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1.  Real Time Detection of Protein Trafficking with High Throughput Flow Cytometry (HTFC) and Fluorogen Activating Protein (FAP) Base Biosensor 
We combined fluorogen activating protein (FAP) technology with high-throughput flow cytometry to detect real-time protein trafficking to and from the plasma membrane in living cells. The hybrid platform allows drug discovery for trafficking receptors, such as G-protein coupled receptors, receptor tyrosine kinases and ion channels, that were previously not suitable for high throughput screening by flow cytometry.. The system has been validated using the β2-adrenergic receptor (β2AR) system and extended to other GPCRs. When a chemical library containing ~1,200 off-patent drugs was screened against cells expressing FAP tagged β2AR, all known β2AR active ligands in the library were successfully identified, together with a few compounds that were later confirmed to regulate receptor internalization in a non-traditional manner. The unexpected discovery of new ligands by this approach indicates the potential of using this protocol for GPCR de-orphanization. In addition, screens of multiplexed targets promise improved efficiency with minor protocol modification.
doi:10.1002/0471142956.cy0943s67
PMCID: PMC3961059  PMID: 24510772
High throughput flow cytometer; Fluorogen activating protein; G protein coupled receptor; Receptor trafficking; Live cell assay
2.  Carbon monoxide down-regulates α4β1 integrin-specific ligand binding and cell adhesion: a possible mechanism for cell mobilization 
BMC Immunology  2014;15(1):52.
Background
Carbon monoxide (CO), a byproduct of heme degradation, is attracting growing attention from the scientific community. At physiological concentrations, CO plays a role as a signal messenger that regulates a number of physiological processes. CO releasing molecules are under evaluation in preclinical models for the management of inflammation, sepsis, ischemia/reperfusion injury, and organ transplantation. Because of our discovery that nitric oxide signaling actively down-regulates integrin affinity and cell adhesion, and the similarity between nitric oxide and CO-dependent signaling, we studied the effects of CO on integrin signaling and cell adhesion.
Results
We used a cell permeable CO releasing molecule (CORM-2) to elevate intracellular CO, and a fluorescent Very Late Antigen-4 (VLA-4, α4β1-integrin)-specific ligand to evaluate the integrin state in real-time on live cells. We show that the binding of the ligand can be rapidly down-modulated in resting cells and after inside-out activation through several Gαi-coupled receptors. Moreover, cell treatment with hemin, a natural source of CO, resulted in comparable VLA-4 ligand dissociation. Inhibition of VLA-4 ligand binding by CO had a dramatic effect on cell-cell interaction in a VLA-4/VCAM-1-dependent cell adhesion system.
Conclusions
We conclude that the CO signaling pathway can rapidly down-modulate binding of the VLA-4 -specific ligand. We propose that CO-regulated integrin deactivation provides a basis for modulation of immune cell adhesion as well as rapid cell mobilization, for example as shown for splenic monocytes in response to surgically induced ischemia of the myocardium.
doi:10.1186/s12865-014-0052-1
PMCID: PMC4221689  PMID: 25367365
Carbon monoxide; Hemin; Integrin; Affinity; Conformation; Cell adhesion
3.  An Automated High-Throughput Cell-Based Multiplexed Flow Cytometry Assay to Identify Novel Compounds to Target Candida albicans Virulence-Related Proteins 
PLoS ONE  2014;9(10):e110354.
Although three major classes of systemic antifungal agents are clinically available, each is characterized by important limitations. Thus, there has been considerable ongoing effort to develop novel and repurposed agents for the therapy of invasive fungal infections. In an effort to address these needs, we developed a novel high-throughput, multiplexed screening method that utilizes small molecules to probe candidate drug targets in the opportunistic fungal pathogen Candida albicans. This method is amenable to high-throughput automated screening and is based upon detection of changes in GFP levels of individually tagged target proteins. We first selected four GFP-tagged membrane-bound proteins associated with virulence or antifungal drug resistance in C. albicans. We demonstrated proof-of-principle that modulation of fluorescence intensity can be used to assay the expression of specific GFP-tagged target proteins to inhibitors (and inducers), and this change is measurable within the HyperCyt automated flow cytometry sampling system. Next, we generated a multiplex of differentially color-coded C. albicans strains bearing C-terminal GFP-tags of each gene encoding candidate drug targets incubated in the presence of small molecules from the Prestwick Chemical Library in 384-well microtiter plate format. Following incubation, cells were sampled through the HyperCyt system and modulation of protein levels, as indicated by changes in GFP-levels of each strain, was used to identify compounds of interest. The hit rate for both inducers and inhibitors identified in the primary screen did not exceed 1% of the total number of compounds in the small-molecule library that was probed, as would be expected from a robust target-specific, high-throughput screening campaign. Secondary assays for virulence characteristics based on null mutant strains were then used to further validate specificity. In all, this study presents a method for the identification and verification of new antifungal drugs targeted to fungal virulence proteins using C. albicans as a model fungal pathogen.
