High-Throughput Flow Cytometry Drug Combination Discovery with Novel Synergy Analysis Software, SynScreen.

Classical therapeutic regimens are subject to toxicity, low efficacy, and/or the development of drug resistance. Thus, the discovery of synergistic drug combinations would permit treatment with lower, tolerable dosages of each agent and restored sensitivity. We describe the development and use of the SynScreen software application, which allows for visual and mathematical determinations of compound concentrations that produce super-additive effects. This software uses nonlinear regression fits of dose responses to determine synergism by the Bliss independence and Loewe additivity analysis models. We demonstrate the utility of SynScreen with data analysis from in vitro high-throughput flow cytometry (HTFC) combination screens with repurposed drugs and multiplexed synergy analysis of multiple biologic parameters in parallel. The applicability of SynScreen was confirmed by testing open-source data sets used in published drug combination literature. A key benefit of SynScreen for high-throughput drug combination screening is that observed measurements are graphically depicted in comparison with a three-dimensional surface that represents the theoretical responses at which Bliss additivity would occur. These images and summary tables for the calculated drug interactions are automatically exported. This allows for substantial data sets to be visually assessed, expediting the quick identification of efficacious drug combinations and thereby facilitating the design of confirmatory studies and clinical trials.

High-Throughput Flow Cytometry Screening of Multidrug Efflux Systems.

The resistance nodulation cell division (RND) family of proteins are inner membrane transporters that associate with periplasmic adaptor proteins and outer membrane porins to affect substrate transport from the cytosol and periplasm in Gram-negative bacteria. Various structurally diverse compounds are substrates of RND transporters. Along with their notable role in antibiotic resistance, these transporters are essential for niche colonization, quorum sensing, and virulence as well as for the removal of fatty acids and bile salts. As such, RNDs are an attractive target for antimicrobial development. However, while enhancing the utility of antibiotics with an RND inhibitor is an appealing concept, only a small core of chemotypes has been identified as efflux pump inhibitors (EPIs). Thus, our key objective is the development and validation of an efflux profiling and discovery strategy for RND model systems. Here we describe a flow cytometric dye accumulation assay that uses fluorescein diacetate (FDA) to interrogate the model Gram-negative pathogens Escherichia coli, Franscisella tularensis, and Burkholderia pseudomallei. Fluorochrome retention is increased in the presence of known efflux inhibitors and in RND deletion strains. The assay can be used in a high-throughput format to evaluate efflux of dye-substrate candidates and to screen chemical libraries for novel EPIs. Triaged compounds that inhibit efflux in pathogenic strains are tested for growth inhibition and antibiotic potentiation using microdilution culture plates in a select agent Biosafety Level-3 (BSL3) environment. This combined approach demonstrates the utility of flow cytometric analysis for efflux activity and provides a useful platform in which to characterize efflux in pathogenic Gram-negative bacteria. Screening small molecule libraries for novel EPI candidates offers the potential for the discovery of new classes of antibacterial compounds.

A High-Throughput Flow Cytometry Assay for Identification of Inhibitors of 3',5'-Cyclic Adenosine Monophosphate Efflux.

Assays to identify small molecule inhibitors of cell transporters have long been used to develop potential therapies for reversing drug resistance in cancer cells. In flow cytometry, these approaches rely on the use of fluorescent substrates of transporters. Compounds which prevent the loss of cell fluorescence have typically been pursued as inhibitors of specific transporters, but further drug development has been largely unsuccessful. One possible reason for this low success rate could be a substantial overlap in substrate specificities and functions between transporters of different families. Additionally, the fluorescent substrates are often synthetic dyes that exhibit promiscuity among transporters as well. Here, we describe an assay in which a fluorescent analog of a natural metabolite, 3',5'-cyclic adenosine monophosphate (F-cAMP), is actively effluxed by malignant leukemia cells. The F-cAMP is loaded into the cell cytoplasm using a procedure based on the osmotic lysis of pinocytic vesicles. The flow cytometric analysis of the fluorescence retained in F-cAMP-loaded cells incubated with various compounds can subsequently identify inhibitors of cyclic AMP efflux (ICE).

