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1.  An overview of the challenges in designing, integrating, and delivering BARD: a public chemical biology resource and query portal across multiple organizations, locations, and disciplines 
Journal of biomolecular screening  2014;19(5):614-627.
Recent industry-academic partnerships involve collaboration across disciplines, locations, and organizations using publicly funded “open-access” and proprietary commercial data sources. These require effective integration of chemical and biological information from diverse data sources, presenting key informatics, personnel, and organizational challenges. BARD (BioAssay Research Database) was conceived to address these challenges and to serve as a community-wide resource and intuitive web portal for public-sector chemical biology data. Its initial focus is to enable scientists to more effectively use the NIH Roadmap Molecular Libraries Program (MLP) data generated from 3-year pilot and 6-year production phases of the Molecular Libraries Probe Production Centers Network (MLPCN), currently in its final year. BARD evolves the current data standards through structured assay and result annotations that leverage the BioAssay Ontology (BAO) and other industry-standard ontologies, and a core hierarchy of assay definition terms and data standards defined specifically for small-molecule assay data. We have initially focused on migrating the highest-value MLP data into BARD and bringing it up to this new standard. We review the technical and organizational challenges overcome by the inter-disciplinary BARD team, veterans of public and private sector data-integration projects, collaborating to describe (functional specifications), design (technical specifications), and implement this next-generation software solution.
PMCID: PMC4468040  PMID: 24441647
chemical and biological data and database; public data sources; “open innovation”; PubChem; web portal; data standards; definitions; assay protocols; data migration; analytical and transactional processing; data warehouse; visualization; community adoption
2.  Small molecule, NSC95397, inhibits the CtBP1-protein partner interaction and CtBP1-mediated transcriptional repression 
Journal of biomolecular screening  2014;20(5):663-672.
Carboxyl-terminal binding protein (CtBP) is a transcriptional co-repressor that suppresses multiple pro-apoptotic and epithelial genes. CtBP is overexpressed in many human cancers and its overexpression increases stem cell-like features, epithelial-mesenchymal transition, and cancer cell survival. Knockdown of CtBP increases apoptosis independent of p53 and dramatically inhibits tumorigenesis in mouse models. Therefore, targeting CtBP with small molecules that disrupt its interaction with transcription factor partners may be an effective cancer therapy. To elicit its co-repressing effect, CtBP binds to a conserved peptide motif in each transcription factor partner. We developed an AlphaScreen high throughput screening assay to monitor the interaction between CtBP and E1A (which mimics the interaction between CtBP and its transcriptional partners). We screened the LOPAC library of 1280 bioactive compounds and identified NSC95397, which inhibits the CtBP-E1A interaction (IC50 = 2.9 μM). The inhibitory activity of NSC95397 was confirmed using two secondary assays and a counterscreen. NSC95397 also behaved as a weak substrate of CtBP dehydrogenase activity and did not inhibit another dehydrogenase, LDH. Finally, NSC95397 was able to disrupt CtBP-mediated transcriptional repression of a target gene. These studies present a new possibility for the development of a therapeutic agent targeting tumors through disrupting the CtBP transcriptional complex.
PMCID: PMC4486263  PMID: 25477201
CtBP1; NSC95397; E1A; AlphaScreen; Protein-Protein Interaction
3.  Cell Treatment and Lysis in 96-Well Filter-Bottom Plates for Screening Bcr-Abl Activity and Inhibition in Whole-Cell Extracts 
Journal of biomolecular screening  2010;15(4):434-440.
Although conventional high-throughput screens performed in vitro with purified protein kinases are powerful tools to discover new kinase inhibitors, they are far from ideal for determining efficacy in vivo. As a complementary approach, cell-based, target-driven secondary screens may help predict in vivo compound potency and specificity as well as evaluate bioavailability and toxicity. Here the authors report a simple protocol for treating K562 Bcr-Abl-expressing cells with small-molecule kinase inhibitors in 96-well filter-bottom plates followed by in-plate cell lysis. The lysates were assayed via a solid-phase kinase assay, allowing determination of apparent IC50 for known Bcr-Abl inhibitors as well as facilitating the screening of a small kinase inhibitor library. This approach may have further applications in generating lysates for analyzing kinase activity and inhibition in other nonadherent suspension cell lines.
