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1.  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
2.  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
3.  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
4.  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.
PMCID: PMC4158006  PMID: 22751729
stem cells; cancer and cancer drugs; high-content screening; nuclear hormone regulation; cytokeratin
5.  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.
PMCID: PMC4132978  PMID: 22498909
Cell-based assays; Gene Expression; Reporter gene assays; Cancer and cancer drugs; Endocrine Diseases; Transcription factors
6.  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.
PMCID: PMC4112551  PMID: 22751730
fluorescence polarization; high-throughput screening; glutamate carboxypeptidase II; prostate-specific membrane antigen; metallopeptidase
7.  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.
PMCID: PMC4096792  PMID: 22904201
stem cells; cardiac disease; cell-based assays
8.  Screening for Inhibitors of an Essential Chromatin Remodeler in Mouse Embryonic Stem Cells by Monitoring Transcriptional Regulation 
Journal of biomolecular screening  2012;17(9):1221-1230.
The SWI/SNF-like adenosine triphosphate (ATP)–dependent chromatin remodeling complex, esBAF, is both necessary and, in some contexts, sufficient to induce the pluripotent state. Furthermore, mutations in various BAF subunits are associated with cancer. Little is known regarding the precise mechanism(s) by which this complex exerts its activities. Thus, it is unclear which protein interactions would be important to disrupt to isolate a relevant readout of mechanism. To address this, we developed a gene expression–based assay to identify inhibitors of the native esBAF complex. Specifically, a quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) assay was developed in mouse embryonic stem (ES) cells to monitor expression of Bmi1, a developmentally important gene repressed by the esBAF complex. The assay was miniaturized to a 384-well format and used to screen a diverse collection of compounds, including novel products of diversity-oriented synthesis (DOS). Confirmed hits were validated using a knock-in ES cell reporter line in which luciferase is inserted into the Bmi1 locus. Several of the validated hits regulate a panel of target genes in a manner similar to the BAF chromatin-remodeling complex. Together these data indicate that expression-based screening using qRT-PCR is a successful approach to identify compounds targeting the regulation of key developmental genes in ES cells.
PMCID: PMC4097195  PMID: 22853929
esBAF; chromatin; qRT-PCR; expression-based screening; stem cells
9.  An In Vivo Zebrafish Screen Identifies Organophosphate Antidotes with Diverse Mechanisms of Action 
Journal of biomolecular screening  2012;18(1):108-115.
Organophosphates are a class of highly toxic chemicals that includes many pesticides and chemical weapons. Exposure to organophosphates, either through accidents or acts of terrorism, poses a significant risk to human health and safety. Existing antidotes, in use for over 50 years, have modest efficacy and undesirable toxicities. Therefore, discovering new organophosphate antidotes is a high priority. Early life stage zebrafish exposed to organophosphates exhibit several phenotypes that parallel the human response to organophosphates, including behavioral deficits, paralysis, and eventual death. Here, we have developed a high-throughput zebrafish screen in a 96-well plate format to find new antidotes that counteract organophosphate-induced lethality. In a pilot screen of 1200 known drugs, we identified 16 compounds that suppress organophosphate toxicity in zebrafish. Several in vitro assays coupled with liquid chromatography/tandem mass spectrometry–based metabolite profiling enabled determination of mechanisms of action for several of the antidotes, including reversible acetylcholinesterase inhibition, cholinergic receptor antagonism, and inhibition of bioactivation. Therefore, the in vivo screen is capable of discovering organophosphate antidotes that intervene in distinct pathways. These findings suggest that zebrafish screens might be a broadly applicable approach for discovering compounds that counteract the toxic effects of accidental or malicious poisonous exposures.
PMCID: PMC4053346  PMID: 22960781
Xenopus; zebrafish; in vivo screening; mass spectrometry; high-content screening
10.  Flow Cytometry Enables A High-Throughput Homogeneous Fluorescent Antibody-Binding Assay for Cytotoxic T Cell Lytic Granule Exocytosis 
Journal of biomolecular screening  2012;18(4):420-429.