A high-throughput phenotypic screen of cytotoxic T lymphocyte lytic granule exocytosis reveals candidate immunosuppressants.

We screened the National Institutes of Health's Molecular Libraries Small Molecule Repository for inhibitors of cytotoxic T lymphocyte (CTL) lytic granule exocytosis by measuring binding of an antibody in the extracellular solution to a lysosomal membrane protein (LAMP-1) that is transferred to the plasma membrane by exocytosis. We used TALL-104 human leukemic CTLs stimulated with soluble chemicals. Using high-throughput cluster cytometry to screen 364,202 compounds in a 1536-well plate format, we identified 2404 initial hits: 161 were confirmed on retesting, and dose-response measurements were performed. Seventy-five of those compounds were obtained, and 48 were confirmed active. Experiments were conducted to determine the molecular mechanism of action (MMOA) of the active compounds. Fifteen blocked increases in intracellular calcium >50%. Seven blocked phosphorylation of extracellular signal-regulated kinase (ERK) by upstream mitogen-activated protein kinase kinases >50%. One completely blocked the activity of the calcium-dependent phosphatase calcineurin. None blocked ERK catalytic activity. Eight blocked more than one pathway. For 8 compounds, we were unable to determine an MMOA. The activity of 1 of these compounds was confirmed from powder resupply. We conclude that a screen based on antibody binding to CTLs is a good means of identifying novel candidate immunosuppressants with either known or unknown MMOAs.

Flow cytometry enables a high-throughput homogeneous fluorescent antibody-binding assay for cytotoxic T cell lytic granule exocytosis.

We developed a homogeneous phenotypic fluorescence end-point 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 Ca(2+); 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 mitogen-activated protein (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.

High-throughput screen for the chemical inhibitors of antiapoptotic bcl-2 family proteins by multiplex flow cytometry.

The human Bcl-2 family includes six antiapoptotic members (Bcl-2, Bcl-B, Bcl-W, Bcl-X(L), Bfl-1, and Mcl-1) and many proapoptotic members, wherein a balance between the two determines cell life or death in many physiological and disease contexts. Elevated expression of various antiapoptotic Bcl-2 members is commonly observed in cancers, and chemical inhibitors of these proteins have been shown to promote apoptosis of malignant cells in culture, in animal models, and in human clinical trials. All six antiapoptotic members bind a helix from the proapoptotic family member Bim, thus quenching Bim's apoptotic signal. Here, we describe the use of a multiplex, high-throughput flow cytometry assay for the discovery of small molecule modulators that disrupt the interaction between the antiapoptotic members of the Bcl-2 family and Bim. The six antiapoptotic Bcl-2 family members were expressed as glutathione-S-transferase fusion proteins and bound individually to six glutathione bead sets, with each set having a different intensity of red fluorescence. A fluorescein-conjugated Bcl-2 homology region 3 (BH3) peptide from Bim was employed as a universal ligand. Flow cytometry measured the amount of green peptide bound to each bead set in a given well, with inhibitory compounds resulting in a decrease of green fluorescence on one or more bead set(s). Hits and cheminformatically selected analogs were retested in a dose-response series, resulting in three "active" compounds for Bcl-B. These three compounds were validated by fluorescence polarization and isothermal titration calorimetry. We discuss some of the lessons learned about screening a chemical library provided by the National Institutes of Health Small Molecule Repository (∼195,000 compounds) using high-throughput flow cytometry.

High-throughput multiplex flow cytometry screening for botulinum neurotoxin type a light chain protease inhibitors.

Given their medical importance, proteases have been studied by diverse approaches and screened for small molecule protease inhibitors. Here, we present a multiplexed microsphere-based protease assay that uses high-throughput flow cytometry to screen for inhibitors of the light chain protease of botulinum neurotoxin type A (BoNTALC). Our assay uses a full-length substrate and several deletion mutants screened in parallel to identify small molecule inhibitors. The use of multiplex flow cytometry has the advantage of using full-length substrates, which contain already identified distal-binding elements for the BoNTALC, and could lead to a new class of BoNTALC inhibitors. In this study, we have screened 880 off patent drugs and bioavailable compounds to identify ebselen as an in vitro inhibitor of BoNTALC. This discovery demonstrates the validity of our microsphere-based approach and illustrates its potential for high-throughput screening for inhibitors of proteases in general.

