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1.  Literature Search and Review 
doi:10.1089/adt.2010.0805.lr
PMCID: PMC2974850
2.  A Homogeneous Method to Measure Nucleotide Exchange by α-Subunits of Heterotrimeric G-Proteins Using Fluorescence Polarization 
Abstract
The mainstay of assessing guanosine diphosphate release by the α-subunit of a heterotrimeric G-protein is the [35S]guanosine 5′-O-(3-thiotriphosphate) (GTPγS) radionucleotide-binding assay. This assay requires separation of protein-bound GTPγS from free GTPγS at multiple time points followed by quantification via liquid scintillation. The arduous nature of this assay makes it difficult to quickly characterize multiple mutants, determine the effects of individual variables (e.g., temperature and Mg2+ concentration) on nucleotide exchange, or screen for small molecule modulators of Gα nucleotide binding/cycling properties. Here, we describe a robust, homogeneous, fluorescence polarization assay using a red-shifted fluorescent GTPγS probe that can rapidly determine the rate of GTPγS binding by Gα subunits.
doi:10.1089/adt.2010.0286
PMCID: PMC2957273  PMID: 20662737
3.  Interview with Jill Jarecki, PhD 
doi:10.1089/adt.2014.1505
PMCID: PMC4142773  PMID: 25147905
4.  A Sensitive Luminescent Assay for the Histone Methyltransferase NSD1 and Other SAM-Dependent Enzymes 
Abstract
A major focus of our pediatric cancer research is the discovery of chemical probes to further our understanding of the biology of leukemia harboring fusion proteins arising from chromosomal rearrangements, and to develop novel specifically targeted therapies. The NUP98-NSD1 fusion protein occurs in a highly aggressive subtype of acute myeloid leukemia after rearrangement of the genes NUP98 and NSD1. The methyltransferase activity of NSD1 is retained in the fusion, and it gives rise to abnormally high levels of methylation at lysine 36 on histone 3, enforcing oncogene activation. Therefore, inhibition of the methyltransferase activity of NUP98-NSD1 may be considered a viable therapeutic strategy. Here, we report the development and validation of a highly sensitive and robust luminescence-based assay for NSD1 and other methyltransferases that use S-adenosylmethionine (SAM) as a methyl donor. The assay quantifies S-adenosylhomocysteine (SAH), which is produced during methyl transfer from SAM. SAH is converted enzymatically to adenosine monophosphate (AMP); in the process, adenosine triphosphate (ATP) is consumed and the amount of ATP remaining is measured using a luminescent assay kit. The assay was validated by pilot high-throughput screening (HTS), dose-response confirmation of hits, and elimination of artifacts through counterscreening against SAH detection in the absence of NSD1. The known methyltransferase inhibitor suramin was identified, and profiled for selectivity against the histone methyltransferases EZH2, SETD7, and PRMT1. HTS using the luminescent NSD1 assay described here has the potential to deliver selective NSD1 inhibitors that may serve as leads in the development of targeted therapies for NUP98-NSD1-driven leukemias.
doi:10.1089/adt.2014.583
PMCID: PMC4060815  PMID: 24927133
5.  Evaluation of Cytotoxicity and DNA Damage Response with Analysis of Intracellular ATM Signaling Pathways 
Abstract
Maintenance of genome integrity by preventing and overcoming DNA damage is critical for cell survival. Deficiency or aberrancy in the DNA damage response, for example, through ataxia telangiectasia mutated (ATM) signaling, lead to pathophysiological perturbations in organs throughout the body. Therefore, control of DNA damage is of major interest for development of therapeutic agents. Such efforts will greatly benefit from convenient and simple diagnostic and/or drug development tools to demonstrate whether ATM and related genes have been activated and to then determine whether these have been returned to normal levels of activity because pathway members sense and also repair DNA damage. To overcome difficulties in analyzing differences in multitudinous ATM pathway members following DNA damage, we measured ATM promoter activity with a fluorescent td-Tomato reporter gene to interrogate the global effects of ATM signaling pathways. In cultured HuH-7 cell line derived from human hepatocellular carcinoma, cis-platinum, acetaminophen, or hydrogen peroxide caused DNA strand breaks and ATM pathway activation as shown by γH2AX expression, which in turn, led to rapid and sustained increases in ATM promoter activity. This assay of ATM promoter activity identified biological agents capable of controlling cellular DNA damage in toxin-treated HuH-7 cells and in mice after onset of drug-induced acute liver failure. Therefore, the proposed assay of ATM promoter activity in HuH-7 cells was appropriately informative for treating DNA damage. High-throughput screens using ATM promoter activation will be helpful for therapeutic development in DNA damage-associated abnormal ATM signaling in various cell types and organs.
