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1.  [No title available] 
PMCID: PMC4013825  PMID: 24436077
2.  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
doi:10.1177/1087057113501081
PMCID: PMC3963394  PMID: 23989453
3.  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.
doi:10.1177/1087057113496848
PMCID: PMC3867592  PMID: 23860224
Phenotypic drug discovery; cell-based assay; Quantitative HTS; Interferon signal enhancer; Statin
4.  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.
doi:10.1177/1087057113501198
PMCID: PMC3901053  PMID: 23989452
H9c2 cardiomyocytes; human primary myocytes; lipid accumulation; Nile red fluorescence; 1536-well High throughput screening; High content analysis; phenotypic screening
5.  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.
doi:10.1177/1087057113495296
PMCID: PMC3830658  PMID: 23832868
6.  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.
doi:10.1177/1087057113485427
PMCID: PMC4242713  PMID: 23580666
7.  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.
doi:10.1177/1087057113503553
PMCID: PMC3884769  PMID: 24045582
phenotypic screening; high-content screening; image-based screening; drug profiling
8.  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.
doi:10.1177/1087057112468765
PMCID: PMC4210376  PMID: 23190737
Superoxide; reactive oxygen species; dihydroethidium; hydroethidine; fluorescence; spectroscopy
9.  Differential determinants of cancer cell insensitivity to anti-mitotic drugs discriminated by a one-step cell imaging assay 
Journal of biomolecular screening  2013;18(9):1062-1071.
Cancer cells can be drug resistant due to genetic variation at multiple steps in the drug response pathway, including drug efflux pumping, target mutation and blunted apoptotic response. These are not discriminated by conventional cell survival assays. Here, we report a rapid and convenient high content cell-imaging assay that measures multiple physiological changes in cells responding to anti-mitotic small-molecule drugs. Our one-step, no-wash assay uses three dyes to stain living cells and is much more accurate for scoring weakly adherent mitotic and apoptotic cells than conventional antibody-based assays. We profiled responses of 33 cell lines to 8 anti-mitotic drugs at multiple concentrations and time points using this assay, and deposited our data and assay protocols into a public database (http://lincs.hms.harvard.edu/). Our data discriminated between alternative mechanisms that compromise drug sensitivity to Paclitaxel, and revealed an unexpected bell-shaped dose-response curve for BI2536, a highly selective inhibitor of Polo-like kinases. Our approach can be generalized, is scalable and should therefore facilitate identification of molecular biomarkers for mechanisms of drug insensitivity in high-throughput screens and other assays.
doi:10.1177/1087057113493804
PMCID: PMC3783590  PMID: 23788527
High-content screening; live cell imaging assay; image analysis; cancer cells; drug sensitivity; anti-mitotic drugs
10.  A high-content screening assay for small molecule modulators of oncogene-induced senescence 
Journal of biomolecular screening  2013;18(9):10.1177/1087057113491827.
Cellular senescence is a state of stable cell growth arrest. Activation of oncogenes such as RAS in mammalian cells typically triggers cellular senescence. Oncogene-induced senescence (OIS) is an important tumor suppression mechanism, and suppression of OIS contributes to cell transformation. Oncogenes trigger senescence through a multitude of incompletely understood downstream signaling events that frequently involve protein kinases. To identify target proteins required for RAS-induced senescence, we developed a small molecule screen in primary human fibroblasts undergoing senescence induced by oncogenic RAS (H-RasG12V). Using a high-content imaging system to monitor two hallmarks of senescence, senescence-associated β-galactosidase activity expression and inhibition of proliferation, we screened a library of known small molecule kinase inhibitors for those that suppressed OIS. Identified compounds were subsequently validated and confirmed using a third marker of senescence, senescence-associated heterochromatin foci. In summary, we have established a novel high-content screening platform that may be useful for elucidating signaling pathways mediating OIS by targeting critical pathway components.
doi:10.1177/1087057113491827
PMCID: PMC3870888  PMID: 23733845
11.  A high throughput splicing assay identifies new classes of inhibitors of human and yeast spliceosomes 
Journal of biomolecular screening  2013;18(9):1110-1120.