PMCID: PMC4471859  PMID: 20237206
cell lysis; secondary assay; Bcr-Abl; imatinib; lead identification
4.  A Multiplexed Cell-Based Assay for the Identification of Modulators of Pre-Membrane Processing as a Target against Dengue Virus 
Journal of Biomolecular Screening  2015;20(5):616-626.
The DenV pre-membrane protein (prM) is a crucial chaperone for the viral envelope protein, preventing premature fusion with vesicles during viral export. prM molecules in immature particles are cleaved by host proteases, leading to mature fusogenic virions. Blockade of prM cleavage would restrict fusion and represents a novel druggable opportunity against DenV. We have thus established a cell-based platform to monitor prM processing that relies on an engineered two-tag scaffold that travels to the cell surface through the secretory pathway. The assay discriminates between a single cell-surface tag when prM is cleaved and two tags when it is not, as detected through fluorescent-coupled antibodies by flow cytometry. The assay, miniaturized into a 96-well plate format, was multiplexed with the HIV-1 envelope boundary, also cleaved in the same pathway. A pilot screen against 1280 compounds was executed, leading to the identification of a potential active and corroborating the robustness of our assay for large-scale screening. We describe for the first time a cell-based assay that monitors DenV prM processing within the classical secretory pathway, which was exploited to identify a potential novel drug against DenV.
PMCID: PMC4438100  PMID: 25724189
dengue virus; high-throughput screen; cell-based assay; multiplexing; prM processing
5.  Development of an HTS Compatible Assay for Discovery of RORα Modulators using AlphaScreen® Technology 
Journal of biomolecular screening  2011;16(2):183-191.
The retinoid acid receptor-related orphan receptors (RORs) represent important targets for treatment of metabolic and immune disorders. Here we describe the application of AlphaScreen® technology to develop an HTS compatible assay to facilitate the discovery of RORα modulators. Using the ligand binding domain (LBD) of RORα and a peptide derived from the NR1 box of the nuclear receptor coactivator PGC-1α, a 384-well format assay was developed exhibiting high sensitivity, requiring only low nanomolar concentration of reagents. Recently it was shown that oxysterols such as 7α-hydroxycholesterol (7α-OHC) function as modulators of the RORs. In this assay 7α-OHC produced a dose-dependent response with an EC50 of 162 nM, Z’ factor of 0.6 and a S/B ratio of 4.2 demonstrating that the assay is HTS compatible. Validation of the assay was afforded by screening against the Sigma LOPAC1280™ library in 384-well format. In summary, the results presented here demonstrate that this assay can be used to screen large chemical libraries to discover novel modulators of RORα.
PMCID: PMC4434603  PMID: 21297105
nuclear receptor (NR); retinoid acid receptor-related orphan receptor (ROR); peroxisome proliferator-activated receptor γ-coactivator-1α (PGC-1α); 7α-hydroxycholesterol (7α-OHC); AlphaScreen® technology; assay performance; high-throughput screening (HTS)
6.  A High-Throughput Cell-Based Gaussia Luciferase Reporter Assay for Identifying Modulators of Fibulin-3 Secretion 
Journal of biomolecular screening  2012;18(6):647-658.
An R345W mutation in fibulin-3 causes its inefficient secretion, increased intracellular steady-state levels, and the macular dystrophy, Malattia Leventinese (ML), a disease similar to age-related macular degeneration. It is unknown whether R345W causes ML through increased intracellular levels, by the secretion of a potentially aggregation-prone protein, or both. To identify small molecules that alter the secretion of fibulin-3, we developed ARPE19 retinal cell lines that inducibly express wild-type (WT) or R345W fibulin-3 fused to an enhanced Gaussia luciferase (eGLuc2). Screening of the Library of Pharmacologically Active Compounds demonstrated that these cell lines and the GLuc assay are suitable for high-throughput chemical screening. Two estrogen-related compounds enhanced fibulin-3 secretion, whereas a diverse series of small molecules reduced fibulin-3 secretion. A counterscreen identified compounds that did not substantially alter the secretion of unfused eGLuc2, demonstrating at least partial selectivity for fibulin-3. A secondary assay using untagged fibulin-3 confirmed that the top three inhibitory compounds reduced R345W fibulin-3 secretion. Interestingly, in untagged fibulin-3 studies, one compound, phorbol 12-myristate 13-acetate, reduced R345W fibulin-3 secretion while minimally enhancing WT fibulin-3 secretion, the desired activity and selectivity we sought for ML. The identified compounds could serve as tools for probing the etiology of fibulin-3–related diseases.