doi:10.1089/adt.2014.571
PMCID: PMC4060777  PMID: 24927134
6.  Development of an HTS-Compatible Assay for the Discovery of ASK1 Signalosome Inhibitors Using AlphaScreen Technology 
Abstract
Genetic target validation studies have demonstrated that the apoptosis signal-regulating kinase 1 (ASK1) represents an important target for the treatment of rheumatoid arthritis, cardiac diseases, and several neurodegenerative disorders. To identify small-molecule inhibitors of ASK1, we have developed a high-throughput screening-compatible, homogenous, biochemical assay using AlphaScreen technology. This novel assay design utilizes purified stress-activated ASK1 signalosome complex, and it monitors phosphorylation of its full-length native substrate, MKK6. The assay has been optimized in a 384-well format and validated by screening the Sigma LOPAC library. The results presented here demonstrate that the assay is sensitive and robust with a Z′ factor value of 0.88±0.04 and a signal-to-background ratio of 11, indicating that this assay can be used to screen large chemical libraries to discover novel inhibitors of ASK1.
doi:10.1089/adt.2013.558
PMCID: PMC4025566  PMID: 24831789
7.  A Phenotypic High Throughput Screening Assay for the Identification of Pharmacoperones for the Gonadotropin Releasing Hormone Receptor 
Abstract
We describe a phenotypic high throughput screening (HTS) calcium flux assay designed to identify pharmacoperones for the gonadotropin releasing hormone receptor (GnRHR). Pharmacoperones are target-specific, small molecules that diffuse into cells, rescue misfolded protein mutants, and restore them to function. Rescue is based on correcting the trafficking of mutants that would otherwise be retained in the endoplasmic reticulum and unable to function correctly. This approach identifies drugs with a significant degree of novelty, relying on cellular mechanisms that are not currently exploited. Development of such assays is important, since the extensive use of agonist/antagonist screens alone means that useful chemical structures may be present in existing libraries but have not been previously identified using existing methods. Our assay utilizes cell lines stably expressing a GnRHR mutant under the control of a tetracycline (OFF) transactivator. This allows us to quantitate the level of functional and properly trafficked G protein coupled receptors present in each test well. Furthermore, since we are able to turn receptor expression on and off, we can rapidly eliminate the majority of false positives from our screening results. Our data show that this approach is likely to be successful in identifying hits from large chemical libraries.
doi:10.1089/adt.2014.576
PMCID: PMC4025569  PMID: 24831790
8.  Evaluation of Impermeant, DNA-Binding Dye Fluorescence as a Real-Time Readout of Eukaryotic Cell Toxicity in a High Throughput Screening Format 
Abstract
Interpretation of high throughput screening (HTS) data in cell-based assays may be confounded by cytotoxic properties of screening compounds. Therefore, assessing cell toxicity in real time during the HTS process itself would be highly advantageous. Here, we investigate the potential of putatively impermeant, fluorescent, DNA-binding dyes to give cell toxicity readout during HTS. Amongst 19 DNA-binding dyes examined, three classes were identified that were (1) permeant, (2) cytotoxic, or (3) neither permeant nor cytotoxic during 3-day incubation with a macrophage cell line. In the last class, four dyes (SYTOX Green, CellTox Green, GelGreen, and EvaGreen) gave highly robust cytotoxicity data in 384-well screening plates. As proof of principle, successful combination with a luminescence-based assay in HTS format was demonstrated. Here, both intracellular growth of Legionella pneumophila (luminescence) and host cell viability (SYTOX Green exclusion) were assayed in the same screening well. Incorporation of membrane-impermeant, DNA-binding, fluorescent dyes in HTS assays should prove useful by allowing evaluation of cytotoxicity in real time, eliminating reagent addition steps and effort associated with endpoint cell viability analysis, and reducing the need for follow-up cytotoxicity screening.
