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1.  Literature Search and Review 
PMCID: PMC2974850
2.  Zebrafish Heart Failure Models for the Evaluation of Chemical Probes and Drugs 
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.
PMCID: PMC3870487  PMID: 24351044
3.  Development and Validation of Fluorescence-Based and Automated Patch Clamp–Based Functional Assays for the Inward Rectifier Potassium Channel Kir4.1 
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.
PMCID: PMC3870600  PMID: 24266659
4.  Bioluminescent Cell-Based NAD(P)/NAD(P)H Assays for Rapid Dinucleotide Measurement and Inhibitor Screening 
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.
PMCID: PMC4270152  PMID: 25506801
5.  Expression and Purification of Recombinant Human Apolipoprotein A-II in Pichia pastoris 
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.
PMCID: PMC3804080  PMID: 24116940
6.  Development of a High-Throughput Screening–Compatible Cell-Based Functional Assay to Identify Small Molecule Probes of the Galanin 3 Receptor (GalR3) 
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.
PMCID: PMC3804082  PMID: 24116939
7.  Development of a High-Throughput Screening Cancer Cell-Based Luciferase Refolding Assay for Identifying Hsp90 Inhibitors 
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.
PMCID: PMC3931435  PMID: 24127661
8.  Development of Multiple Cell-Based Assays for the Detection of Histone H3 Lys27 Trimethylation (H3K27me3) 
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.
PMCID: PMC3777651  PMID: 23992119
9.  Assay Development and Multivariate Scoring for High-Content Discovery of Chemoprotectants of Endoplasmic-Reticulum-Stress-Mediated Amylin-Induced Cytotoxicity in Pancreatic Beta Cells 
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.
PMCID: PMC4146389  PMID: 25181410
10.  Interview with Jill Jarecki, PhD 
PMCID: PMC4142773  PMID: 25147905
11.  A Cell-Based Functional Assay Using a Green Fluorescent Protein-Based Calcium Indicator dCys-GCaMP 
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.
PMCID: PMC4142787  PMID: 25105973
12.  L-Type Ca2+ Channel Responses to Bay K 8644 in Stem Cell-Derived Cardiomyocytes Are Unusually Dependent on Holding Potential and Charge Carrier 
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.
PMCID: PMC4142808  PMID: 25147907
13.  Assays for the Identification and Prioritization of Drug Candidates for Spinal Muscular Atrophy 
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.
PMCID: PMC4142828  PMID: 25147906
14.  A Comparison of Assay Performance Between the Calcium Mobilization and the Dynamic Mass Redistribution Technologies for the Human Urotensin Receptor 
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.
PMCID: PMC4142844  PMID: 25147908
15.  A Time-Resolved Fluorescence Resonance Energy Transfer Assay for High-Throughput Screening of 14-3-3 Protein–Protein Interaction Inhibitors 
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.
PMCID: PMC3751221  PMID: 23906346
16.  A Highly Specific Cell-Based High-Throughput Screening Assay for Ligands of Cyclic Adenosine Monophosphate Receptor Protein in Gram-Negative Bacteria 
Quorum sensing is a cell–cell communication process in bacteria that involves the production, release, and subsequent detection of chemical signal molecules called autoinducers. In Vibrio cholerae, multiple input signals activate the expression of the quorum sensing regulator HapR, which acts to repress the expression of virulence factors. We have shown that CRP, the cyclic adenosine monophosphate (cAMP) receptor protein, enhances quorum sensing by activating the biosynthesis of cholera autoinducer 1, the major signaling molecule that contributes to the activation of HapR. Thus, proquorum sensing CRP agonists could inhibit virulence and lead to new drugs to treat severe cholera. In this study, we show that expression of the quorum sensing-regulated luxCDABE operon can be used as a robust readout for CRP activity. Further, we describe and validate a highly specific cell-based luminescence high-throughput screening assay for proquorum sensing CRP ligands. A pilot screen of 9,425 compounds yielded a hit rate of 0.02%, one hit being cAMP itself. The Z′ value for this assay was 0.76 and its coefficient of variance 8% for the positive control compound. To our knowledge, this is the first cell-based assay for ligands of the highly conserved CRP protein of Gram-negative bacteria. The use of this assay to screen large chemical libraries could identify lead compounds to treat cholera, as well as small molecules to probe ligand–receptor interactions in the CRP molecule.