PMCID: PMC4435789  PMID: 23230284
fibulin-3; Gaussia luciferase; Malattia Leventinese; high-throughput chemical screening; LOPAC; fibulin-3–dependent gliomas
7.  Discovering New Medicines Targeting Helicases: Challenges and Recent Progress 
Journal of biomolecular screening  2013;18(7):761-781.
Helicases are ubiquitous motor proteins that separate and/or rearrange nucleic acid duplexes in reactions fueled by adenosine triphosphate (ATP) hydrolysis. Helicases encoded by bacteria, viruses, and human cells are widely studied targets for new antiviral, antibiotic, and anticancer drugs. This review summarizes the biochemistry of frequently targeted helicases. These proteins include viral enzymes from herpes simplex virus, papillomaviruses, polyomaviruses, coronaviruses, the hepatitis C virus, and various flaviviruses. Bacterial targets examined include DnaB-like and RecBCD-like helicases. The human DEAD-box protein DDX3 is the cellular antiviral target discussed, and cellular anticancer drug targets discussed are the human RecQ-like helicases and eIF4A. We also review assays used for helicase inhibitor discovery and the most promising and common helicase inhibitor chemotypes, such as nucleotide analogues, polyphenyls, metal ion chelators, flavones, polycyclic aromatic polymers, coumarins, and various DNA binding pharmacophores. Also discussed are common complications encountered while searching for potent helicase inhibitors and possible solutions for these problems.
PMCID: PMC4427233  PMID: 23536547
motor protein; ATPase; RNA binding proteins; molecular probes; antivirals; antibiotic; anticancer
8.  Identification and optimization of PDE10A inhibitors using fragment-based screening by nanocalorimetry and X-ray crystallography 
Journal of biomolecular screening  2013;19(4):497-507.
Fragment-based lead discovery (FBLD) is a technique in which, small, low-complexity chemical fragments of 6 to 15 heavy atoms are screened for binding to or inhibiting activity of the target. Hits are then linked and/ or elaborated into tightly binding ligands, ideally yielding early lead compounds for drug discovery. 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 isothermal titration calorimetry. Here we use enthalpy arrays, which are arrays of nanocalorimeters, to perform an enzyme activity-based fragment screen for competitive inhibitors of phosphodiesterase 10A (PDE10A). Two dozen fragments with KI <2 mM were identified and moved to crystal soaking trials. All soak experiments yielded high resolution diffraction with two-thirds of the fragments yielding high-resolution co-crystal structures with PDE10A. The structural information was used to elaborate fragment hits, yielding leads with KI <1 µM. This study shows how array calorimetry can be used as a prescreening method for fragment-based lead discovery with enzyme targets and paired successfully with an x-ray crystallography secondary screen.
PMCID: PMC4007584  PMID: 24375910
nanocalorimetry; enzyme assay; label-free assay; fragment-based lead discovery; X-ray crystallography
9.  Identification of agents that promote endoplasmic reticulum stress using an assay that monitors luciferase secretion 
Journal of biomolecular screening  2013;19(4):575-584.
Disruption of protein processing in the secretory pathway is a measurable hallmark of endoplasmic reticulum (ER) stress. Activation of ER stress-mediated pathways has been implicated in numerous diseases including cancer. To identify agents that induce ER stress, we established a screen for compounds that reduce secretion of the reporter protein Gaussia luciferase (GLUC). Given the clinically validated importance of targeting ER stress-mediated pathways in the treatment of multiple myeloma (MM), we used this hematological malignancy as a model for validating our screening system. From a screen of 2000 marketed drugs and natural compounds in KMS11 and ARP1 MM cells, we identified 97 agents that reduced GLUC secretion in both cell lines by at least 30%. In order to confirm inducers of ER stress, we applied a secondary screen that assessed splicing of the unfolded protein response (UPR) transcription factor XBP1. One agent, theaflavin-3,3′–digallate (TF-3), was chosen based on its history of safe human consumption and further validated through studies of ER stress-related pathways including the UPR and apoptosis. Given these promising results, this screen could be a useful tool to identify agents targeting ER stress-related mechanisms in other cellular systems wherein ER stress plays a role in disease etiology.