doi:10.1371/journal.pone.0110354
PMCID: PMC4211665  PMID: 25350399
4.  The Mathematics of a Successful Deconvolution: A Quantitative Assessment of Mixture-Based Combinatorial Libraries Screened Against Two Formylpeptide Receptors 
Molecules (Basel, Switzerland)  2013;18(6):6408-6424.
In the past 20 years, synthetic combinatorial methods have fundamentally advanced the ability to synthesize and screen large numbers of compounds for drug discovery and basic research. Mixture-based libraries and positional scanning deconvolution combine two approaches for the rapid identification of specific scaffolds and active ligands. Here we present a quantitative assessment of the screening of 32 positional scanning libraries in the identification of highly specific and selective ligands for two formylpeptide receptors. We also compare and contrast two mixture-based library approaches using a mathematical model to facilitate the selection of active scaffolds and libraries to be pursued for further evaluation. The flexibility demonstrated in the differently formatted mixture-based libraries allows for their screening in a wide range of assays.
doi:10.3390/molecules18066408
PMCID: PMC4106117  PMID: 23722730
combinatorial libraries; mixture-based libraries; harmonic mean mixture model; mathematical modeling; formylpeptide receptors
5.  Rapid Scanning Structure-Activity Relationships in Combinatorial Data Sets: Identification of Activity Switches 
We present a general approach to describe the structure-activity relationships (SAR) of combinatorial data sets with activity for two biological endpoints with emphasis on the rapid identification of substitutions that have a large impact on activity and selectivity. The approach uses Dual-Activity Difference (DAD) maps that represent a visual and quantitative analysis of all pairwise comparisons of one, two, or more substitutions around a molecular template. Scanning the SAR of data sets using DAD maps allows the visual and quantitative identification of activity switches defined as specific substitutions that have an opposite effect on the activity of the compounds against two targets. The approach also rapidly identifies single- and double-target R-cliffs, i.e., compounds where a single or double substitution around the central scaffold dramatically modifies the activity for one or two targets, respectively. The approach introduced in this report can be applied to any analogue series with two biological activity endpoints. To illustrate the approach, we discuss the SAR of 106 pyrrolidine bis-diketopiperazines tested against two formylpeptide receptors obtained from positional scanning deconvolution methods of mixture-based libraries.
doi:10.1021/ci400192y
PMCID: PMC3715655  PMID: 23705689
combinatorial chemistry; data mining; data visualization; Dual-Activity Difference (DAD) maps; formylpeptide receptors; multi-target activity landscapes; R-cliffs; structure-activity relationships; positional scanning libraries
6.  Selective Chemical Inhibition of agr Quorum Sensing in Staphylococcus aureus Promotes Host Defense with Minimal Impact on Resistance 
PLoS Pathogens  2014;10(6):e1004174.
Bacterial signaling systems are prime drug targets for combating the global health threat of antibiotic resistant bacterial infections including those caused by Staphylococcus aureus. S. aureus is the primary cause of acute bacterial skin and soft tissue infections (SSTIs) and the quorum sensing operon agr is causally associated with these. Whether efficacious chemical inhibitors of agr signaling can be developed that promote host defense against SSTIs while sparing the normal microbiota of the skin is unknown. In a high throughput screen, we identified a small molecule inhibitor (SMI), savirin (S. aureus virulence inhibitor) that disrupted agr-mediated quorum sensing in this pathogen but not in the important skin commensal Staphylococcus epidermidis. Mechanistic studies employing electrophoretic mobility shift assays and a novel AgrA activation reporter strain revealed the transcriptional regulator AgrA as the target of inhibition within the pathogen, preventing virulence gene upregulation. Consistent with its minimal impact on exponential phase growth, including skin microbiota members, savirin did not provoke stress responses or membrane dysfunction induced by conventional antibiotics as determined by transcriptional profiling and membrane potential and integrity studies. Importantly, savirin was efficacious in two murine skin infection models, abating tissue injury and selectively promoting clearance of agr+ but not Δagr bacteria when administered at the time of infection or delayed until maximal abscess development. The mechanism of enhanced host defense involved in part enhanced intracellular killing of agr+ but not Δagr in macrophages and by low pH. Notably, resistance or tolerance to savirin inhibition of agr was not observed after multiple passages either in vivo or in vitro where under the same conditions resistance to growth inhibition was induced after passage with conventional antibiotics. Therefore, chemical inhibitors can selectively target AgrA in S. aureus to promote host defense while sparing agr signaling in S. epidermidis and limiting resistance development.