The spliceosome is the macromolecular machine responsible for pre-mRNA splicing, an essential step in eukaryotic gene expression. During splicing a myriad of subunits join and leave the spliceosome as it works on the pre-mRNA substrate. Strikingly, there are very few small molecules known to interact with the spliceosome. Splicing inhibitors are needed to capture transient spliceosome conformations and probe important functional components. Such compounds may also have chemotherapeutic applications, as links between splicing and cancer are increasingly uncovered. To identify new splicing inhibitors, we developed a high throughput assay for in vitro splicing using an RT-qPCR readout. In a pilot screen of 3,080 compounds we identified three small molecules that inhibit splicing in HeLa extract by interfering with different stages of human spliceosome assembly. Two of the compounds similarly impact spliceosomes in yeast extracts, suggesting selective targeting of conserved components. By examining related molecules, we identified chemical features required for the activity of two of the splicing inhibitors. In addition to verifying our assay procedure and paving the way to larger screens, these studies establish new compounds as chemical probes for investigating the splicing machinery.
doi:10.1177/1087057113493117
PMCID: PMC3902173  PMID: 23771823
pre-mRNA splicing; spliceosome; RT-qPCR; inhibitor; high-throughput assay
12.  Plasmid-based shRNA lentiviral particle production for RNAi applications 
Journal of biomolecular screening  2014;19(9):1309-1313.
Lentiviral vectors have become mainstream gene transfer vehicles for their ability to delivery and integrate into host cells. In RNA interference (RNAi) applications, lentiviral constructs constitutively express dsRNA molecules usually as short hairpin RNA (shRNA) enabling long-term gene silencing and when pseudotyped with a broad host glycoprotein envelope; allows a multitude of cell types to be transduced. Their successful use ultimately relies on the production of lentiviral particles in high-titer and uniformity. Typical methods require the transfection of three or more plasmids in which essential viral elements have been encoded separated so as to remain replication deficient. These transfection procedures are of critical importance; however, methods often vary among laboratories making it difficult to assess the overall efficiency of lentiviral particle production. In this report, we focused exclusively on this step and compared the overall impact of the commercial transfection reagent FuGENE 6 to FuGENE HD. We found that FuGENE HD resulted in at least 5-fold improvement in viral particle titer as assessed by the p24 standard ELISA assay. We present the complete optimized workflow and demonstrate this utility in which a single modification of this transfection step improved the lentiviral particle production.
doi:10.1177/1087057114539390
PMCID: PMC4170048  PMID: 24939963
shRNA; FuGENE 6; FuGENE HD; RNAi; lentiviral particles; viral titer; plasmid; DNA
13.  TR-FRET-Based High-Throughput Screening Assay for Identification of UBC13 Inhibitors 
Journal of biomolecular screening  2011;17(2):163-176.
UBC13 is a non-canonical Ubiquitin Conjugating Enzyme (E2) that has been implicated in a variety of cellular signaling processes due to its ability to catalyze formation of Lysine 63-linked polyubiquitin chains on various substrates. In particular, UBC13 is required for signaling by a variety of receptors important in immune regulation, making it a candidate target for inflammatory diseases. UBC13 is also critical for double-strand DNA repair, and thus a potential radiosensitizer and chemosensitizer target for oncology. We developed a high-throughput screening (HTS) assay for UBC13 based on the method of time-resolved fluorescence resonance energy transfer (TR-FRET). The TR-FRET assay combines fluorochrome (Fl)-conjugated ubiquitin (fluorescence acceptor) with terbium (Tb)-conjugated ubiquitin (fluorescence donor), such that the assembly of mixed chains of Fl- and Tb-ubiquitin creates a robust TR-FRET signal. We defined conditions for optimized performance of the TR-FRET assay in both 384 and 1536-well formats. Chemical library screens (total 456,865 compounds) were conducted in high-throughput mode using various compound collections, affording superb Z' scores (typically > 0.7) and thus validating the performance of the assays. Altogether, the HTS assays described here are suitable for large-scale, automated screening of chemical libraries in search of compounds with inhibitory activity against UBC13.
doi:10.1177/1087057111423417
PMCID: PMC4172584  PMID: 22034497
14.  A High Content Screening (HCS) Assay for the Identification of Chemical Inducers of PML Oncogenic Domains (PODs) 
Journal of biomolecular screening  2011;16(2):251-258.