We developed a homogeneous phenotypic fluorescence endpoint assay for cytotoxic T lymphocyte lytic granule exocytosis. This flow cytometric assay measures binding of an antibody to a luminal epitope of a lysosomal membrane protein (LAMP-1) that is exposed by exocytosis to the extracellular solution. Washing to remove unbound antibody is not required. Confirming the assay’s ability to detect novel active compounds, we screened at a concentration of 50 μM a synthetic diversity library of 91 compounds in a 96-well plate format, identifying 17 compounds that blocked by 90% or more. The actions of six structurally related tetracyano-hexahydroisoindole compounds that inhibited by ~90% at a concentration of 10 μM were investigated further. Four reduced elevations in intracellular Ca2+; it is likely that depolarization of the cells’ membrane potential underlies the effect for at least two of the compounds. Another compound was found to be a potent inhibitor of the activation of the MAP kinase ERK. Finally, we transferred the assay to a 384-well format and screened the Prestwick Compound Library using high-throughput flow cytometry. Our results indicate that our assay will likely be a useful means of screening libraries for novel compounds with important biological activities.
PMCID: PMC4043149  PMID: 23160568
Cytotoxic T lymphocytes; exocytosis; flow cytometry; high-throughput screen; phenotypic assay
11.  A spectrophotometric assay for quantitative measurement of aminoacyl-tRNA synthetase activity 
Journal of biomolecular screening  2012;18(4):490-497.
Aminoacyl-tRNA synthetases are enzymes that charge specific tRNAs with their cognate amino acids and play an essential role in the initial steps of protein synthesis. Because these enzymes are attractive targets for drug development in many microorganisms, there is a pressing need for assays suitable for compound screening. We developed 1) a high throughput assay for measuring aminoacyl-tRNA synthetase activity and 2) an accompanying method for preparing the tRNA substrate. The assay can be performed in 96-well plates and relies on malachite green detection of pyrophosphate (Pi) as an indicator of aminoacyl-tRNA synthetase activity. Analysis of Trypanosoma brucei isoleucyl-tRNA synthetase (IleRS) activity showed that the assay exhibits sensitivity to picomoles of product, and yielded a Z′-factor of 0.56. We show that this assay is applicable to other aminoacyl-tRNA synthetases and to enzyme inhibition studies. Using this assay, we found that the compound NSC616354 inhibits recombinant IleRS with an IC50 of 0.6μM. Enzymology studies were also performed with rIleRS and its Km and kcat determined as 3.97 × 10−5 mol/L and 142 S−1, respectively. This assay will facilitate the screening of compounds to identify inhibitors of aminoacyl-tRNA synthetases.
PMCID: PMC3774597  PMID: 23134734
aminoacyl-tRNA synthetase; high throughput assay; malachite green; tRNA; aminoacylation
12.  Development and validation of a high throughput intrinsic ATPase activity assay for the discovery of MEKK2 inhibitors 
Journal of biomolecular screening  2012;18(4):388-399.
The kinase MEKK2 (MAP3K2) has recently been implicated in tumor growth and metastasis. Thus, selective inhibition of MEKK2 may be a novel strategy for cancer therapy. In order to identify inhibitors of MEKK2 kinase activity, we have developed a novel activity assay for MEKK2 based on the discovery that recombinant purified MEKK2 has intrinsic ATPase activity. This MEKK2 ATPase assay was validated for enzyme identity and enzymatic purity by multiple methods including mass spectrometry analysis, testing different sources of MEKK2 and comparing ATPase assay IC50 data for multiple inhibitors to literature values and to IC50 data generated using MEKK2 binding and transphosphorylation assays. Taken together, these data indicated that genuine MEKK2 activity was being measured in this assay and no other ATPases contributed to the signal. A miniaturized version of the assay was validated for high throughput screening and compound libraries were screened. The screening hits generated comparable potencies in the MEKK2 intrinsic ATPase, binding and transphosphorylation assays. We identified a novel MEKK2 inhibitor and confirmed that crizotinib and bosutinib are potent in vitro inhibitors of MEKK2 activity with IC50 values of <100 nM. Thus, this assay has utility for the discovery of small molecule inhibitors of MEKK2 activity.