Author Summary
New approaches are needed to lessen the burden of antibiotic resistant bacterial infections. One strategy is to develop therapies that target virulence which rely on host defense elements to clear the bacteria rather than direct antimicrobial killing. Quorum sensing is a bacterial signaling mechanism that often regulates virulence in medically relevant bacterial pathogens. Therefore, drugs that inhibit quorum sensing can promote host defense by rendering the pathogenic bacteria avirulent and/or less fit for survival within the host. Our work addressed this strategy in the pathogen Staphylococcus aureus which is the major cause of acute bacterial skin and soft tissue infections. We conducted a high throughput screen to identify compounds that could inhibit signaling by the quorum sensing operon, agr. We found a compound that we termed savirin (S. aureus virulence inhibitor) that could inhibit signaling by this operon. The drug helped the innate immune system in animals to clear bacteria that express this operon without affecting clearance of bacteria that do not have this operon. We addressed the mechanism of action of this compound and whether resistance or tolerance to this compound would likely develop. Our data indicate for the first time that host defense against S. aureus skin infections can be enhanced by chemical inhibition of agr-mediated quorum sensing.
doi:10.1371/journal.ppat.1004174
PMCID: PMC4055767  PMID: 24945495
7.  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.
doi:10.1177/1087057112466697
PMCID: PMC4043149  PMID: 23160568
Cytotoxic T lymphocytes; exocytosis; flow cytometry; high-throughput screen; phenotypic assay
8.  Fluorescent substrates for flow cytometric evaluation of efflux inhibition in ABCB1, ABCC1, and ABCG2 transporters 
Analytical biochemistry  2013;437(1):77-87.
ATP binding cassette (ABC) transmembrane efflux pumps such as P-glycoprotein (ABCB1), multidrug resistance protein 1 (ABCC1), and breast cancer resistance protein (ABCG2) play an important role in anti-cancer drug resistance. A large number of structurally and functionally diverse compounds act as substrates or modulators of these pumps. In vitro assessment of the affinity of drug candidates for multidrug resistance proteins is central to predict in vivo pharmacokinetics and drug–drug interactions. The objective of this study was to identify and characterize new substrates for these transporters. As part of a collaborative project with Life Technologies, 102 fluorescent probes were investigated in a flow cytometric screen of ABC transporters. The primary screen compared substrate efflux activity in parental cell lines with their corresponding highly expressing resistant counterparts. The fluorescent compound library included a range of excitation/emission profiles and required dual laser excitation as well as multiple fluorescence detection channels. A total of 31 substrates with active efflux in one or more pumps and practical fluorescence response ranges were identified and tested for interaction with eight known inhibitors. This screening approach provides an efficient tool for identification and characterization of new fluorescent substrates for ABCB1, ABCC1, and ABCG2.
doi:10.1016/j.ab.2013.02.018
PMCID: PMC3785545  PMID: 23470221
Efflux inhibition; ABCB1; ABCC1; ABCG2; Fluorescent substrate; Flow cytometry
9.  Identification of Isoxsuprine Hydrochloride as a Neuroprotectant in Ischemic Stroke through Cell-Based High-Throughput Screening 
PLoS ONE  2014;9(5):e96761.