PML is a tumor suppressor that promotes apoptosis through both p53-dependent and - independent mechanisms, participates in Rb-mediated cell cycle arrest, inhibits neoangiogenesis, and contributes to maintenance of genomic stability. PML also plays a role in host defense against viruses, conferring antiviral activity. When active, PML localizes to subnuclear structures named PML oncogenic domains (PODs) or PML nuclear bodies (PML-NBs), whereas inactive PML is located diffusely throughout the nucleus of cells, thus providing a morphological indicator. Known activators of PML include arsenicals and interferons, however, these agents induce a plethora of toxic effects, limiting their effectiveness. The objective of the current study was to develop a high content screening (HCS) assay for the identification of chemical activators of PML. We describe methods for automated analysis of POD formation using high throughput microscopy (HTM) to localize PML immunofluorescence in conjunction with image analysis software for POD quantification. Using this HCS assay in 384 well format, we performed pilot screens of a small synthetic chemical library and mixture-based combinatorial libraries, demonstrating the robust performance of the assay. HCS counter-screening assays were also developed for hit characterization, based on immunofluorescence analyses of the subcellular location of phosphorylated H2AX or phosphorylated CHK1, which increase in a punctate nuclear pattern in response to DNA damage. Thus, the HCS assay devised here represents a high throughput screen that can be utilized to discover POD-inducing compounds that may restore the tumor suppressor activity of PML in cancers or possibly promote anti-viral states.
doi:10.1177/1087057110394181
PMCID: PMC4172586  PMID: 21233309
PML; POD; nuclear bodies; apoptosis; high content screening
15.  High-Throughput Screening AlphaScreen Assay for Identification of Small-Molecule Inhibitors of Ubiquitin E3 Ligase SCFSkp2-Cks1 
Journal of biomolecular screening  2013;18(8):910-920.
Decreased levels of cell cycle inhibitor p27Kip1 due to excessive degradation occur in a variety of aggressive human tumors. Since reduced p27Kip1 expression has been associated with a poor prognosis in many human cancers and resistance to certain antitumor therapies, elevation of p27Kip1 expression could improve prognosis and prevent excessive cell proliferation. SCFSkp2 is one of the major ubiquitin E3 ligases responsible for degradation of p27Kip1. Ubiquitination of p27Kip1 also requires a small adaptor protein, Cks1, which facilitates substrate recruitment by bridging the interaction between Skp2 and p27Kip1. It has been shown previously that a direct interaction between Cks1 and Skp2 is required for p27Kip1 degradation. Accordingly, perturbation of the Skp2-Cks1 interaction may represent an attractive target for pharmacological intervention. Here we describe a high-throughput AlphaScreen assay for discovering small-molecule inhibitors of the Skp2-Cks1 protein-protein interaction in vitro. Two compounds (NSC689857 and NSC681152) were identified and validated through a structure-activity relationship analysis. Both compounds were also shown to inhibit p27Kip1 ubiquitination in vitro. These studies demonstrate that disruption of the Skp2-Cks1 interaction provides a viable strategy to prevent p27Kip1 ubiquitination and may potentially be useful for the control of excessive degradation of this cell cycle inhibitor in tumor cells.
doi:10.1177/1087057113485789
PMCID: PMC4168015  PMID: 23589337
E3 ligase; inhibitor; Skp2; Cks1; p27kip1; ubiquitin; proteolysis
16.  A High-Content Assay to Identify Small-Molecule Modulators of a Cancer Stem Cell Population in Luminal Breast Cancer 
Journal of biomolecular screening  2012;17(9):1211-1220.
Breast cancers expressing hormone receptors for estrogen (ER) and progesterone (PR) represent ~70% of all cases and are treated with both ER-targeted and chemotherapies, with near 40% becoming resistant. We have previously described that in some ER+ tumors, the resistant cells express cytokeratin 5 (CK5), a putative marker of breast stem and progenitor cells. CK5+ cells have lost expression of ER and PR, express the tumor-initiating cell surface marker CD44, and are relatively quiescent. In addition, progestins, which increase breast cancer incidence, expand the CK5+ subpopulation in ER+PR+ breast cancer cell lines. We have developed models to induce and quantitate CK5+ER−PR− cells, using CK5 promoter-driven luciferase (Fluc) or green fluorescent protein (GFP) reporters stably transduced into T47D breast cancer cells (CK5Pro-GFP or CK5Pro-Luc). We validated the CK5Pro-GFP-T47D model for high-content screening in 96-well microplates and performed a pilot screen using a focused library of 280 compounds from the National Institutes of Health clinical collection. Four hits were obtained that significantly abrogated the progestin-induced CK5+ cell population, three of which were members of the retinoid family. Hence, this approach will be useful in discovering small molecules that could potentially be developed as combination therapies, preventing the acquisition of a drug-resistant subpopulation.
doi:10.1177/1087057112452138
PMCID: PMC4158006  PMID: 22751729
stem cells; cancer and cancer drugs; high-content screening; nuclear hormone regulation; cytokeratin
17.  Identification of Small-Molecule Frequent Hitters from AlphaScreen High-Throughput Screens 
Journal of Biomolecular Screening  2014;19(5):715-726.