PMCID: PMC4338999  PMID: 24371212
Gaussia luciferase; multiple myeloma; endoplasmic reticulum stress; protein secretion
10.  A High-Throughput Screen with Isogenic PTEN+/+ and PTEN−/− Cells Identifies CID1340132 as a Novel Compound That Induces Apoptosis in PTEN and PIK3CA Mutant Human Cancer Cells 
Journal of biomolecular screening  2011;16(4):383-393.
The PTEN tumor suppressor gene is one of the most commonly mutated genes in human cancer. Because inactivation of PTEN is a somatic event, PTEN mutations represent an important genetic difference between cancer cells and normal cells and therefore a potential anticancer drug target. However, it remains a substantial challenge to identify compounds that target loss-of-function events such as mutations of tumor suppressors. In an effort to identify small molecules that preferentially kill cells with mutations of PTEN, the authors developed and implemented a high-throughput, paired cell-based screen composed of parental HCT116 cells and their PTEN gene-targeted derivatives. From 138 758 compounds tested, two hits were identified, and one, N′-[(1-benzyl-1H-indol-3-yl)methylene]benzenesulfonohydrazide (CID1340132), was further studied using a variety of cell-based models, including HCT116, MCF10A, and HEC1A cells with targeted deletion of either their PTEN or PIK3CA genes. Preferential killing of PTEN and PIK3CA mutant cells was accompanied by DNA damage, inhibition of DNA synthesis, and apoptosis. taken together, these data validate a cell-based screening approach for identifying lead compounds that target cells with specific tumor suppressor gene mutations and describe a novel compound with preferential killing activity toward PTEN and PIK3CA mutant cells.
PMCID: PMC4381441  PMID: 21335596
PTEN; PIK3CA; human somatic cell gene targeting; phenotypic screen; DNA damage; high-throughput screen; synthetic lethality
11.  High-Content Analysis of Pro-Apoptotic EphA4 Dependence Receptor Functions using Small Molecule Libraries 
Journal of biomolecular screening  2012;17(6):785-795.
Small molecule compounds (SMCs) can provide an inexpensive and selective approach to modifying biological responses. High-content analysis (HCA) of SMC libraries can help identify candidate molecules that inhibit or activate cellular responses. In particular, regulation of cell death has important implications for many pathological conditions. Dependence receptors are a new classification of pro-apoptotic membrane receptors that, unlike classic death receptors, initiate apoptotic signals in the absence of their ligands. EphA4 has recently been identified as a dependence receptor that may have important functions in conditions as disparate as cancer biology and CNS injury and disease. To screen potential candidate SMCs that inhibit or activate EphA4-induced cell death, HCA of a SMC library was performed using stable EphA4-expressing NIH3T3 cells. Our results describe a high-content method for screening dependence receptor-signaling pathways, and demonstrate that several candidate SMCs can inhibit EphA4-mediated cell death.
PMCID: PMC4380140  PMID: 22492230
Ephrins; Eph receptors; Dependence Receptor; Apoptosis; High-Content Screen
12.  Identification of Potent Inhibitors of the Trypanosoma brucei Methionyl-tRNA Synthetase via High Throughput Orthogonal Screening 
Journal of biomolecular screening  2014;20(1):122-130.
Improved therapies for the treatment of Trypanosoma brucei (T. brucei), the etiological agent of the neglected tropical disease human African trypanosomiasis, are urgently needed. We targeted T. brucei methionyl-tRNA synthetase (MetRS), an aminoacyl-tRNA synthase (aaRS), which is considered an important drug target due to its role in protein synthesis, cell survival and its significant differences in structure from its mammalian ortholog. Previous work using RNA interference of MetRS demonstrated growth inhibition of T. brucei, further validating it as an attractive target. We report the development and implementation of two orthogonal high throughput screening assays to identify inhibitors of T. brucei MetRS. First, a chemiluminescence assay was implemented in 1536-well plate format and used to monitor ATP depletion during the aminoacylation reaction. Hit confirmation then used a counterscreen in which AMP production was assessed using fluorescence polarization technology. In addition, a miniaturized cell viability assay was used to triage cytotoxic compounds. Finally, lower throughput assays involving whole parasite growth inhibition of both human and parasite MetRS were used to analyze compound selectivity and efficacy. The outcome of this HTS campaign has led to the discovery of nineteen potent and selective T. brucei MetRS inhibitors.