PMCID: PMC3751313  PMID: 23906348
17.  Discovering Small Molecule Ligands of Vascular Endothelial Growth Factor That Block VEGF–KDR Binding Using Label-Free Microarray-Based Assays 
We present here a label-free microarray-based assay platform that we used to identify inhibitors of vascular endothelial growth factor (VEGF)–kinase-insertion domain receptor (KDR) binding. Supported by a combination of special ellipsometry-based optical detection and small molecule microarrays (SMM), this platform consists of three assays: (1) the first assay detects binding of a target protein with SMM and identifies ligands to the protein as inhibitor candidates; (2) the second assay detects binding of a receptor protein with identical SMM and subsequent binding of the target protein (a sandwich assay) to identify the ligands to the receptor protein that do not interfere with the target-receptor binding; (3) the third assay detects binding of the target protein to the receptor protein in the presence of the ligands of the target protein identified from the first assay, with the receptor protein immobilized to a solid surface through the ligands identified in the second assay, to yield dose–response curves. Using this platform, we screened 7,961 compounds from the National Cancer Institute and found 12 inhibitors to VEGF–KDR (VEGFR2) interactions with IC50 ranging from 0.3 to 60 μM. The inhibitory potency of these inhibitors found in the microarray-based assay was confirmed by their inhibition of VEGF-induced VEGFR2 phosphorylation in a cell-based assay.
PMCID: PMC3685389  PMID: 23772553
18.  Interview with John W. Kozarich, PhD 
PMCID: PMC3709828  PMID: 23772549
19.  The Tenth Annual Ion Channel Retreat, Vancouver, Canada, June 25–27, 2012 
Ten years after Aurora Biomed (Vancouver, British Columbia, Canada) hosted the inaugural Ion Channel Retreat, this event is recognized as a leading conference for ion channel researchers. Held annually in Vancouver, this meeting consistently provides an outlet for researchers to share their findings while learning about new concepts, methods, and technologies. Researchers use this forum to discuss and debate a spectrum of topics from ion channel research and technology to drug discovery and safety. The Retreat covered key subjects in the ion channel industry, including ion channels as disease targets, transient receptor protein channels as pain and disease targets, ion channels as pain targets, ion channel structure and function, ion channel screening technologies, cardiac safety and toxicology, and cardiac function and pharmacology.
PMCID: PMC3656625  PMID: 23679851
20.  Assay Development for Histone Methyltransferases 
Epigenetic modifications play a crucial role in human diseases. Unlike genetic mutations, however, they do not change the underlying DNA sequences. Epigenetic phenomena have gained increased attention in the field of cancer research, with many studies indicating that they are significantly involved in tumor establishment and progression. Histone methyltransferases (HMTs) are a large group of enzymes that specifically methylate protein lysine and arginine residues, especially in histones, using S-adenosyl-l-methionine (SAM) as the methyl donor. However, in general, HMTs have no widely accepted high-throughput screening (HTS) assay format, and reference inhibitors are not available for many of the enzymes. In this study, we describe the application of a miniaturized, radioisotope-based reaction system: the HotSpotSM platform for methyltransferases. Since this platform employs tritiated SAM as a cofactor, it can be applied to the assay of any HMT. The key advantage of this format is that any substrate can be used, including peptides, proteins, or even nucleosomes, without the need for labeling or any other modifications. Using this platform, we have determined substrate specificities, characterized enzyme kinetics, performed compound profiling for both lysine and arginine methyltransferases, and carried out HTS for a small-library LOPAC against DOT1L. After hit confirmation and profiling, we found that suramin inhibited DOT1L, NSD2, and PRMT4 with IC50 values at a low μM range.