Although small-molecule drug discovery efforts have focused largely on enzyme, receptor, and ion-channel targets, there has been an increase in such activities to search for protein-protein interaction (PPI) disruptors by applying high-throughout screening (HTS)–compatible protein-binding assays. However, a disadvantage of these assays is that many primary hits are frequent hitters regardless of the PPI being investigated. We have used the AlphaScreen technology to screen four different robust PPI assays each against 25,000 compounds. These activities led to the identification of 137 compounds that demonstrated repeated activity in all PPI assays. These compounds were subsequently evaluated in two AlphaScreen counter assays, leading to classification of compounds that either interfered with the AlphaScreen chemistry (60 compounds) or prevented the binding of the protein His-tag moiety to nickel chelate (Ni2+-NTA) beads of the AlphaScreen detection system (77 compounds). To further triage the 137 frequent hitters, we subsequently confirmed by a time-resolved fluorescence resonance energy transfer assay that most of these compounds were only frequent hitters in AlphaScreen assays. A chemoinformatics analysis of the apparent hits provided details of the compounds that can be flagged as frequent hitters of the AlphaScreen technology, and these data have broad applicability for users of these detection technologies.
doi:10.1177/1087057113516861
PMCID: PMC4153540  PMID: 24371213
AlphaScreen; protein-protein interaction; assay development; frequent hitter; drug discovery; high-throughput screening
18.  A time-table organizer for the planning and implementation of screenings in manual or semi-automation mode 
Journal of biomolecular screening  2013;18(8):938-942.
We have designed a software to facilitate the planning and execution of screenings performed manually or in semi-automation mode, which follow a sequential sequence of events. Many assays involve multiple steps, often including time-sensitive stages, thus complicating the proper implementation to ensure that all plates are treated equally in order to achieve reliable outcomes. The Excel Macro-Enabled Workbook presented in this study analyzes and breaks down the timings for all tasks, calculates the maximum number of plates that suit the desired parameters, and allows for optimization based on tolerance of time delay and equal treatment of plates when possible. The generated Gantt charts allow for visual inspection of the screening process, and provide timings in tabulated form to assist the user to conduct the experiments as projected by the software. The program can be downloaded from http://sourceforge.net/projects/sams-hts/.
doi:10.1177/1087057113487556
PMCID: PMC3740013  PMID: 23653394
Gantt chart; high throughput screening; time optimization; manual screening
19.  High Throughput Screen for Pharmacoperones of the Vasopressin Type 2 Receptor 
Journal of biomolecular screening  2013;18(8):930-937.
Pharmacoperone drugs correct the folding of misfolded protein mutants and restore function (i.e. “rescue”) by correcting the routing of (otherwise) misrouted mutants. Assays for pharmacoperones have not been applied to screen large libraries previously. Currently most pharmacoperones possess intrinsic agonist or antagonist activities since these were identified using high throughput screens aimed at discovering direct agonists or antagonists. Here we describe an ultra-high throughput compatible no-wash assay system designed to specifically identify pharmacoperones of the vasopressin type 2 (V2) receptor (V2R). Development of such assays is important and novel since useful chemical structures with the ability to control cellular trafficking, but lacking intrinsic agonist or antagonist properties have not likely been identified using existing screens. In the described assay, the level of functional hV2R (mutant) present in each test well is quantitated by stimulation with saturating levels of agonist followed by use of a luminescent-based cyclic adenosine monophosphate (cAMP) assay. This allows the assay to identify compounds which increase the trafficking of mutant hV2R[L83Q] in our model system.
doi:10.1177/1087057113483559
PMCID: PMC3735853  PMID: 23640875
vasopressin type 2 receptor (V2R); pharmacoperone; chemical library; GPCRs; protein
20.  Evaluation of a Luciferase-based Reporter Assay as a Screen for Inhibitors of Estrogen-ERα-induced Proliferation of Breast Cancer Cells 
Journal of biomolecular screening  2012;17(7):921-932.