Identification of inhibitors of vacuolar proton-translocating ATPase pumps in yeast by high-throughput screening flow cytometry.

Fluorescence intensity of the pH-sensitive carboxyfluorescein derivative 2,7-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) was monitored by high-throughput flow cytometry in living yeast cells. We measured fluorescence intensity of BCECF trapped in yeast vacuoles, acidic compartments equivalent to lysosomes where vacuolar proton-translocating ATPases (V-ATPases) are abundant. Because V-ATPases maintain a low pH in the vacuolar lumen, V-ATPase inhibition by concanamycin A alkalinized the vacuole and increased BCECF fluorescence. Likewise, V-ATPase-deficient mutant cells had greater fluorescence intensity than wild-type cells. Thus, we detected an increase of fluorescence intensity after short- and long-term inhibition of V-ATPase function. We used yeast cells loaded with BCECF to screen a small chemical library of structurally diverse compounds to identify V-ATPase inhibitors. One compound, disulfiram, enhanced BCECF fluorescence intensity (although to a degree beyond that anticipated for pH changes alone in the mutant cells). Once confirmed by dose-response assays (EC(50)=26 microM), we verified V-ATPase inhibition by disulfiram in secondary assays that measured ATP hydrolysis in vacuolar membranes. The inhibitory action of disulfiram against V-ATPase pumps revealed a novel effect previously unknown for this compound. Because V-ATPases are highly conserved, new inhibitors identified could be used as research and therapeutic tools in cancer, viral infections, and other diseases where V-ATPases are involved.

Preclinical development of a neutral, estrogen receptor-targeted, tridentate 99mTc(I)-estradiol-pyridin-2-yl hydrazine derivative for imaging of breast and endometrial cancers.

UNLABELLED: Breast and endometrial cancers are the most common invasive malignancies in women, with more than 217,000 new diagnoses per year in the United States. These cancers are often classified into 2 subtypes based on the expression of the classical estrogen receptor. In this study, we describe a new structural class of neutral tridentate 99mTc(I)-estradiol-pyridin-2-yl hydrazine derivatives for potential use in breast and endometrial cancer imaging. METHODS: The 99mTc(I)-estradiol-pyridin-2-yl hydrazine derivative was synthesized via the Sonogashira cross-coupling reaction and radiolabeled via the tricarbonyl approach. Radiochemical purity was assessed by high-performance liquid chromatography. Cell-binding studies were performed with human breast adenocarcinoma MCF-7 cells. The in vivo biodistribution of the 99mTc(I) derivative was evaluated in virgin female C57BL/6 mice in defined phases of the estrous cycle. Biodistribution and SPECT/CT studies were performed with mice bearing MCF-7 and primary human endometrial tumors. RESULTS: Radiochemical analysis demonstrated that the postpurification purity of the 99mTc(I)-estradiol-pyridin-2-yl hydrazine derivative was > or =95%, with a specific activity of 99mTc of 47.5 TBq/mmol. Cell-binding studies yielded a dissociation constant (mean +/- SEM) of 11 +/- 1.5 nM. In vivo studies revealed that receptor-mediated uptake was present in all phases of the estrous cycle in reproductive organs and mammary glands but was highest during the diestrous phase of the estrous cycle. Despite high nonspecific uptake in the liver, significant receptor-mediated uptake was observed in target tissues and estrogen receptor-expressing tumors (0.67% for MCF-7 tumors and 0.77% for endometrial tumors). Tumor uptake was reduced by approximately 50% on coinjection with 17beta-estradiol. CONCLUSION: We have characterized a novel neutral tridentate 99mTc(I)-estradiol-pyridin-2-yl hydrazine derivative for potential use in breast and endometrial cancer imaging. This study represents the first step on a path toward the design of estrogen-based Tc-labeled tracers with improved targeting and SPECT imaging characteristics.

