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1.  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
2.  Identification of a selective small molecule inhibitor series targeting the Eyes Absent 2 (Eya2) phosphatase activity 
Eya proteins are essential co-activators of the Six family of homeobox transcription factors and also contain a unique protein tyrosine phosphatase activity, belonging to the haloacid dehalogenase family of phosphatases. The phosphatase activity of Eya is important for a subset of Six1-mediated transcription, making this a unique type of transcriptional control. It is also responsible for directing cells to the repair instead of apoptosis pathway upon DNA damage. Furthermore, the phosphatase activity of Eya is critical for transformation, migration, invasion, and metastasis of breast cancer cells. Thus, inhibitors of the Eya phosphatase activity may be anti-tumorigenic and anti-metastatic, as well as sensitize cancer cells to DNA damage inducing therapies. In this paper, we identified a previously unknown chemical series using high throughput screening that inhibits the Eya2 phosphatase activity with IC50s ranging from 1.8 to 79 μM. Compound activity was confirmed using an alternative malachite green assay and H2AX, a known Eya substrate. Importantly, these Eya2 phosphatase inhibitors show specificity and do not significantly inhibit several other cellular phosphatases. Our studies identify the first selective Eya2 phosphatase inhibitors that can potentially be developed into chemical probes for functional studies of Eya phosphatase or into anti-cancer drugs in the future.
doi:10.1177/1087057112453936
PMCID: PMC3893891  PMID: 22820394
Phosphatase; Eyes Absent 2; Eya2; Eya2 inhibitor; Six1
3.  Evaluation of Cholesterol Reduction Activity of Methyl-β-cyclodextrin Using Differentiated Human Neurons and Astrocytes 
Journal of biomolecular screening  2012;17(9):1243-1251.
Recent advances in stem cell technology have enabled large scale production of human cells such as cardiomyocytes, hepatocytes and neurons for evaluation of pharmacological effect and toxicity of drug candidates. The assessment of compound efficacy and toxicity using human cells should lower the high clinical attrition rates of drug candidates by reducing the impact of species differences on drug efficacy and toxicity from animal studies. Methyl-β-cyclodextrin (MBCD) has shown to reduce lysosomal cholesterol accumulation in skin fibroblasts derived from patients with Niemann Pick type C disease and in the NPC1−/− mouse model. However, the compound has never been tested in human differentiated neurons. We have determined the cholesterol reduction effect of MBCD in neurons differentiated from human neural stem cells and commercially available astrocytes. The use of NSCs for producing differentiated neurons in large quantities can significantly reduce the production time and enhance the reproducibility of screening results. The EC50 values of MBCD on cholesterol reduction in human neurons and astrocytes were 66.9 and 110.7 µM, respectively. The results indicate that human neurons differentiated from the NSCs and human astrocytes are useful tools for evaluating pharmacological activity and toxicity of drug candidates to predict their clinical efficacy.
doi:10.1177/1087057112456877
PMCID: PMC3530257  PMID: 22923786
induced pluripotent stem cells; neural stem cells; human neurons; astrocytes; skin fibroblasts; methyl-β-cyclodextrin
4.  An AlphaScreen™ Based High-throughput Screen to Identify Inhibitors of Hsp90 and Cochaperone Interaction 
Journal of biomolecular screening  2009;14(3):273-281.
Hsp90 has emerged as an important anti-cancer drug target because of its essential role in promoting the folding and maturation of many oncogenic proteins. Here we describe the development of the first high throughput screen, based on AlphaScreen™ technology, to identify a novel type of Hsp90 inhibitors that interrupt its interaction with the cochaperone HOP. The assay uses the 20-mer C-terminal peptide of Hsp90 and the TPR2A domain of HOP. Assay specificity was demonstrated by measuring different interactions using synthetic peptides, with measured IC50s in good agreement with reported values. The assay is stable over 12 hours and tolerates DMSO up to 5%. We first validated the assay by screening against 20,000 compounds in 384-well format. After further optimization into a 1536-well format, it was screened against a NCGC library of 76,134 compounds, with a signal-to-background (S/B) ratio of 78 and Z’ factor of 0.77. The present assay can be used for discovery of novel small molecule Hsp90 inhibitors that can be used as chemical probes to investigate the role of cochaperones in Hsp90 function. Such molecules have the potential to be developed into novel anti-cancer drugs, for use alone or in combination with other Hsp90 inhibitors.
doi:10.1177/1087057108330114
PMCID: PMC3066041  PMID: 19211782
heat shock protein 90 (Hsp90); Hsp organizing protein (HOP); tetratricopeptide repeat (TPR); AlphaScreen™; high-throughput screening (HTS)
5.  Automated High-Content Screening for Compounds That Disassemble the Perinucleolar Compartment 
Journal of biomolecular screening  2009;14(9):1045-1053.