doi:10.1089/adt.2014.577
PMCID: PMC4026211  PMID: 24831788
9.  Three-Dimensional Cell Culture Systems and Their Applications in Drug Discovery and Cell-Based Biosensors 
Abstract
Three-dimensional (3D) cell culture systems have gained increasing interest in drug discovery and tissue engineering due to their evident advantages in providing more physiologically relevant information and more predictive data for in vivo tests. In this review, we discuss the characteristics of 3D cell culture systems in comparison to the two-dimensional (2D) monolayer culture, focusing on cell growth conditions, cell proliferation, population, and gene and protein expression profiles. The innovations and development in 3D culture systems for drug discovery over the past 5 years are also reviewed in the article, emphasizing the cellular response to different classes of anticancer drugs, focusing particularly on similarities and differences between 3D and 2D models across the field. The progression and advancement in the application of 3D cell cultures in cell-based biosensors is another focal point of this review.
doi:10.1089/adt.2014.573
PMCID: PMC4026212  PMID: 24831787
10.  A BSL-4 High-Throughput Screen Identifies Sulfonamide Inhibitors of Nipah Virus 
Abstract
Nipah virus is a biosafety level 4 (BSL-4) pathogen that causes severe respiratory illness and encephalitis in humans. To identify novel small molecules that target Nipah virus replication as potential therapeutics, Southern Research Institute and Galveston National Laboratory jointly developed an automated high-throughput screening platform that is capable of testing 10,000 compounds per day within BSL-4 biocontainment. Using this platform, we screened a 10,080-compound library using a cell-based, high-throughput screen for compounds that inhibited the virus-induced cytopathic effect. From this pilot effort, 23 compounds were identified with EC50 values ranging from 3.9 to 20.0 μM and selectivities >10. Three sulfonamide compounds with EC50 values <12 μM were further characterized for their point of intervention in the viral replication cycle and for broad antiviral efficacy. Development of HTS capability under BSL-4 containment changes the paradigm for drug discovery for highly pathogenic agents because this platform can be readily modified to identify prophylactic and postexposure therapeutic candidates against other BSL-4 pathogens, particularly Ebola, Marburg, and Lassa viruses.
doi:10.1089/adt.2013.567
PMCID: PMC3994909  PMID: 24735442
11.  A Phenotypic Screening Approach to Identify Anticancer Compounds Derived from Marine Fungi 
Abstract
This study covers the isolation, testing, and identification of natural products with anticancer properties. Secondary metabolites were isolated from fungal strains originating from a variety of marine habitats. Strain culture protocols were optimized with respect to growth media composition and fermentation conditions. From these producers, isolated compounds were screened for their effect on the viability and proliferation of a subset of the NCI60 panel of cancer cell lines. Active compounds of interest were identified and selected for detailed assessments and structural elucidation using nuclear magnetic resonance. This revealed the majority of fungal-derived compounds represented known anticancer chemotypes, confirming the integrity of the process and the ability to identify suitable compounds. Examination of effects of selected compounds on cancer-associated cell signaling pathways used phospho flow cytometry in combination with 3D fluorescent cell barcoding. In parallel, the study addressed the logistical aspects of maintaining multiple cancer cell lines in culture simultaneously. A potential solution involving microbead-based cell culture was investigated (BioLevitator, Hamilton). Selected cell lines were cultured in microbead and 2D methods and cell viability tests showed comparable compound inhibition in both methods (R2=0.95). In a further technology assessment, an image-based assay system was investigated for its utility as a possible complement to ATP-based detection for quantifying cell growth and viability in a label-free manner.
doi:10.1089/adt.2013.564
PMCID: PMC3994914  PMID: 24735443
12.  Development of Fluorescent Substrates and Assays for the Key Autophagy-Related Cysteine Protease Enzyme, ATG4B 
Abstract
The cysteine protease ATG4B plays a role in key steps of the autophagy process and is of interest as a potential therapeutic target. At an early step, ATG4B cleaves proLC3 isoforms to form LC3-I for subsequent lipidation to form LC3-II and autophagosome membrane insertion. ATG4B also cleaves phosphatidylethanolamine (PE) from LC3-II to regenerate LC3-I, enabling its recycling for further membrane biogenesis. Here, we report several novel assays for monitoring the enzymatic activity of ATG4B. An assay based on mass spectrometric analysis and quantification of cleavage of the substrate protein LC3-B was developed and, while useful for mechanistic studies, was not suitable for high throughput screening (HTS). A doubly fluorescent fluorescence resonance energy transfer (FRET) ligand YFP-LC3B-EmGFP (FRET-LC3) was constructed and shown to be an excellent substrate for ATG4B with rates of cleavage similar to that for LC3B itself. A HTS assay to identify candidate inhibitors of ATG4B utilizing FRET-LC3 as a substrate was developed and validated with a satisfactory Z′ factor and high signal-to-noise ratio suitable for screening small molecule libraries. Pilot screens of the 1,280-member library of pharmacologically active compounds (LOPAC™) and a 3,481-member library of known drugs (KD2) gave hit rates of 0.6% and 0.5% respectively, and subsequent titrations confirmed ATG4B inhibitory activity for three compounds, both in the FRET and mass spectrometry assays. The FRET- and mass spectrometry–based assays we have developed will allow for both HTS for inhibitors of ATG4B and mechanistic approaches to study inhibition of a major component of the autophagy pathway.