PMCID: PMC3656627  PMID: 23557020
21.  Development of a High-Throughput Screening–Compatible Assay for the Discovery of Inhibitors of the AF4-AF9 Interaction Using AlphaScreen Technology 
Rearrangements of the mixed-lineage leukemia (MLL) gene occur predominately in pediatric leukemia cases and are generally predictors of a poor prognosis. These chromosomal rearrangements result in fusion of the protein MLL to one of more than 60 protein partners. MLL fusions are potent inducers of leukemia through activation of oncogene expression; therefore, targeting this transcriptional activation function may arrest MLL-rearranged (MLL-R) leukemia. Leukemic cell lines harboring the most common fusion protein, MLL-AF4, require the direct interaction of AF4 with the transcription factor AF9 to survive and self-renew; disrupting this interaction with a cell-penetrating AF4-derived peptide results in cell death, suggesting that the AF4-AF9 interaction could be a viable target for a novel MLL-R leukemia therapy. Here we describe the use of AlphaScreen technology to develop a high-throughput screening (HTS) assay to detect nonpeptidic inhibitors of AF4-AF9 binding. The assay is economical, requiring only low nanomolar concentrations of biotinylated AF4-derived peptide and FLAG-tagged AF9 in low-volume 384-well plates. A Z′-factor of 0.71 and a signal-to-background ratio of 21.3 showed the assay to be robust, and sensitivity to inhibition was demonstrated with competing AF4-derived peptides. Two pilot screens comprising 5,680 compounds served as validation for HTS at Nemours and the Broad Institute. Assay artifacts were excluded using a counterscreen comprising a biotinylated FLAG peptide. This is the first reported HTS-compatible assay to identify compounds that inhibit a key binding interaction of an MLL fusion partner, and the results presented here demonstrate suitability for screening large chemical libraries in high-density, low-volume plate formats.
PMCID: PMC3656630  PMID: 23679849
22.  An Arrayed Genome-Scale Lentiviral-Enabled Short Hairpin RNA Screen Identifies Lethal and Rescuer Gene Candidates 
RNA interference technology is becoming an integral tool for target discovery and validation.; With perhaps the exception of only few studies published using arrayed short hairpin RNA (shRNA) libraries, most of the reports have been either against pooled siRNA or shRNA, or arrayed siRNA libraries. For this purpose, we have developed a workflow and performed an arrayed genome-scale shRNA lethality screen against the TRC1 library in HeLa cells. The resulting targets would be a valuable resource of candidates toward a better understanding of cellular homeostasis. Using a high-stringency hit nomination method encompassing criteria of at least three active hairpins per gene and filtered for potential off-target effects (OTEs), referred to as the Bhinder–Djaballah analysis method, we identified 1,252 lethal and 6 rescuer gene candidates, knockdown of which resulted in severe cell death or enhanced growth, respectively. Cross referencing individual hairpins with the TRC1 validated clone database, 239 of the 1,252 candidates were deemed independently validated with at least three validated clones. Through our systematic OTE analysis, we have identified 31 microRNAs (miRNAs) in lethal and 2 in rescuer genes; all having a seed heptamer mimic in the corresponding shRNA hairpins and likely cause of the OTE observed in our screen, perhaps unraveling a previously unknown plausible essentiality of these miRNAs in cellular viability. Taken together, we report on a methodology for performing large-scale arrayed shRNA screens, a comprehensive analysis method to nominate high-confidence hits, and a performance assessment of the TRC1 library highlighting the intracellular inefficiencies of shRNA processing in general.