All solid malignancies share characteristic traits, including unlimited cellular proliferation, evasion of immune regulation, and the propensity to metastasize. The authors have previously described that a subnuclear structure, the perinucleolar compartment (PNC), is associated with the metastatic phenotype in solid tumor cancer cells. The percentage of cancer cells that contain PNCs (PNC prevalence) is indicative of the malignancy of a tumor both in vitro and in vivo, and thus PNC prevalence is a marker that reflects metastatic capability in a population of tumor cells. Although the function of the PNC remains to be determined, the PNC is highly enriched with small RNAs and RNA binding proteins. The initial chemical biology studies using a set of anticancer drugs that disassemble PNCs revealed a direct association of the structure with DNA. Therefore, PNC prevalence reduction as a phenotypic marker can be used to identify compounds that target cellular processes required for PNC maintenance and hence used to elucidate the nature of the PNC function. Here the authors report the development of an automated high-content screening assay that is capable of detecting PNC prevalence in prostate cancer cells (PC-3M) stably expressing a green fluorescent protein (GFP)–fusion protein that localizes to the PNC. The assay was optimized using known PNC-reducing drugs and non-PNC-reducing cytotoxic drugs. After optimization, the fidelity of the assay was probed with a collection of 8284 compounds and was shown to be robust and capable of detecting known and novel PNC-reducing compounds, making it the first reported high-content phenotypic screen for small changes in nuclear structure.
doi:10.1177/1087057109343120
PMCID: PMC2857721  PMID: 19762548
high-content screening; nucleus; cancer; perinucleolar compartment; small molecules
6.  A Cell-based PDE4 Assay in 1536-well Plate format for High Throughput Screening 
Journal of biomolecular screening  2008;13(7):609-618.
The cyclic nucleotide phosphodiesterases (PDEs) are intracellular enzymes that catalyze the hydrolysis of 3', 5'-cyclic nucleotides, such as cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP), to their corresponding 5'-nucleotide monophosphates. These enzymes play an important role in controlling cellular concentrations of cyclic nucleotides and thus regulate a variety of cellular signaling events. PDEs are emerging as drug targets for several diseases including asthma, cardiovascular disease, ADHD, Parkinson’s disease, and Alzheimer’s disease. Though biochemical assays with purified recombinant PDE enzymes and cAMP or cGMP substrate are commonly used for compound screening, cell-based assays would provide a better assessment of compound activity in a more physiological context. Here we report the development and validation of a new cell-based PDE4 assay using a constitutively active GPCR as a driving force for cAMP production and a cyclic nucleotide gated (CNG) cation channel as a biosensor in 1536-well plates.
doi:10.1177/1087057108319977
PMCID: PMC2661206  PMID: 18591513
phosphodiesterase; PDE IV; cyclic nucleotide gated ion channels; cell-based assay; high throughput screening
7.  Quantitative High Throughput Screening Using a Live Cell cAMP Assay Identifies Small Molecule Agonists of the TSH Receptor 
Journal of biomolecular screening  2008;13(2):120-127.
The thyroid stimulating hormone receptor (TSHR) belongs to the glycoprotein hormone receptor subfamily of seven-transmembrane spanning receptors. TSHR is expressed in thyroid follicular cells and is activated by TSH, which regulates growth and function of these cells. Recombinant TSH is used in diagnostic screens for thyroid cancer, especially in patients after thyroid cancer surgery. Currently, no selective small molecule agonist of the TSHR is available. To screen for novel TSHR agonists, we miniaturized a cell-based cAMP assay into 1536-well plate format. This assay uses a HEK293 cell line stably expressing the TSHR and a cyclic nucleotide gated ion channel (CNG), which functions as a biosensor. From a quantitative high-throughput screen of 73,180 compounds in parallel with a parental cell line (without the TSHR), 276 primary active compounds were identified. The activities of the selected active compounds were further confirmed in an orthogonal HTRF cAMP-based assay. 49 compounds in several structural classes have been confirmed as small molecule TSHR agonists that will serve as starting compounds for chemical optimization and studies of thyroid physiology in health and disease.
doi:10.1177/1087057107313786
PMCID: PMC2653065  PMID: 18216391
Thyroid-stimulating hormone TSH; TSHR; TSHR agonist; quantitative high throughput screening; qHTS; HTS; probe identification; CNG; PubChem

Results 1-7 (7)