PMCID: PMC3723327  PMID: 23134735
MEKK2; MAP3K2; ATPase; crizotinib; bosutinib
13.  A High Throughput Assay to Identify Small Molecule Modulators of Alternative pre-mRNA Splicing 
Journal of biomolecular screening  2012;18(2):180-190.
Alternative splicing (AS) is an efficient mechanism that involves the generation of transcriptome and protein diversity from a single gene. Defects in pre-mRNA splicing are an important cause of numerous diseases, including cancer. AS of pre-mRNA as a target for cancer therapy has not been well studied. We have reported previously that a splicing factor, polypyrimidine tract-binding protein (PTB) is overexpressed in ovarian tumors, compared to matched normal controls, and knockdown (KD) of PTB expression by shRNA impairs ovarian tumor cell growth, colony formation and invasiveness. Given the complexity of PTB’s molecular functions, a chemical method for controlling PTB activity might provide a therapeutic and experimental tool. However, no commercially available PTB inhibitors have yet been described. To expand our ability to find novel inhibitors, we developed a robust, fluorometric, cell-based high throughput screening HTS assay in 96-well plates that reports on the splicing activity of PTB. In an attempt to use the cells for large-scale chemical screens to identify PTB modulators, we established cell lines stably expressing the reporter gene. Our results suggest that this high throughput assay could be used to identify small molecule modulators of PTB activity. Based on these findings and the role that upregulated PTB has on cell proliferation and malignant properties of tumors targeting PTB for inhibition with small molecules offers a promising strategy for cancer therapy.
PMCID: PMC3541474  PMID: 22972848
PTB; ovarian cancer; fluorescence methods; cell-based HTS; alternative splicing
14.  A High-Throughput Screen for Wnt/β-catenin Signaling Pathway Modulators in Human iPSC-derived Neural Progenitors 
Journal of biomolecular screening  2012;17(9):1252-1263.
Wnt/β-catenin signaling has emerged as a central player in pathways implicated in the pathophysiology and treatment of neuropsychiatric disorders. To identify potential novel therapeutics for these disorders, high-throughput screening (HTS) assays reporting on Wnt/β-catenin signaling in disease relevant contexts are needed. The use of human patient-derived induced pluripotent stem cell (iPSC) models provides ideal disease relevant context if these stem cell cultures can be adapted for HTS-compatible formats. Here, we describe a sensitive, HTS-compatible Wnt/β-catenin signaling reporter system generated in homogeneous, expandable neural progenitor cells (NPCs) derived from human iPSCs. We validated this system by demonstrating dose responsive stimulation by several known Wnt/β-catenin signaling pathway modulators, including Wnt3a, a glycogen synthase kinase-3 (GSK3) inhibitor, and the bipolar disorder therapeutic lithium. These responses were robust and reproducible over time across many repeated assays. We then conducted a screen of ~1,500 compounds from a library of FDA-approved drugs and known bioactives, and confirmed HTS hits, revealing multiple chemical and biological classes of novel small molecule probes of Wnt/β-catenin signaling. Generating this type of pathway-selective, cell-based phenotypic assays in human iPSC-derived neural cells will advance the field of human experimental neurobiology toward the goal of identifying and validating targets for neuropsychiatric disorder therapeutics.
PMCID: PMC3903585  PMID: 22923789
induced pluripotent stem cell (iPSC); neural progenitor cell (NPC); Wnt/β-catenin signaling; neuropsychiatric disorders; human neurons
15.  Agents that Stabilize Mutated von Hippel Lindau Protein Result in Differential Post-Translational Modification and Subcellular Localization 
Journal of biomolecular screening  2012;17(5):572-580.
von Hippel Lindau (VHL) disease is an autosomal dominant inherited disorder that results in multiple organ systems being affected. Treatment is mainly surgical, however, effective systemic therapies are needed. We developed and tested a cell-based screening tool to identify compounds that stabilize or upregulate full-length, point mutated VHL.
The 786-0 cell line was infected with full-length W117A mutated VHL linked to a C-terminal Venus fluorescent protein. This VHL-W117A-Venus line was used to screen the Prestwick drug library and was tested against the known proteasome inhibitors MG132 and bortezomib. Western blot validation and evaluation of downstream functional readouts, including HIF and GLUT1 levels, were performed.