doi:10.1089/adt.2013.561
PMCID: PMC3994995  PMID: 24735444
13.  Zebrafish Heart Failure Models for the Evaluation of Chemical Probes and Drugs 
Abstract
Heart failure is a complex disease that involves genetic, environmental, and physiological factors. As a result, current medication and treatment for heart failure produces limited efficacy, and better medication is in demand. Although mammalian models exist, simple and low-cost models will be more beneficial for drug discovery and mechanistic studies of heart failure. We previously reported that aristolochic acid (AA) caused cardiac defects in zebrafish embryos that resemble heart failure. Here, we showed that cardiac troponin T and atrial natriuretic peptide were expressed at significantly higher levels in AA-treated embryos, presumably due to cardiac hypertrophy. In addition, several human heart failure drugs could moderately attenuate the AA-induced heart failure by 10%–40%, further verifying the model for drug discovery. We then developed a drug screening assay using the AA-treated zebrafish embryos and identified three compounds. Mitogen-activated protein kinase kinase inhibitor (MEK-I), an inhibitor for the MEK-1/2 known to be involved in cardiac hypertrophy and heart failure, showed nearly 60% heart failure attenuation. C25, a chalcone derivative, and A11, a phenolic compound, showed around 80% and 90% attenuation, respectively. Time course experiments revealed that, to obtain 50% efficacy, these compounds were required within different hours of AA treatment. Furthermore, quantitative polymerase chain reaction showed that C25, not MEK-I or A11, strongly suppressed inflammation. Finally, C25 and MEK-I, but not A11, could also rescue the doxorubicin-induced heart failure in zebrafish embryos. In summary, we have established two tractable heart failure models for drug discovery and three potential drugs have been identified that seem to attenuate heart failure by different mechanisms.
doi:10.1089/adt.2013.548
PMCID: PMC3870487  PMID: 24351044
14.  Development and Validation of Fluorescence-Based and Automated Patch Clamp–Based Functional Assays for the Inward Rectifier Potassium Channel Kir4.1 
Abstract
The inward rectifier potassium (Kir) channel Kir4.1 plays essential roles in modulation of neurotransmission and renal sodium transport and may represent a novel drug target for temporal lobe epilepsy and hypertension. The molecular pharmacology of Kir4.1 is limited to neurological drugs, such as fluoxetine (Prozac©), exhibiting weak and nonspecific activity toward the channel. The development of potent and selective small-molecule probes would provide critically needed tools for exploring the integrative physiology and therapeutic potential of Kir4.1. A fluorescence-based thallium (Tl+) flux assay that utilizes a tetracycline-inducible T-Rex-HEK293-Kir4.1 cell line to enable high-throughput screening (HTS) of small-molecule libraries was developed. The assay is dimethyl sulfoxide tolerant and exhibits robust screening statistics (Z′=0.75±0.06). A pilot screen of 3,655 small molecules and lipids revealed 16 Kir4.1 inhibitors (0.4% hit rate). 3,3-Diphenyl-N-(1-phenylethyl)propan-1-amine, termed VU717, inhibits Kir4.1-mediated thallium flux with an IC50 of ∼6 μM. An automated patch clamp assay using the IonFlux HT workbench was developed to facilitate compound characterization. Leak-subtracted ensemble “loose patch” recordings revealed robust tetracycline-inducible and Kir4.1 currents that were inhibited by fluoxetine (IC50=10 μM), VU717 (IC50=6 μM), and structurally related calcium channel blocker prenylamine (IC50=6 μM). Finally, we demonstrate that VU717 inhibits Kir4.1 channel activity in cultured rat astrocytes, providing proof-of-concept that the Tl+ flux and IonFlux HT assays can enable the discovery of antagonists that are active against native Kir4.1 channels.