PMCID: PMC4378865  PMID: 25163684
aminoacyl-tRNA synthetases; high-throughput screening; human African trypanosomiasis; orthogonal screening; Trypanosoma brucei
13.  A New Experimental Model for Assessing Drug Efficacy against Trypanosoma cruzi Infection Based on Highly Sensitive In Vivo Imaging 
The protozoan Trypanosoma cruzi is the causative agent of Chagas disease, one of the world’s major neglected infections. Although development of improved antiparasitic drugs is considered a priority, there have been no significant treatment advances in the past 40 years. Factors that have limited progress include an incomplete understanding of pathogenesis, tissue tropism, and disease progression. In addition, in vivo models, which allow parasite burdens to be tracked throughout the chronic stage of infection, have been lacking. To address these issues, we have developed a highly sensitive in vivo imaging system based on bioluminescent T. cruzi, which express a red-shifted luciferase that emits light in the tissue-penetrating orange-red region of the spectrum. The exquisite sensitivity of this noninvasive murine model has been exploited to monitor parasite burden in real time throughout the chronic stage, has allowed the identification of the gastrointestinal tract as the major niche of long-term infection, and has demonstrated that chagasic heart disease can develop in the absence of locally persistent parasites. Here, we review the parameters of the imaging system and describe how this experimental model can be incorporated into drug development programs as a valuable tool for assessing efficacy against both acute and chronic T. cruzi infections.
PMCID: PMC4361455  PMID: 25296657
Chagas disease; trypanosomes; drugs; imaging
14.  A New System for Profiling Drug-Induced Calcium Signal Perturbation in Human Embryonic Stem Cell–Derived Cardiomyocytes 
Journal of Biomolecular Screening  2015;20(3):330-340.
The emergence of human stem cell–derived cardiomyocyte (hSCCM)–based assays in the cardiovascular (CV) drug discovery sphere requires the development of improved systems for interrogating the rich information that these cell models have the potential to yield. We developed a new analytical framework termed SALVO (synchronization, amplitude, length, and variability of oscillation) to profile the amplitude and temporal patterning of intra- and intercellular calcium signals in hSCCM. SALVO quantified drug-induced perturbations in the calcium signaling “fingerprint” in spontaneously contractile hSCCM. Multiparametric SALVO outputs were integrated into a single index of in vitro cytotoxicity that confirmed the rank order of perturbation as astemizole > thioridazine > cisapride > flecainide > valdecoxib > sotalol > nadolol ≈ control. This rank order of drug-induced Ca2+ signal disruption is in close agreement with the known arrhythmogenic liabilities of these compounds in humans. Validation of the system using a second set of compounds and hierarchical cluster analysis demonstrated the utility of SALVO to discriminate drugs based on their mechanisms of action. We discuss the utility of this new mechanistically agnostic system for the evaluation of in vitro drug cytotoxicity in hSCCM syncytia and the potential placement of SALVO in the early stage drug screening framework.
PMCID: PMC4361473  PMID: 25367900
calcium signaling; cell imaging; human; cardiac; stem cells; drug discovery
15.  Quantification of Histone H3 Lys27 Trimethylation (H3K27me3) by High-Throughput Microscopy Enables Cellular Large-Scale Screening for Small-Molecule EZH2 Inhibitors 
Journal of Biomolecular Screening  2015;20(2):190-201.
EZH2 inhibition can decrease global histone H3 lysine 27 trimethylation (H3K27me3) and thereby reactivates silenced tumor suppressor genes. Inhibition of EZH2 is regarded as an option for therapeutic cancer intervention. To identify novel small-molecule (SMOL) inhibitors of EZH2 in drug discovery, trustworthy cellular assays amenable for phenotypic high-throughput screening (HTS) are crucial. We describe a reliable approach that quantifies changes in global levels of histone modification marks using high-content analysis (HCA). The approach was validated in different cell lines by using small interfering RNA and SMOL inhibitors. By automation and miniaturization from a 384-well to 1536-well plate, we demonstrated its utility in conducting phenotypic HTS campaigns and assessing structure-activity relationships (SAR). This assay enables screening of SMOL EZH2 inhibitors and can advance the mechanistic understanding of H3K27me3 suppression, which is crucial with regard to epigenetic therapy. We observed that a decrease in global H3K27me3, induced by EZH2 inhibition, comprises two distinct mechanisms: (1) inhibition of de novo DNA methylation and (II) inhibition of dynamic, replication-independent H3K27me3 turnover. This report describes an HCA assay for primary HTS to identify, profile, and optimize cellular active SMOL inhibitors targeting histone methyltransferases, which could benefit epigenetic drug discovery.