A multifunctional androgen receptor screening assay using the high-throughput Hypercyt flow cytometry system.

The androgen receptor (AR) is a steroid hormone receptor which regulates transcription of androgen-sensitive genes and is responsible for the development and maintenance of male secondary sexual characteristics. Chemicals that interfere with AR activity may lead to pathological conditions in androgen-sensitive tissues. A variety of reporter systems have been developed, driven by androgen-sensitive promoters, which screen for chemicals that modulate androgenic activity. We have developed a flexible, high-throughput AR transcriptional activation assay, designated the Multifunctional Androgen Receptor Screening (MARS) assay, to facilitate the identification of novel modulators of AR transcriptional activity using flow cytometry. Androgen-independent human prostate cancer-derived PC3 cells were transiently cotransfected with an expression vector for the wild-type human AR and an androgen-sensitive promoter regulating the expression of destabilized enhanced GFP (dsEGFP). The transfected cells were stimulated with established androgenic and antiandrogenic compounds and assessed for increased or decreased dsEGFP expression. To screen for antagonists of AR transcription, the AR agonist R1881 was coadministered at submaximal concentrations with potential AR antagonists. The assay was formatted for high-throughput screening using the HyperCyt flow cytometry system. Agents with established androgenic and antiandrogenic activity were used for validation of the MARS assay. AR agonists were found to potently induce dsEGFP. Furthermore, AR agonists induced dsEGFP expression in a dose-dependent manner. Alternatively, AR antagonists blocked dsEGFP expression when coadministered with low-dose R1881, which also occurred in a dose-dependent manner. Modulators of AR transcriptional activity can be successfully identified by the MARS assay, utilizing a rapid, flexible, sensitive, and high-throughput format. Dose-response curves can be successfully generated for these compounds, allowing for an assessment of potency. Because of its simplicity and high-throughput compatibility, the MARS assay and HyperCyt system combined with flow cytometric analysis represents a valuable and novel addition to the current repertoire of AR transcriptional activation screening assays.

GPR30: a novel therapeutic target in estrogen-related disease.

Estrogen is a crucial hormone in human physiology that regulates a multitude of biological processes. It is also an important target in many diseases such as cancer and skeletal, neurological and immunological conditions. The actions of estrogen have traditionally been ascribed to one of two closely related classical nuclear hormone receptors, ERalpha and ERbeta, which are best characterized for regulating gene expression. Recent studies have revealed the contribution of a novel estrogen receptor GPR30, which belongs to the family of seven-transmembrane G-protein-coupled receptors, to many of the rapid biological responses to estrogen. Many drugs, such as tamoxifen and fulvestrant, which seem to selectively inhibit the activities of the classical estrogen receptors, are in widespread clinical use. However, recent results indicate that these same drugs activate multiple cellular-signaling pathways via GPR30. Unraveling the pharmacological profiles and specificities of ERalpha, ERbeta and GPR30 will be vital for understanding not only the physiological roles of each receptor but also for the development of the next generation of receptor-specific drugs.

High-throughput flow cytometry to detect selective inhibitors of ABCB1, ABCC1, and ABCG2 transporters.

Up-regulation of pump (transporter) expression and selection of resistant cancer cells result in cancer multidrug resistance to diverse substrates of these transporters. While more than 48 members of the ATP binding cassette (ABC) transporter superfamily have been identified, up to now only three human ABC transporters-ABCB1, ABCC1, and ABCG2-have unambiguously been shown to contribute to cancer multidrug resistance. The use of low-toxicity and high-specificity agents as a targeted transporter inhibition strategy is necessary to effectively overcome multiple drug resistance. An objective of the present studies was to develop and validate HyperCyt (IntelliCyt, Albuquerque, NM) flow cytometry high-throughput screeening assays to assess the specificity of test compounds that inhibited transporters as an integral part of the screen. Two separate duplex assays were constructed: one in which ABCB1 and ABCG2 transporters were evaluated in parallel using fluorescent J-aggregate-forming lipophilic cation 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolcarbocyanine iodide as substrate, and the other in which ABCB1 and ABCC1 transporters were evaluated in parallel using fluorescent calcein acetoxymethyl ester as substrate. ABCB1-expressing cells were color-coded to allow their distinction from cells expressing the alternate transporter. The assays were validated in a screen of the Prestwick Chemical Library (Illkirch, France). Three novel selective inhibitors of the ABCC1 transporter were identified in the screen, and the activity of each was confirmed in follow-up chemosensitivity shift and reversal studies. This high-throughput screening assay provides an efficient approach for identifying selective inhibitors of individual ABC transporters, promising as probes of transporter function and therapeutic tools for treating chemotherapy-resistant cancers.