doi:10.1089/adt.2013.544
PMCID: PMC3870600  PMID: 24266659
15.  Bioluminescent Cell-Based NAD(P)/NAD(P)H Assays for Rapid Dinucleotide Measurement and Inhibitor Screening 
Abstract
The central role of nicotinamide adenine dinucleotides in cellular energy metabolism and signaling makes them important nodes that link the metabolic state of cells with energy homeostasis and gene regulation. In this study, we describe the implementation of cell-based bioluminescence assays for rapid and sensitive measurement of those important redox cofactors. We show that the sensitivity of the assays (limit of detection ∼0.5 nM) enables the selective detection of total amounts of nonphosphorylated or phosphorylated dinucleotides directly in cell lysates. The total amount of NAD+NADH or NADP+NADPH levels can be detected in as low as 300 or 600 cells/well, respectively. The signal remains linear up to 5,000 cells/well with the maximum signal-to-background ratios ranging from 100 to 200 for NAD+NADH and from 50 to 100 for NADP+NADPH detection. The assays are robust (Z′ value >0.7) and the inhibitor response curves generated using a known NAD biosynthetic pathway inhibitor FK866 correlate well with the reported data. More importantly, by multiplexing the dinucleotide detection assays with a fluorescent nonmetabolic cell viability assay, we show that dinucleotide levels can be decreased dramatically (>80%) by FK866 treatment before changes in cell viability are detected. The utility of the assays to identify modulators of intracellular nicotinamide adenine dinucleotide levels was further confirmed using an oncology active compound library, where novel dinucleotide regulating compounds were identified. For example, the histone deacetylase inhibitor entinostat was a potent inhibitor of cellular nicotinamide adenine dinucleotides, whereas the selective estrogen receptor modulator raloxifene unexpectedly caused a twofold increase in cellular nicotinamide adenine dinucleotide levels.
doi:10.1089/adt.2014.605
PMCID: PMC4270152  PMID: 25506801
16.  Expression and Purification of Recombinant Human Apolipoprotein A-II in Pichia pastoris 
Abstract
Apolipoprotein A-II (ApoA-II) is the second most abundant protein constituent of high-density lipoprotein (HDL). The physiologic role of ApoA-II is poorly defined. ApoA-II may inhibit lecithin:cholesterol acyltransferase and cholesteryl-ester-transfer protein activities, but may increase the hepatic lipase activity. ApoA-II may also inhibit the hepatic cholesteryl uptake from HDL probably through the scavenger receptor class B type I depending pathway. Interpretation of data from transgenic and knockout mice of genes involved in lipoprotein metabolism has been often complicated as clinical implications because of species difference. So it is important to obtain human ApoA-II for further studies about its functions. In our studies, Pichia pastoris expression system was first used to express a high-level secreted recombinant human ApoA-II (rhApoA-II). We have cloned the cDNA encoding human ApoA-II and achieved its high-level secreting expression with a yield of 65 mg/L of yeast culture and the purification process was effective and easy to handle. The purified rhApoA-II can be used to further study its biological activities.
doi:10.1089/adt.2013.511
PMCID: PMC3804080  PMID: 24116940
17.  Development of a High-Throughput Screening–Compatible Cell-Based Functional Assay to Identify Small Molecule Probes of the Galanin 3 Receptor (GalR3) 
Abstract
The galanin 3 receptor (GalR3) belongs to the large G protein–coupled receptor (GPCR) family of proteins. GalR3 and two other closely related receptors, GalR1 and GalR2, together with their endogenous ligand galanin, are involved in a variety of physiological and pathophysiological processes. GalR3 in particular has been strongly implicated in addiction and mood-related disorders such as anxiety and depression. It has been the target of many drug discovery programs within the pharmaceutical industry, but despite the significant resources and effort devoted to discovery of galanin receptor subtype selective small molecule modulators, there have been very few reports for the discovery of such molecules. GalR3 has proven difficult to enable in cell-based functional assays due to its apparent poor cell surface expression in recombinant systems. Here, we describe the generation of a modified GalR3 that facilitates its cell surface expression while maintaining wild-type receptor pharmacology. The modified GalR3 has been used to develop a high-throughput screening–compatible, cell-based, cAMP biosensor assay to detect selective small molecule modulators of GalR3. The performance of the assay has been validated by challenging it against a test library of small molecules with known pharmacological activities (LOPAC; Sigma Aldrich). This approach will enable identification of GalR3 selective modulators (chemical probes) that will facilitate dissection of the biological role(s) that GalR3 plays in normal physiological processes as well as in disease states.