PMCID: PMC4361481  PMID: 25409661
high-content analysis; EZH2; KMT6; histone methyltransferase; chromatin modulators
16.  Using Weighted Entropy to Rank Chemicals in Quantitative High Throughput Screening Experiments 
Journal of biomolecular screening  2013;19(3):344-353.
Quantitative high throughput screening (qHTS) experiments can simultaneously produce concentration-response profiles for thousands of chemicals. In a typical qHTS study, a large chemical library is subjected to a primary screen in order to identify candidate hits for secondary screening, validation studies or prediction modeling. Different algorithms, usually based on the Hill equation logistic model, have been used to classify compounds as active or inactive (or inconclusive). However, observed concentration-response activity relationships may not adequately fit a sigmoidal curve. Furthermore, it is unclear how to prioritize chemicals for follow-up studies given the large uncertainties that often accompany parameter estimates from nonlinear models. Weighted Shannon entropy can address these concerns by ranking compounds according to profile-specific statistics derived from estimates of the probability mass distribution of response at the tested concentration levels. This strategy can be used to rank all tested chemicals in the absence of a pre-specified model structure or the approach can complement existing activity call algorithms by ranking the returned candidate hits. The weighted entropy approach was evaluated here using data simulated from the Hill equation model. The procedure was then applied to a chemical genomics profiling data set interrogating compounds for androgen receptor agonist activity.
PMCID: PMC4029130  PMID: 24056003
quantitative high throughput screening; information theory; concentration response; Tox21
17.  Significance of Lipid Composition in a Blood Brain Barrier-Mimetic PAMPA Assay 
Journal of biomolecular screening  2013;19(3):437-444.
Endothelial cells forming the blood-brain barrier limit drug access into the brain, due to tight junctions, membrane drug transporters, and unique lipid composition. Passive permeability, thought to mediate drug access, is typically tested using porcine whole brain lipid. However human endothelial cell lipid composition differs. This investigation evaluated the influence of lipid composition on passive permeability across artificial membranes. Permeability of CNS-active drugs across an immobilized lipid membrane was determined using three lipid models: crude extract from whole pig brain, human brain microvessel lipid, and microvessel lipid plus cholesterol. Lipids were immobilized on polyvinylidene difluoride, forming donor and receiver chambers, in which drug concentration were measured after 2 hr. The log of effective permeability was then calculated using the measured concentrations. Permeability of small, neutral compounds was unaffected by lipid composition. Several structurally diverse drugs were highly permeable in porcine whole brain lipid but 1–2 orders of magnitude less permeable across human brain endothelial cell lipid. Inclusion of cholesterol had the greatest influence on bulky amphipathic compounds such as glucuronide conjugates. Lipid composition markedly influences passive permeability. This was most apparent for charged or bulky compounds. These results demonstrate the importance of using species-specific lipid models in passive permeability assays.
PMCID: PMC4078735  PMID: 23945876
PAMPA; lipid composition; passive permeability; blood brain barrier; central nervous system
18.  [No title available] 
PMCID: PMC4013825  PMID: 24436077
19.  A screening based approach to circumvent tumor microenvironment-driven intrinsic resistance to BCR-ABL+ inhibitors in Ph+ acute lymphoblastic leukemia 
Journal of biomolecular screening  2013;19(1):158-167.
Signaling by the BCR-ABL fusion kinase drives Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ ALL) and chronic myelogenous leukemia (CML). Despite their clinical activity in many patients with CML, the BCR-ABL kinase inhibitors (BCR-ABL-KIs) imatinib, dasatinib, and nilotinib provide only transient leukemia reduction in patients in Ph+ ALL. While host-derived growth factors present in the leukemia microenvironment have been invoked to explain this drug resistance, their relative contribution remains uncertain. Using genetically-defined murine Ph+ ALL cells, we identified Interleukin 7 (IL-7) as the dominant host-factor that attenuates response to BCR-ABL-KIs. To identify potential combination drugs that could overcome this IL-7-dependent BCR-ABL-KI-resistant phenotype, we screened a small molecule library including FDA-approved drugs. Among the validated hits, the well-tolerated anti-malarial drug dihydroartemisinin (DHA) displayed potent activity in vitro and modest in vivo monotherapy activity against engineered murine BCR-ABL-KI–resistant Ph+ ALL. Strikingly, co-treatment with DHA and dasatinib in vivo strongly reduced primary leukemia burden and improved long-term survival in a murine model that faithfully captures the BCR-ABL-KI-resistant phenotype of human Ph+ ALL. This co-treatment protocol durably cured 90% of treated animals, suggesting that this cell-based screening approach efficiently identified drugs that could be rapidly moved to human clinical testing
PMCID: PMC3963394  PMID: 23989453
20.  High-Throughput Screening Normalized To Biological Response: Application To Antiviral Drug Discovery 
Journal of biomolecular screening  2013;19(1):10.1177/1087057113496848.