Bortezomib, MG132, and the Prestwick compounds 8-azaguanine, thiostrepton and thioguanosine were found to reliably upregulate VHL-W117A-Venus in 786-0 cells. 8-azaguanine was found to downregulate HIF2α levels, and was augmented by the presence of VHL W117A. VHL p30 band intensities varied as a function of compound used, suggesting alternate post-translational processing. In addition, nuclear-cytoplasmic localization of pVHL varied amongst the different compounds.
786-0 cells containing VHL-W117A-Venus can be successfully used to identify compounds that upregulate VHL levels, and that have a differential effect on pVHL intracellular localization and posttranslational processing. Further screening efforts will broaden the number of pharmacophores available to develop therapeutic agents that will upregulate and refunctionalize mutated VHL.
PMCID: PMC3895461  PMID: 22357874
VHL upregulation; proteostasis; high-throughput screen; Prestwick
16.  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.
PMCID: PMC3893891  PMID: 22820394
Phosphatase; Eyes Absent 2; Eya2; Eya2 inhibitor; Six1
17.  A Selective ATP-binding Cassette Sub-family G Member 2 Efflux Inhibitor Revealed Via High-Throughput Flow Cytometry 
Chemotherapeutics tumor resistance is a principal reason for treatment failure and clinical and experimental data indicate that multidrug transporters such as ATP-binding Cassette (ABC) B1 and ABCG2 play a leading role by preventing cytotoxic intracellular drug concentrations. Functional efflux inhibition of existing chemotherapeutics by these pumps continues to present a promising approach for treatment. A contributing factor to the failure of existing inhibitors in clinical applications is limited understanding of specific substrate/inhibitor/pump interactions. We have identified selective efflux inhibitors by profiling multiple ABC transporters against a library of small molecules to find molecular probes to further explore such interactions. In our primary screening protocol using JC-1 as a dual-pump fluorescent reporter substrate we identified a piperazine substituted pyrazolo[1,5-a]pyrimidine substructure with promise for selective efflux inhibition. As a result of a focused SAR-driven chemistry effort we describe compound 1 (CID44640177), an efflux inhibitor with selectivity toward ABCG2 over ABCB1. Compound 1 is also shown to potentiate the activity of mitoxantrone in vitro as well as preliminarily in vivo in an ABCG2 over-expressing tumor model. At least two analogs significantly reduce tumor size in combination with the chemotherapeutic topotecan. To our knowledge, low nanomolar chemoreversal activity coupled with direct evidence of efflux inhibition for ABCG2 is unprecedented.
PMCID: PMC3623016  PMID: 22923785
Multi-drug resistance; ABC Transporter; ABCG2; ABCB1; Efflux inhibition
18.  High-Throughput FRET Assay Yields Allosteric SERCA Activators 
Using fluorescence resonance energy transfer (FRET), we performed a high-throughput screen (HTS) in a reconstituted membrane system, seeking compounds that reverse inhibition of sarco-/endoplasmic reticulum Ca-ATPase (SERCA) by its endogenous regulator, phospholamban (PLB). Such compounds have long been sought to correct aberrant Ca2+ regulation in heart failure. Donor-SERCA was reconstituted in phospholipid membranes with or without acceptor-PLB, and FRET was measured in a steady-state fluorescence microplate reader. A 20,000-compound library was tested in duplicate. Compounds that decreased FRET by more than three standard deviations were considered hits. From 43 primary hits (0.2%), 31 (72%) were found to be false positives upon more thorough testing. The remaining 12 hits were tested in assays of Ca-ATPase activity, and six of these activated SERCA significantly, by as much as 60%, and several also enhanced cardiomyocyte contractility. These compounds directly activated SERCA from heart and other tissues. These results validate our FRET approach and set the stage for medicinal chemistry and pre-clinical testing. We were concerned about the high rate of false positives, resulting from the low precision of steady-state fluorescence. Preliminary studies with a novel fluorescence lifetime plate reader show 20-fold higher precision. This instrument can dramatically increase the quality of future HT.