doi:10.1089/adt.2013.526
PMCID: PMC3804082  PMID: 24116939
18.  Development of a High-Throughput Screening Cancer Cell-Based Luciferase Refolding Assay for Identifying Hsp90 Inhibitors 
Abstract
The 90 kDa heat-shock protein (Hsp90) and other cochaperones allow for proper folding of nascent or misfolded polypeptides. Cancer cells exploit these chaperones by maintaining the stability of mutated and misfolded oncoproteins and allowing them to evade proteosomal degradation. Inhibiting Hsp90 is an attractive strategy for cancer therapy, as the concomitant degradation of multiple oncoproteins may lead to effective anti-neoplastic agents. Unfortunately, early clinical trials have been disappointing with N-terminal Hsp90 inhibitors, as it is unclear whether the problems that plague current Hsp90 inhibitors in clinical trials are related to on-target or off-target activity. One approach to overcome these pitfalls is to identify structurally diverse scaffolds that improve Hsp90 inhibitory activity in the cancer cell milieu. Utilizing a panel of cancer cell lines that express luciferase, we have designed an in-cell Hsp90-dependent luciferase refolding assay. The assay was optimized using previously identified Hsp90 inhibitors and experimental novobiocin analogues against prostate, colon, and lung cancer cell lines. This assay exhibits good interplate precision (% CV), a signal-to-noise ratio (S/N) of ≥7, and an approximate Z-factor ranging from 0.5 to 0.7. Novobiocin analogues that revealed activity in this assay were examined via western blot experiments for client protein degradation, a hallmark of Hsp90 inhibition. Subsequently, a pilot screen was conducted using the Prestwick library, and two compounds, biperiden and ethoxyquin, revealed significant activity. Here, we report the development of an in-cell Hsp90-dependent luciferase refolding assay that is amenable across cancer cell lines for the screening of inhibitors in their specific milieu.
doi:10.1089/adt.2012.498
PMCID: PMC3931435  PMID: 24127661
19.  Development of Multiple Cell-Based Assays for the Detection of Histone H3 Lys27 Trimethylation (H3K27me3) 
Abstract
Posttranslational modification of histone proteins in eukaryotes plays an important role in gene transcription and chromatin structure. Dysregulation of the enzymes involved in histone modification has been linked to many cancer forms, making this target class a potential new area for therapeutics. A reliable assay to monitor small-molecule inhibition of various epigenetic enzymes should play a critical role in drug discovery to fight cancer. However, it has been challenging to develop cell-based assays for high-throughput screening (HTS) and compound profiling. Recently, two homogeneous cell-based assay kits using the AlphaLISA® and LanthaScreen® technologies to detect trimethyl histone H3 Lysine 27 have become commercially available, and a heterogeneous cell assay with modified dissociation-enhanced lanthanide fluorescence immunoassay (DELFIA®) format has been reported. To compare their pros and cons, we evaluated, optimized, and validated these three assay formats in three different cell lines and compared their activities with traditional Western blot detection of histone methylation inhibition by using commercial and in-house small-molecule inhibitors. Our data indicate that, although all four formats produced acceptable results, the homogeneous AlphaLISA assay was best suited for HTS and compound profiling due to its wider window and ease of automation. The DELFIA and Western blot assays were useful as validation tools to confirm the cell activities and eliminate potential false-positive compounds.
doi:10.1089/adt.2013.515
PMCID: PMC3777651  PMID: 23992119
20.  Assay Development and Multivariate Scoring for High-Content Discovery of Chemoprotectants of Endoplasmic-Reticulum-Stress-Mediated Amylin-Induced Cytotoxicity in Pancreatic Beta Cells 
Abstract
The underlying pathogenesis of type-II diabetes mellitus is in the dysfunction and selective loss of pancreatic islet β-cells, which ultimately leads to underproduction of endogenous insulin. Amylin, a 37-amino-acid human hormone that is cosecreted with insulin, helps regulate gastric emptying and maintain blood glucose homeostasis through improved postprandial satiety. It is hypothesized that amylin protofibrils cause selective loss of pancreatic β-cells in a manner similar to amyloid β aggregation in Alzheimer's disease. β-Cell death occurs in vitro when isolated human or rodent β-cells are exposed to micromolar concentrations of amylin, but the exact mechanism of selective β-cell loss in vivo remains unknown. Therefore, pursuing small-molecule drug discovery for chemoprotectants of amylin-induced β-cell toxicity is a viable phenotypic target that can lead to potential pharmacotherapies for the preservation of β-cell mass, delaying insulin dependence and allowing additional opportunities for lifestyle intervention. Additionally, chronic endoplasmic reticulum (ER) stress induced by chronic hyperglycemia and hyperlipidemia is a potentiating factor of amylin-induced β-cell loss. Herein, we describe a high-content/high-throughput screening (HTS) assay for the discovery of small molecules that are chemoprotective of amylin-induced, ER-stress-potentiated β-cell loss. We also put forth a general method for construction of a robust well-level multivariate scoring system using partial least squares regression analysis to improve high-content assay performance and to streamline the association of complex high-content data into HTS activity databases where univariate responses are typical.