Estrogens, acting through estrogen receptor α (ERα), stimulate breast cancer proliferation, making ERα an attractive drug target. Since 384-well format screens for inhibitors of proliferation can be challenging for some cells, inhibition of luciferase-based reporters is often used as a surrogate end-point. To identify novel small molecules inhibitors of 17β-estradiol (E2)-ERα-stimulated cell proliferation, we established a cell-based screen for inhibitors of E2-ERα induction of an estrogen response element ((ERE)3-luciferase) reporter. 75 “hits” were evaluated in tiered follow-up assays to identify where hits failed to progress and evaluate their effectiveness as inhibitors of E2-ERα induced proliferation of breast cancer cells. Only 8 of 75 hits from the luciferase screen inhibited estrogen-induced proliferation of ERα positive MCF-7 and T47D cells, but not control ERα negative MDA-MB-231 cells. While 12% of compounds inhibited E2-ERα-stimulated proliferation in only one of the ERα positive cell lines, 40% of compounds were toxic and inhibited growth of all the cell lines, and ~37% exhibited little or no ability to inhibit E2-ERα-stimulated cell proliferation. Representative compounds were evaluated in more detail and a lead ERα inhibitor was identified.
doi:10.1177/1087057112442960
PMCID: PMC4132978  PMID: 22498909
Cell-based assays; Gene Expression; Reporter gene assays; Cancer and cancer drugs; Endocrine Diseases; Transcription factors
21.  A high-throughput screen of the GTPase activity of Escherichia coli EngA to find an inhibitor of bacterial ribosome biogenesis 
Journal of biomolecular screening  2013;18(7):830-836.
The synthesis of ribosomes is an essential process, which is aided by a variety of transacting factors in bacteria. Among these is a group of GTPases essential for bacterial viability and emerging as promising targets for new antibacterial agents. Herein, we describe a robust high-throughput screening process for inhibitors of one such GTPase, the Escherichia coli EngA protein. The primary screen employed an assay of phosphate production in 384-well density. Reaction conditions were chosen to maximize sensitivity for the discovery of competitive inhibitors while maintaining a strong signal amplitude and low noise. In a pilot screen of 31,800 chemical compounds, 44 active compounds were identified. Further, we describe the elimination of non-specific inhibitors that were detergent-sensitive or reactive as well as those that interfered with the high-throughput phosphate assay. Four inhibitors survived these common counter-screens for non-specificity but these chemicals were also inhibitors of the unrelated enzyme dihydrofolate reductase, suggesting that they too were promiscuously active. The high-throughput screen of the EngA protein described here provides a meticulous pilot study in the search for specific inhibitors of GTPases involved in ribosome biogenesis.
doi:10.1177/1087057113486001
PMCID: PMC4061131  PMID: 23606650 CAMSID: cams4460
EngA; GTPase; ribosome biogenesis; enzyme screen
22.  A High Throughput Screening Assay for Fungicidal Compounds against 
Journal of biomolecular screening  2013;19(2):270-277.
Cryptococcus neoformans is a pathogenic fungus that causes meningitis world-wide, particularly in HIV-infected individuals. Although amphotericin B is the “gold standard” treatment for cryptococcal meningitis, the toxicity and inconvenience of intravenous injection emphasizes a need for development of new anti-cryptocccal drugs. Recent data from humans and animal studies suggested that a nutrient-deprived host environment may exist in cryptococcal meningitis. Thus, a screening assay for identifying fungicidal compounds under nutrient-deprived conditions may provide an alternative strategy to develop new anti-cryptococcal drugs for this disease. A high throughput fungicidal assay was developed using a profluorescent dye, alamarBlue, to detect residual metabolic activity of C. neoformans under nutrient-limiting conditions. Screening a library of pharmaceutically active compounds (LOPAC) with this assay identified a potential chemical scaffold, 10058-F4 that exhibited fungicidal activity in the low micromolar range. These results thus demonstrate the feasibility of this alamarBlue-based assay for high throughput screening of fungicidal compounds under nutrient-limiting conditions for new anti-cryptococcal drug development.
doi:10.1177/1087057113496847
PMCID: PMC4017337  PMID: 23896686
Cryptococcus neoformans; fungicidal screen; high throughput screen; alamarBlue assay
23.  Development of a High-Throughput Fluorescence Polarization Assay to Identify Novel Ligands of Glutamate Carboxypeptidase II 
Journal of biomolecular screening  2012;17(8):1030-1040.
