict their clinical efficacy.
induced pluripotent stem cells; neural stem cells; human neurons; astrocytes; skin fibroblasts; methyl-β-cyclodextrin
The secretory and transmembrane isoforms of Prostatic acid phosphatase (PAP) can dephosphorylate extracellular adenosine 5′-monophosphate (AMP) to adenosine, classifying PAP as an ectonucleotidase. Currently, there are no compounds that inhibit PAP in living cells. To identify small molecule modulators of PAP, we used a 1,536-well based quantitative high-throughput fluorogenic assay to screen the Library of Pharmacologically Active Compounds (LOPAC1280) arrayed as eight-concentration dilution series. This fluorogenic assay used difluoro-4-methylumbelliferyl phosphate (DiFMUP) as substrate and collected data in kinetic mode. Candidate hits were subsequently tested in an orthogonal absorbance-based biochemical assay that used AMP as substrate. From these initial screens, three inhibitors of secretory human (h) and mouse (m)PAP were identified: 8-(4-chlorophenylthio) cAMP (pCPT-cAMP), calmidazolium chloride and nalidixic acid. These compounds did not inhibit recombinant alkaline phosphatase. Of these compounds, only pCPT-cAMP and a related cyclic nucleotide analog [8-(4-chlorophenylthio) cGMP; pCPT-cGMP] inhibited the ectonucleotidase activity of transmembrane PAP in a cell-based assay. These cyclic nucleotides are structurally similar to AMP but cannot be hydrolyzed by PAP. In summary, we identified two cyclic nucleotide analogs that inhibit secretory and transmembrane PAP in vitro and in live cells.
ectonucleotidase; prostatic acid phosphatase; ACPP; pain; nociception
Caspases are central to the execution of programmed cell death and their activation constitutes the biochemical hallmark of apoptosis. In this article, we report the successful adaptation of a high content assay method utilizing the DEVD-NucView488™ fluorogenic substrate, and for the first time, we show caspase activation in live cells induced either by drugs or siRNA. The fluorogenic substrate was found to be non-toxic over an exposure period of several days; during which we demonstrate automated imaging and quantification of caspase activation of the same cell population as a function of time. Overexpression of the anti-apoptotic protein Bcl-XL, alone or in combination with the inhibitor Z-VAD-FMK, attenuated caspase activation in HeLa cells exposed to Doxorubicin, Etoposide or cell death siRNA. Our method was further validated against two well characterized NSCLC cell lines reported to be sensitive (H3255) or refractory (H2030) to Erlotinib; where we show a differential time dependent activation was observed for H3255 and no significant changes in H2030, consistent with their respective chemosensitivity profile. In summary, our results demonstrate the feasibility of using this newly adapted and validated high content assay to screen chemical or RNAi libraries for the identification of previously uncovered enhancers and suppressors of the apoptotic machinery in live cells.
High content assay; RNAi HT screening; Chemical HT screening; caspase; apoptosis; cancer; live cells