doi:10.1089/adt.2014.591
PMCID: PMC4146389  PMID: 25181410
21.  A Cell-Based Functional Assay Using a Green Fluorescent Protein-Based Calcium Indicator dCys-GCaMP 
Abstract
Measurement of the changes in intracellular Ca2+ levels is an important assay for drug discovery. In this report, we describe a novel Ca2+ indicator, dCys-GCaMP, based on the green fluorescent protein and the development of a rapid and simple cell-based functional assay using this new Ca2+ indicator. We demonstrated the sensitivity and reliability of the assay by measuring the cellular responses to the agonists, antagonists, channel blockers, and modulators of the ionotropic N-methyl-D-aspartate (NMDA) subtype of glutamate receptors. HEK293 cells coexpressing the NMDA receptor and dCys-GCaMP displayed a strong increase in fluorescence intensity when stimulated with the agonist glutamate. This increase in the fluorescence signal was agonist concentration dependent and could be blocked by NMDAR antagonists and channel blockers. The pharmacological parameters measured with the dCys-GCaMP assay are in close agreement with those derived from conventional assays with synthetic dye fluo-4 and literature values. In addition, we showed that this assay could be used on G protein-coupled receptors as well, as exemplified by studies on the α1A adrenergic receptor. A limited scale evaluation of the assay performance in a 96-well compound screening format suggests that the dCys-GCaMP assay could be easily adapted to a high-throughput screening environment. The most important advantage of this new assay over the conventional fluo-4 and aequorin assays is the elimination of the dye-loading or substrate-loading process.
doi:10.1089/adt.2014.584
PMCID: PMC4142787  PMID: 25105973
22.  L-Type Ca2+ Channel Responses to Bay K 8644 in Stem Cell-Derived Cardiomyocytes Are Unusually Dependent on Holding Potential and Charge Carrier 
Abstract
Human stem cell-derived cardiomyocytes provide a cellular model for the study of electrophysiology in the human heart and are finding a niche in the field of safety pharmacology for predicting proarrhythmia. The cardiac L-type Ca2+ channel is an important target for some of these safety studies. However, the pharmacology of this channel in these cells is altered compared to native cardiac tissue, specifically in its sensitivity to the Ca2+ channel activator S-(−)-Bay K 8644. Using patch clamp electrophysiology, we examined the effects of S-(−)-Bay K 8644 in three separate stem cell-derived cardiomyocyte cell lines under various conditions in an effort to detect more typical responses to the drug. S-(−)-Bay K 8644 failed to produce characteristically large increases in current when cells were held at −40 mV and Ca2+ was used as the charge carrier, although high-affinity binding and the effects of the antagonist isomer, R-(+)-Bay K 8644, were intact. Dephosphorylation of the channel with acetylcholine failed to restore the sensitivity of the channel to the drug. Only when the holding potential was shifted to a more hyperpolarized (−60 mV) level, and external Ca2+ was replaced by Ba2+, could large increases in current amplitude be observed. Even under these conditions, increases in current amplitude varied dramatically between different cell lines and channel kinetics following drug addition were generally atypical. The results indicate that the pharmacology of S-(−)-Bay K 8644 in stem cell-derived cardiomyocytes varies by cell type, is unusually dependent on holding potential and charge carrier, and is different from that observed in primary human heart cells.