The process of conducting cell-based phenotypic screens can result in datasets from small libraries or portions of large libraries, making accurate hit picking from multiple datasets important for efficient drug discovery. Here, we describe a screen design and data analysis approach that allows for normalization not only between quadrants and plates but also between screens or batches in a robust, quantitative fashion enabling hit-selection from multiple datasets. We independently screened the Microsource Spectrum and NCI Diversity Set II libraries using a cell-based phenotypic HTS assay that uses interferon stimulated response element (ISRE)-driven luciferase-reporter assay to identify interferon (IFN) signal enhancers. Inclusion of a per-plate, per-quadrant IFN dose-response standard curve enabled conversion of ISRE activity to effective IFN concentrations. We identified 45 hits based on a combined z-score ≥ 2.5 from the two libraries, and 25 of 35 available hits were validated in a compound concentration-response assay when tested using fresh compound. The results provide a basis for further analysis of chemical structure in relation to biological function. Together, the results establish an HTS method that can be extended to screening for any class of compounds that influence a quantifiable biological response for which a standard is available.
PMCID: PMC3867592  PMID: 23860224
Phenotypic drug discovery; cell-based assay; Quantitative HTS; Interferon signal enhancer; Statin
21.  Identification of Inhibitors of Triacylglyceride Accumulation in Muscle Cells: Comparing HTS Results from 1536-well Plate-Based and High-Content Platforms 
Excess caloric consumption leads to triacylglyceride (TAG) accumulation in tissues that do not typically store fat, such as skeletal muscle. This ectopic accumulation alters cells, contributing to the pathogenesis of metabolic syndrome, a major health problem worldwide. We developed a 1536-well assay to measure intracellular TAG accumulation in differentiating H9c2 myoblasts. For this assay, cells were incubated with oleic acid to stimulate TAG accumulation prior to adding compounds. We used Nile red as a fluorescent dye to quantify TAG content with a microplate-reader. The cell nuclei were counterstained with DAPI nuclear stain to assess cell count and filter cytotoxic compounds. In parallel, we developed an image-based assay in H9c2 cells to measure lipid accumulation levels via high-content analysis, exploiting the dual emission spectra characteristic of Nile red staining of neutral and phospholipids. Using both approaches, we successfully screened ~227,000 compounds from the NIH Library. The screening data from the plate-reader and IC50 values correlated with that from the Opera QEHS cell imager. The 1536-well plate-reader assay is a powerful HTS platform to identify potent inhibitors of TAG accumulation to better understand the molecular pathways involved in lipid metabolism that lead to lipotoxicity.
PMCID: PMC3901053  PMID: 23989452
H9c2 cardiomyocytes; human primary myocytes; lipid accumulation; Nile red fluorescence; 1536-well High throughput screening; High content analysis; phenotypic screening
22.  Development of high-content assays for kidney progenitor cell expansion in transgenic zebrafish 
Journal of biomolecular screening  2013;18(10):10.1177/1087057113495296.
Reactivation of genes normally expressed during organogenesis is a characteristic of kidney regeneration. Enhancing this reactivation could potentially be a therapeutic target to augment kidney regeneration. The inductive events that drive kidney organogenesis in zebrafish are similar to the initial steps in mammalian kidney organogenesis. Therefore, quantifying embryonic signals that drive zebrafish kidney development is an attractive strategy for the discovery of potential novel therapeutic modalities that accelerate kidney regeneration. The Lim1 homeobox protein, Lhx1, is a marker of kidney development that is also expressed in the regenerating kidneys after injury. Utilizing a fluorescent Lhx1a-EGFP transgene whose phenotype faithfully recapitulates that of the endogenous protein we developed a high-content assay for Lhx1a-EGFP expression in transgenic zebrafish embryos employing an artificial intelligence-based image analysis method termed Cognition Network Technology (CNT). Implementation of the CNT assay on high-content readers enabled automated real-time in vivo time-course, dose-response, and variability studies in the developing embryo. The Lhx1a assay was complemented with a kidney-specific secondary CNT assay that enables direct measurements of the embryonic renal tubule cell population. The integration of fluorescent transgenic zebrafish embryos with automated imaging and artificial intelligence-based image analysis provides an in vivo analysis system for structure-activity relationship studies and de novo discovery of novel agents that augment innate regenerative processes.