Stroke is a leading cause of death and disability and treatment options are limited. A promising approach to accelerate the development of new therapeutics is the use of high-throughput screening of chemical libraries. Using a cell-based high-throughput oxygen-glucose deprivation (OGD) model, we evaluated 1,200 small molecules for repurposed application in stroke therapy. Isoxsuprine hydrochloride was identified as a potent neuroprotective compound in primary neurons exposed to OGD. Isoxsuprine, a β2-adrenergic agonist and NR2B subtype-selective N-methyl-D-aspartate (NMDA) receptor antagonist, demonstrated no loss of efficacy when administered up to an hour after reoxygenation in an in vitro stroke model. In an animal model of transient focal ischemia, isoxsuprine significantly reduced infarct volume compared to vehicle (137±18 mm3 versus 279±25 mm3, p<0.001). Isoxsuprine, a peripheral vasodilator, was FDA approved for the treatment of cerebrovascular insufficiency and peripheral vascular disease. Our demonstration of the significant and novel neuroprotective action of isoxsuprine hydrochloride in an in vivo stroke model and its history of human use suggest that isoxsuprine may be an ideal candidate for further investigation as a potential stroke therapeutic.
doi:10.1371/journal.pone.0096761
PMCID: PMC4013073  PMID: 24804769
10.  Targeting the Transposase Domain of the DNA Repair Component Metnase to Enhance Chemotherapy 
Cancer research  2012;72(23):6200-6208.
Previous studies have shown that the DNA repair component Metnase (SETMAR) mediates resistance to DNA damaging cancer chemotherapy. Metnase has a nuclease domain that shares homology with the Transposase family. We therefore virtually screened the tertiary Metnase structure against the 550,000 compound ChemDiv library to identify small molecules that might dock in the active site of the transposase nuclease domain of Metnase. We identified eight compounds as possible Metnase inhibitors. Interestingly, among these candidate inhibitors were quinolone antibiotics and HIV integrase inhibitors, which share common structural features. Previous reports have described possible activity of quinolones as antineoplastic agents. Therefore, we chose the quinolone ciprofloxacin for further study, based on its wide clinical availability and low toxicity. We found that ciprofloxacin inhibits the ability of Metnase to cleave DNA and inhibits Metnase-dependent DNA repair. Ciprofloxacin on its own did not induce DNA damage, but it did reduce repair of chemotherapy-induced DNA damage. Ciprofloxacin increased the sensitivity of cancer cell lines and a xenograft tumor model to clinically relevant chemotherapy. These studies provide a mechanism for the previously postulated antineoplastic activity of quinolones, and suggest that ciprofloxacin might be a simple yet effective adjunct to cancer chemotherapy.
doi:10.1158/0008-5472.CAN-12-0313
PMCID: PMC3972061  PMID: 23090115
11.  High-Throughput Flow Cytometry Compatible Biosensor Based on Fluorogen Activating Protein Technology 
Monitoring the trafficking of multiple proteins simultaneously in live cells is of great interest because many receptor proteins are found to function together with others in the same cell. However, existing fluorescent labeling techniques have restricted the mechanistic study of functional receptor pairs. We have expanded a hybrid system combining fluorogen activating protein (FAP) technology and high-throughput flow cytometry to a new type of biosensor that is robust, sensitive, and versatile. This provides the opportunity to study multiple trafficking proteins in the same cell. Human beta2 adrenergic receptor (β2AR) fused with FAP AM2.2 and murine C-C chemokines receptor type 5 fused with FAP MG13 was chosen for our model system. The function of the receptor and the binding between MG13 and fluorogen MG-2p have been characterized by flow cytometry and confocal microscopy assays. The binding of fluorogen and the FAP pair is highly specific, while both FAP-tagged fusion proteins function similarly to their wild type counterparts. The system has successfully served as a counter screen assay to eliminate false positive compounds identified in a screen against NIH Molecular Libraries Small Molecule Repository targeting regulators of the human β2AR.
doi:10.1002/cyto.a.22242
PMCID: PMC3621705  PMID: 23303704
High-throughput; Flow Cytometry; Fluorogen Activating Protein; Live Cell; Protein Trafficking
12.  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.