PMCID: PMC3619155  PMID: 23198867
23.  An Arrayed RNA Interference Genome-Wide Screen Identifies Candidate Genes Involved in the MicroRNA 21 Biogenesis Pathway 
MicroRNAs (miRNAs) are evolutionary conserved noncoding molecules that regulate gene expression. They influence a number of diverse biological functions, such as development and differentiation. However, their dysregulation has been shown to be associated with disease states, such as cancer. Genes and pathways regulating their biogenesis remain unknown and are highly sought after. For this purpose, we have validated a multiplexed high-content assay strategy to screen for such modulators. Here, we describe its implementation that makes use of a cell-based gain-of-function reporter assay monitoring enhanced green fluorescent protein expression under the control of miRNA 21 (miR-21); combined with measures of both cell metabolic activities through the use of Alamar Blue and cell death through imaged Hoechst-stained nuclei. The strategy was validated using a panel of known genes and enabled us to successfully progress to and complete an arrayed genome-wide short interfering RNA (siRNA) screen against the Ambion Silencer Select v4.0 library containing 64,755 siRNA duplexes covering 21,565 genes. We applied a high-stringency hit analysis method, referred to as the Bhinder–Djaballah analysis method, leading to the nomination of 1,273 genes as candidate inhibitors of the miR-21 biogenesis pathway; after several iterations eliminating those genes with only one active duplex and those enriched in seed sequence mediated off-target effects. Biological classifications revealed four major control junctions among them vesicular transport via clathrin-mediated endocytosis. Altogether, our screen has uncovered a number of novel candidate regulators that are potentially good druggable targets allowing for the discovery and development of small molecules for regulating miRNA function.
PMCID: PMC3619226  PMID: 23153064
24.  Extracellular Matrix Remodeling: The Common Denominator in Connective Tissue DiseasesPossibilities for Evaluation and Current Understanding of the Matrix as More Than a Passive Architecture, but a Key Player in Tissue Failure 
Increased attention is paid to the structural components of tissues. These components are mostly collagens and various proteoglycans. Emerging evidence suggests that altered components and noncoded modifications of the matrix may be both initiators and drivers of disease, exemplified by excessive tissue remodeling leading to tissue stiffness, as well as by changes in the signaling potential of both intact matrix and fragments thereof. Although tissue structure until recently was viewed as a simple architecture anchoring cells and proteins, this complex grid may contain essential information enabling the maintenance of the structure and normal functioning of tissue. The aims of this review are to (1) discuss the structural components of the matrix and the relevance of their mutations to the pathology of diseases such as fibrosis and cancer, (2) introduce the possibility that post-translational modifications (PTMs), such as protease cleavage, citrullination, cross-linking, nitrosylation, glycosylation, and isomerization, generated during pathology, may be unique, disease-specific biochemical markers, (3) list and review the range of simple enzyme-linked immunosorbent assays (ELISAs) that have been developed for assessing the extracellular matrix (ECM) and detecting abnormal ECM remodeling, and (4) discuss whether some PTMs are the cause or consequence of disease. New evidence clearly suggests that the ECM at some point in the pathogenesis becomes a driver of disease. These pathological modified ECM proteins may allow insights into complicated pathologies in which the end stage is excessive tissue remodeling, and provide unique and more pathology-specific biochemical markers.
PMCID: PMC3593693  PMID: 23046407
25.  Prototype for Automatable, Dielectrophoretically-Accessed Intracellular Membrane–Potential Measurements by Metal Electrodes 
Functional access to membrane proteins, for example, ion channels, of individual cells is an important prerequisite in drug discovery studies. The highly sophisticated patch-clamp method is widely used for electrogenic membrane proteins, but is demanding for the operator, and its automation remains challenging. The dielectrophoretically-accessed, intracellular membrane–potential measurement (DAIMM) method is a new technique showing high potential for automation of electrophysiological data recording in the whole-cell configuration. A cell suspension is brought between a mm-scaled planar electrode and a μm-scaled tip electrode, placed opposite to each other. Due to the asymmetric electrode configuration, the application of alternating electric fields (1–5 MHz) provokes a dielectrophoretic force acting on the target cell. As a consequence, the cell is accelerated and pierced by the tip electrode, hence functioning as the internal (working) electrode. We used the light-gated cation channel Channelrhodopsin-2 as a reporter protein expressed in HEK293 cells to characterize the DAIMM method in comparison with the patch-clamp technique.
PMCID: PMC3567700  PMID: 22994967

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