PMCID: PMC3830658  PMID: 23832868
23.  A multiplexed fluorescent assay for independent second messenger systems: Decoding GPCR activation in living cells 
Journal of biomolecular screening  2013;18(7):797-806.
There is a growing need in drug discovery and basic research to measure multiple second messenger components of cell signaling pathways in real time and in relevant tissues and cell types. Many G-protein coupled receptors activate the heterotrimeric protein, Gq, which in turn activates phospholipase C (PLC; Figure 1). PLC cleaves Phosphatidylinositol 4,5-bisphosphate (PIP2) to produce two second messengers: diacylglycerol (DAG), which remains in the plasma membrane, and inositol triphosphate (IP3), which diffuses through the cytosol to release stores of intracellular calcium ions (Ca2+). Our goal was to create a series of multiplex sensors that would make it possible to simultaneously measure two different components of the Gq pathway in living cells. Here we describe new fluorescent sensors for DAG and PIP2 that produce robust changes in green or red fluorescence, and can be combined with one another, or with existing Ca2+ sensors, in a live cell assay. These assays can detect multiple components of Gq signaling, simultaneously in real time, on standard fluorescent plate readers or live cell imaging systems.
PMCID: PMC4242713  PMID: 23580666
24.  Comparison of methods for image-based profiling of cellular morphological responses to small-molecule treatment 
Journal of biomolecular screening  2013;18(10):10.1177/1087057113503553.
Quantitative microscopy has proven a versatile and powerful phenotypic screening technique. Recently, image-based profiling has shown promise as a means for broadly characterizing molecules’ effects on cells in several drug-discovery applications, including target-agnostic screening and predicting a compound’s mechanism of action (MOA). Several profiling methods have been proposed, but little is known about their comparative performance, impeding the wider adoption and further development of image-based profiling. We compared these methods by applying them to a widely applicable assay of cultured cells and measuring the ability of each method to predict the MOA of a compendium of drugs. A very simple method that is based on population means performed as well as methods designed to take advantage of the measurements of individual cells. This is surprising because many treatments induced a heterogeneous phenotypic response across the cell population in each sample. Another simple method, which performs factor analysis on the cellular measurements before averaging them, provided substantial improvement and was able to predict MOA correctly for 94% of the treatments in our ground-truth set. To facilitate the ready application and future development of image-based phenotypic profiling methods, we provide our complete ground-truth and test datasets, as well as open-source implementations of the various methods in a common software framework.
PMCID: PMC3884769  PMID: 24045582
phenotypic screening; high-content screening; image-based screening; drug profiling
25.  Rapid and specific measurements of superoxide using fluorescence spectroscopy 
Journal of biomolecular screening  2012;18(4):498-503.
Superoxide plays a key role in many pathological processes; however, detection of superoxide by one of the most common methods using dihydroethidium may be unspecific due to overlapping fluorescence of the superoxide specific product, 2-OH-ethidium (2OH-E), and the unspecific oxidation product, ethidium. Here, we show new optimized fluorescence spectroscopy protocol that allows rapid and specific detection of superoxide in cell free systems and intact cells using dihydroethydium (DHE). We defined new optimized fluorescent settings to measure superoxide specific product and minimize interference of unspecific DHE oxidation products. Using this protocol we studied real time superoxide production by xanthine oxidase and menadione-treated cultured cells. Specificity of the plate reader-based superoxide measurements was confirmed by the inhibition of fluorescence with superoxide dismutase and HPLC analysis. We show that limitations of the HPLC-based analysis can be overcome by the optimized fluorescence spectroscopy.
PMCID: PMC4210376  PMID: 23190737
Superoxide; reactive oxygen species; dihydroethidium; hydroethidine; fluorescence; spectroscopy

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