PMCID: PMC3721969  PMID: 22923787
calcium pump; calcium transport; phospholamban; reconstituted membrane; fluorescence lifetime
19.  Identification of Novel Compounds That Increase SMN Protein Levels Using an Improved SMN2 Reporter Cell Assay 
Journal of biomolecular screening  2012;17(4):10.1177/1087057111431605.
Spinal muscular atrophy (SMA) is a neurodegenerative disorder that is characterized by progressive loss of motor neuron function. It is caused by the homozygous loss of the SMN1 (survival of motor neuron 1) gene and a decrease in full-length SMN protein. SMN2 is a nearly identical homolog of SMN1 that, due to alternative splicing, expresses predominantly truncated SMN protein. SMN2 represents an enticing therapeutic target. Increasing expression of full-length SMN from the SMN2 gene might represent a treatment for SMA. We describe a newly designed cell-based reporter assay that faithfully and reproducibly measures full-length SMN expression from the SMN2 gene. This reporter can detect increases of SMN protein by an array of compounds previously shown to regulate SMN2 expression and by the overexpression of proteins that modulate SMN2 splicing. It also can be used to evaluate changes at both the transcriptional and splicing level. This assay can be a valuable tool for the identification of novel compounds that increase SMN2 protein levels and the optimization of compounds already known to modulate SMN2 expression. We present here preliminary data from a high-throughput screen using this assay to identify novel compounds that increase expression of SMN2.
PMCID: PMC3870337  PMID: 22233647
spinal muscular atrophy; survival of motor neuron; SMN1; SMN2; cell-based assay; high-content screening; HTS
20.  Identification of Rift Valley Fever Virus Nucleocapsid Protein-RNA Binding Inhibitors Using a High-Throughput Screening Assay 
Journal of biomolecular screening  2012;17(8):1062-1070.
Rift Valley fever virus (RVFV) is an emerging infectious pathogen that causes severe disease in humans and livestock and has the potential for global spread. Currently, there is no proven effective treatment for RVFV infection and there is no licensed vaccine. Inhibition of RNA binding to the essential viral nucleocapsid (N) protein represents a potential anti-viral therapeutic strategy because all of the functions performed by N during infection involve RNA binding. To target this interaction, we developed a fluorescence polarization-based high-throughput drug screening assay and tested 26,424 chemical compounds for their ability to disrupt an N-RNA complex. From libraries of FDA approved drugs, drug-like molecules and natural products extracts we identified several lead compounds that are promising candidates for medicinal chemistry.
PMCID: PMC3520603  PMID: 22644268
nucleocapsid; RNA; Rift Valley fever virus; high-throughput screen; fluorescence polarization
21.  High-Throughput Luciferase Reporter Assay for Small-Molecule Inhibitors of MicroRNA Function 
Journal of biomolecular screening  2012;17(6):822-828.
MicroRNAs (miRNAs) are endogenous, single-stranded, noncoding RNAs of 21 to 23 nucleotides that regulate gene expression, typically by binding the 3′ untranslated regions of target messenger RNAs. It is estimated that miRNAs are involved in the regulation of 30% of all genes and almost every genetic pathway. Recently, the misregulation of miRNAs has been linked to various human diseases including cancer and viral infections, identifying miRNAs as potential targets for drug discovery. Thus, small-molecule modifiers of miRNAs could serve as lead structures for the development of new therapeutic agents and be useful tools in the elucidation of detailed mechanisms of miRNA function. As a result, we have developed a high-throughput screen for potential small-molecule regulators of the liver-specific microRNA miR-122, which is involved in hepatocellular carcinoma development and hepatitis C virus infection. Our small-molecule screen employs a Huh7 human hepatoma cell line stably transfected with a Renilla luciferase sensor for endogenous miR-122. The assay was optimized and validated using an miR-122 antisense agent and a previously identified small-molecule miR-122 inhibitor. The described reporter assay will enable the high-throughput screening of small-molecule miR-122 inhibitors and can be readily extended to other miRNAs.