doi:10.1089/adt.2014.596
PMCID: PMC4142808  PMID: 25147907
23.  Assays for the Identification and Prioritization of Drug Candidates for Spinal Muscular Atrophy 
Abstract
Spinal muscular atrophy (SMA) is an autosomal recessive genetic disorder resulting in degeneration of α-motor neurons of the anterior horn and proximal muscle weakness. It is the leading cause of genetic mortality in children younger than 2 years. It affects ∼1 in 11,000 live births. In 95% of cases, SMA is caused by homozygous deletion of the SMN1 gene. In addition, all patients possess at least one copy of an almost identical gene called SMN2. A single point mutation in exon 7 of the SMN2 gene results in the production of low levels of full-length survival of motor neuron (SMN) protein at amounts insufficient to compensate for the loss of the SMN1 gene. Although no drug treatments are available for SMA, a number of drug discovery and development programs are ongoing, with several currently in clinical trials. This review describes the assays used to identify candidate drugs for SMA that modulate SMN2 gene expression by various means. Specifically, it discusses the use of high-throughput screening to identify candidate molecules from primary screens, as well as the technical aspects of a number of widely used secondary assays to assess SMN messenger ribonucleic acid (mRNA) and protein expression, localization, and function. Finally, it describes the process of iterative drug optimization utilized during preclinical SMA drug development to identify clinical candidates for testing in human clinical trials.
doi:10.1089/adt.2014.587
PMCID: PMC4142828  PMID: 25147906
24.  A Comparison of Assay Performance Between the Calcium Mobilization and the Dynamic Mass Redistribution Technologies for the Human Urotensin Receptor 
Abstract
The popular screening method for urotensin (UT) receptor antagonists is to measure the intracellular calcium concentration with a calcium-sensitive fluorescent dye. This assay format has an inherent limitation on the problem related to the fluorescence interference as it involves fluorescent dyes. In the present study, a label-free assay for the screening of UT receptor antagonists was developed by using dynamic mass redistribution (DMR) assay based on label-free optical biosensor. The addition of urotensin II (UII) stimulated a DMR profile to HEK293 cells stably expressing the human UT receptor (HEK293UT cells) but not on parental cells. The EC50 value of UII in label-free assay was 4.58 nM, which is very similar to that in conventional calcium mobilization assay (4.15 nM). Compared with the calcium mobilization assay for UII (Z′ factor, 0.77), the current label-free assay presented improved Z′ factor (0.81), with a relatively similar S/B ratio (28.0 and 25.6, respectively). The known high-affinity UT receptor antagonists, SB657510, GSK562590, and urantide, exhibited comparable IC50 values but rather less potent in the DMR assay than in calcium mobilization. Our DMR assay was able to present various functional responses, including inverse agonism in SB657510 and GSK1562590 as well as partial agonism in urantide. Moreover, the DMR assay exerted the stable antagonist window upon the minimal agonist stimulus. These results suggest that the label-free cell-based UT receptor assay can be applicable to evaluate the various functional activities of UT receptor-related drug candidates.
doi:10.1089/adt.2014.590
PMCID: PMC4142844  PMID: 25147908
25.  A Time-Resolved Fluorescence Resonance Energy Transfer Assay for High-Throughput Screening of 14-3-3 Protein–Protein Interaction Inhibitors 
Abstract
Protein–protein interaction networks mediate diverse biological processes by regulating various signaling hubs and clusters. 14-3-3 proteins, a family of phosphoserine/threonine-binding molecules, serve as major interaction hubs in eukaryotic cells and have emerged as promising therapeutic targets for various human diseases. In order to identify chemical probes for mechanistic studies and for potential therapeutic development, we have developed highly sensitive bioassays to monitor the interaction of 14-3-3 with a client protein. In this study, we describe a homogenous time-resolved fluorescence resonance energy transfer (TR-FRET) assay to detect the interaction of 14-3-3 with Bad, a proapoptotic member of the Bcl-2 family. Through a series of titration studies in which europium-labeled 14-3-3 serves as an FRET donor and a Dy647-labeled phosphorylated Bad, the peptide acts as an FRET acceptor, we have achieved a robust TR-FRET assay that is suitable for high-throughput screening (HTS) with an excellent signal-to-background ratio of >20 and Z′ values >0.7. This assay was further miniaturized to a 1,536-well format for ultra-HTS (uHTS), and exhibited a similar robust performance. The utility and performance of the assay for uHTS were validated by (i) known inhibitors, including peptide R18 and small molecule FOBISIN101, and (ii) screening of a 51,200 compound library. This simple and robust assay is generally applicable to detect the interaction of 14-3-3 with other client proteins. It provides a sensitive and easy-to-use tool to facilitate the discovery of 14-3-3 protein inhibitors as well as to study 14-3-3-mediated protein–protein interactions.
doi:10.1089/adt.2013.507
PMCID: PMC3751221  PMID: 23906346

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