Distribution and characterization of estrogen receptor G protein-coupled receptor 30 in the rat central nervous system.

The G protein-coupled receptor 30 (GPR 30) has been identified as the non-genomic estrogen receptor, and G-1, the specific ligand for GPR30. With the use of a polyclonal antiserum directed against the human C-terminus of GPR30, immunohistochemical studies revealed GPR30-immunoreactivity (irGPR30) in the brain of adult male and non-pregnant female rats. A high density of irGPR30 was noted in the Islands of Calleja and striatum. In the hypothalamus, irGPR30 was detected in the paraventricular nucleus and supraoptic nucleus. The anterior and posterior pituitary contained numerous irGPR30 cells and terminal-like endings. Cells in the hippocampal formation as well as the substantia nigra were irGPR30. In the brainstem, irGPR30 cells were noted in the area postrema, nucleus of the solitary tract, and dorsal motor nucleus of the vagus; a cluster of cells were prominently labeled in the nucleus ambiguus. Tissue sections processed with pre-immune serum showed no irGPR30, affirming the specificity of the antiserum. G-1 (100 nM) caused a large increase of intracellular calcium concentrations [Ca(2+) ](i) in dissociated and cultured rat hypothalamic neurons, as assessed by microfluorometric Fura-2 imaging. The calcium response to a second application of G-1 showed a marked homologous desensitization. Our result shows a high expression of irGPR30 in the hypothalamic-pituitary axis, hippocampal formation, and brainstem autonomic nuclei; and the activation of GPR30 by G-1 is associated with a mobilization of calcium in dissociated and cultured rat hypothalamic neurons.

Post-high-throughput screening analysis: an empirical compound prioritization scheme.

An empirical scheme to evaluate and prioritize screening hits from high-throughput screening (HTS) is proposed. Negative scores are given when chemotypes found in the HTS hits are present in annotated databases such as MDDR and WOMBAT or for testing positive in toxicity-related experiments reported in TOXNET. Positive scores were given for higher measured biological activities, for testing negative in toxicity-related literature, and for good overlap when profiled against drug-related properties. Particular emphasis is placed on estimating aqueous solubility to prioritize in vivo experiments. This empirical scheme is given as an illustration to assist the decision-making process in selecting chemotypes and individual compounds for further experimentation, when confronted with multiple hits from high-throughput experiments. The decision-making process is discussed for a set of G-protein coupled receptor antagonists and validated on a literature example for dihydrofolate reductase inhibition.

Integration of virtual screening with high-throughput flow cytometry to identify novel small molecule formylpeptide receptor antagonists.

The formylpeptide receptor (FPR) family of G-protein-coupled receptors contributes to the localization and activation of tissue-damaging leukocytes at sites of chronic inflammation. We developed a FPR homology model and pharmacophore (based on the bovine rhodopsin crystal structure and known FPR ligands, respectively) for in silico screening of approximately 480,000 drug-like small molecules. A subset of 4324 compounds that matched the pharmacophore was then physically screened with the HyperCyt flow cytometry platform in high-throughput, no-wash assays that directly measure human FPR binding, with samples (each approximately 2500 cells in 2 microl) analyzed at 40/min. From 52 confirmed hits (1.2% hit rate), we identified 30 potential lead compounds (inhibition constant, Ki= 1-32 microM) representing nine distinct chemical families. Four compounds in one family were weak partial agonists. All others were antagonists. This virtual screening approach improved the physical screening hit rate by 12-fold (versus 0.1% hit-rate in a random compound collection), providing an efficient process for identifying small molecule antagonists.