PMCID: PMC3758890  PMID: 22412086
high-throughput assay; cell-based assay; luciferase; microRNA; small-molecule inhibitor
22.  Fragment-based screening for inhibitors of PDE4A using enthalpy arrays and X-ray crystallography 
Journal of biomolecular screening  2012;17(4):469-480.
Fragment-based screening has typically relied on X-ray or NMR methods to identify low affinity ligands that bind to therapeutic targets. These techniques are expensive in terms of material and time, so it useful to have a higher-throughput method to reliably pre-screen a fragment library to identify a subset of compounds for structural analysis. 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 ITC. Here we have used enthalpy arrays, which are arrays of nanocalorimeters, to perform an enzyme activity based fragment screen for competitive inhibitors of phosphodiesterase 4A (PDE4A). Several inhibitors with KI<2 mM were identified and moved to X-ray crystallization trials. Although the co-crystals did not yield high-resolution data, evidence of binding was observed and the chemical structures of the hits were consistent with motifs of known PDE4 inhibitors. This study shows how array calorimetry can be used as a pre-screening method for fragment-based lead discovery with enzyme targets, and it provides a list of candidate fragments for inhibition of PDE4A.
PMCID: PMC3745005  PMID: 22223051
nanocalorimetry; enzyme assay; label-free assay; fragment-based lead discovery; X-ray crystallography
23.  High-Throughput Screening of a Diversity Collection Using Biodefense Category A and B Priority Pathogens 
Journal of biomolecular screening  2012;17(7):946-956.
One of the objectives of the National Institutes of Allergy and Infectious Diseases (NIAID) Biodefense Program is to identify or develop broad-spectrum antimicrobials for use against bioterrorism pathogens and emerging infectious agents. As a part of that program, our institution has screened the 10 000-compound MyriaScreen Diversity Collection of high-purity druglike compounds against three NIAID category A and one category B priority pathogens in an effort to identify potential compound classes for further drug development. The effective use of a Clinical and Laboratory Standards Institute–based high-throughput screening (HTS) 96-well–based format allowed for the identification of 49 compounds that had in vitro activity against all four pathogens with minimum inhibitory concentration values of ≤16 μg/mL. Adaptation of the HTS process was necessary to conduct the work in higher-level containment, in this case, biosafety level 3. Examination of chemical scaffolds shared by some of the 49 compounds and assessment of available chemical databases indicates that several may represent broad-spectrum antimicrobials whose activity is based on novel mechanisms of action.
PMCID: PMC3700734  PMID: 22653912
anti-infective drugs; automation; cell-based assays; compound repositories; high-content screening
24.  A High-Content Biosensor Based Screen Identifies Cell Permeable Activators and Inhibitors of EGFR Function: Implications in Drug Discovery 
Journal of biomolecular screening  2012;17(7):885-899.
Early success of kinase inhibitors has validated their use as drugs. However, discovery efforts have also suffered from high attrition rates; due to lack of cellular activity. We reasoned that screening for such candidates in live cells would identify novel cell permeable modulators for development. For this purpose, we have used our recently optimized EGFR biosensor (EGFRB) assay to screen for modulators of EGFR activity. Here, we report on its validation under HTS conditions displaying a S/N ratio of 21 and a Z’ value of 0.56; attributes of a robust cell based assay. We performed a pilot screen against a library of 6,912 compounds demonstrating good reproducibility and identifying 82 inhibitors and 66 activators with initial hit rates of 1.2% and 0.95 %, respectively. Follow up dose response studies revealed that 12 out of the 13 known EGFR inhibitors in the library confirmed as hits. ZM-306416, a VEGFR antagonist, was identified as a potent inhibitor of EGFR function. Flurandrenolide, beclomethasone and ebastine were confirmed as activators of EGFR function. Taken together, our results validate this novel approach and demonstrate its utility in the discovery of novel kinase modulators with potential use in the clinic.
PMCID: PMC3615554  PMID: 22573732
EGFR; domain-based biosensor; high content analysis; live cell imaging
25.  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.
PMCID: PMC3530257  PMID: 22923786
induced pluripotent stem cells; neural stem cells; human neurons; astrocytes; skin fibroblasts; methyl-β-cyclodextrin

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