Glutamate carboxypeptidase II (GCPII) is an important target for therapeutic and diagnostic interventions aimed at prostate cancer and neurologic disorders. Here we describe the development and optimization of a high-throughput screening (HTS) assay based on fluorescence polarization (FP) that facilitates the identification of novel scaffolds inhibiting GCPII. First, we designed and synthesized a fluorescence probe based on a urea-based inhibitory scaffold covalently linked to a Bodipy TMR fluorophore (TMRGlu). Next, we established and optimized conditions suitable for HTS and evaluated the assay robustness by testing the influence of a variety of physicochemical parameters (e.g., pH, temperature, time) and additives. Using known GCPII inhibitors, the FP assay was shown to be comparable to benchmark assays established in the field. Finally, we evaluated the FP assay by HTS of a 20 000–compound library. The novel assay presented here is robust, highly reproducible (Z′ = 0.82), inexpensive, and suitable for automation, thus providing an excellent platform for HTS of small-molecule libraries targeting GCPII.
doi:10.1177/1087057112451924
PMCID: PMC4112551  PMID: 22751730
fluorescence polarization; high-throughput screening; glutamate carboxypeptidase II; prostate-specific membrane antigen; metallopeptidase
24.  Inhibition of Microglia Activation as a Phenotypic Assay in Early Drug Discovery 
Complex biological processes such as inflammation, cell death, migration, proliferation, and the release of biologically active molecules can be used as outcomes in phenotypic assays during early stages of drug discovery. Although target-based approaches have been widely used over the past decades, a disproportionate number of first-in-class drugs have been identified using phenotypic screening. This review details phenotypic assays based on inhibition of microglial activation and their utility in primary and secondary screening, target validation, and pathway elucidation. The role of microglia, both in normal as well as in pathological conditions such as chronic neurodegenerative diseases, is reviewed. Methodologies to assess microglia activation in vitro are discussed in detail, and classes of therapeutic drugs known to decrease the proinflammatory and cytotoxic responses of activated microglia are appraised, including inhibitors of glutaminase, cystine/glutamate antiporter, nuclear factor κB, and mitogen-activated protein kinases.
doi:10.1177/1087057113499406
PMCID: PMC4111462  PMID: 23945875
cytokines; reactive oxygen species; nitric oxide; glutamate; screening; cell-based assays
25.  Pro-angiogenic Cell Colonies Grown In Vitro from Human Peripheral Blood Mononuclear Cells 
Journal of biomolecular screening  2012;17(9):1128-1135.
Although multiple culture assays have been designed to identify “endothelial progenitor cells” (EPCs), the phenotype of cells grown in culture often remains undefined. We sought to define and characterize the pro-angiogenic cell population within human peripheral blood mononuclear cells. Mononuclear cells were isolated from peripheral blood and grown under angiogenic conditions for 7 days. Formed colonies (CFU-As) were identified and analyzed for proliferation, mRNA and surface antigen expression, tube-forming ability and chromosomal content. Colonies were composed of a heterogeneous group of cells expressing the leukocyte antigens CD45, CD14, and CD3, as well as the endothelial proteins vascular endothelial (VE) cadherin, von Willebrand's Factor (vWF), CD31 and endothelial nitric oxide synthase (eNOS). Colony cells expressed increased levels of pro-angiogenic growth factors, and they formed tubes in Matrigel. In comparison with colonies from the CFU-Hill assay, our assay resulted in a greater number of colonies (19±9 vs. 13±7; p<0.0001) with a substantial number of cells expressing an endothelial phenotype (20.2±7.4% vs. 2.2±1.2% expressing eNOS, p=0006). Chromosomal analysis indicated the colony cells were bone marrow-derived. We, therefore, describe a colony forming unit assay that measures bone marrow-derived circulating mononuclear cells with the capacity to proliferate and mature into proangiogenic leukocytic and endothelial-like cells. This assay, therefore, reflects circulating, bone marrow-derived pro-angiogenic activity.
doi:10.1177/1087057112457043
PMCID: PMC4096792  PMID: 22904201
stem cells; cardiac disease; cell-based assays

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