doi:10.1177/1087057112456875
PMCID: PMC3623016  PMID: 22923785
Multi-drug resistance; ABC Transporter; ABCG2; ABCB1; Efflux inhibition
13.  Targeting GTPases in Parkinson’s disease: comparison to the historic path of kinase drug discovery and perspectives 
Neurological diseases have placed heavy social and financial burdens on modern society. As the life expectancy of humans is extended, neurological diseases, such as Parkinson’s disease, have become increasingly common among senior populations. Although the enigmas of Parkinson’s diseases await resolution, more vivid pictures on the cause, progression, and control of the illness are emerging after years of research. On the molecular level, GTPases are implicated in the etiology of Parkinson’s disease and are rational pharmaceutical targets for their control. However, targeting individual GTPases, which belong to a superfamily of proteins containing multiple members with a conserved guanine nucleotide binding domain, has proven to be challenging. In contrast, pharmaceutical pursuit of inhibition of kinases, which constitute another superfamily of proteins with more than 500 members, has been fairly successful. We reviewed the breakthroughs in the history of kinase drug discovery to provide guidance for the GTPase field. We summarize recent progress made in the regulation of GTPase activity. We also present an efficient and cost effective approach to drug screening, which uses multiplex flow cytometry and mixture-based positional scanning libraries. These methods allow simultaneous measurements of both the activity and the selectivity of the screened library. Several GTPase activator clusters were identified which showed selectivity against different GTPase subfamilies. While the clusters need to be further deconvoluted to identify individual active compounds, the method described here and the structure information gathered create a foundation for further developments to build upon.
doi:10.3389/fnmol.2014.00052
PMCID: PMC4046578  PMID: 24926233
Parkinson’s; GTPase; kinase; drug; multiplex
14.  Paroxetine Is a Direct Inhibitor of G Protein-Coupled Receptor Kinase 2 and Increases Myocardial Contractility 
ACS chemical biology  2012;7(11):1830-1839.
G protein-coupled receptor kinase 2 (GRK2) is a well-established therapeutic target for the treatment of heart failure. Herein we identify the selective serotonin reuptake inhibitor (SSRI) paroxetine as a selective inhibitor of GRK2 activity both in vitro and in living cells. In the crystal structure of the GRK2·paroxetine-Gβγ complex, paroxetine binds in the active site of GRK2 and stabilizes the kinase domain in a novel conformation in which a unique regulatory loop forms part of the ligand binding site. Isolated cardiomyocytes show increased isoproterenol-induced shortening and contraction amplitude in the presence of paroxetine, and pretreatment of mice with paroxetine before isoproterenol significantly increases left ventricular inotropic reserve in vivo with no significant effect on heart rate. Neither is observed in the presence of the SSRI fluoxetine. Our structural and functional results validate a widely available drug as a selective chemical probe for GRK2 and represent a starting point for the rational design of more potent and specific GRK2 inhibitors.
doi:10.1021/cb3003013
PMCID: PMC3500392  PMID: 22882301
15.  Overview: assays for studying integrin-dependent cell adhesion 
Interaction of the integrin receptors with ligands determines the molecular basis of integrin –dependent cell adhesion. Integrin ligands are typically large proteins with relatively low binding affinities. This makes direct ligand-binding kinetic measurements somewhat difficult. Here we examine several real-time methods, aimed to overcome these experimental limitations and to distinguish the regulation of integrin conformation and affinity. This chapter includes: the use of a small ligand-mimetic probe for studies of inside-out regulation of integrin affinity and unbending, real-time cell aggregation and disaggregation kinetics to probe integrin conformational states and the number of integrin-ligand bonds, as well as the real-time monitoring of ligand -induced epitopes under signaling through G-protein-coupled receptors, and others. Experimental data obtained using these novel methods are summarized in terms of the current model of integrin activation.
doi:10.1007/978-1-61779-166-6_1
PMCID: PMC3805125  PMID: 21909902
Ligand-receptor interaction; Ligand mimetic; Real-time kinetics; Cells adhesion; Inside-out signal; Monoclonal antibodies; Quantitative approaches
16.  Microbial Efflux Pump Inhibition: Tactics and Strategies 
Current pharmaceutical design  2011;17(13):1291-1302.
Traditional antimicrobials are increasingly suffering from the emergence of multidrug resistance among pathogenic microorganisms. To overcome these deficiencies, a range of novel approaches to control microbial infections are under investigation as potential alternative treatments. Multidrug efflux is a key target of these efforts. Efflux mechanisms are broadly recognized as major components of resistance to many classes of chemotherapeutic agents as well as antimicrobials. Efflux occurs due to the activity of membrane transporter proteins widely known as Multidrug Efflux Systems (MES). They are implicated in a variety of physiological roles other than efflux and identifying natural substrates and inhibitors is an active and expanding research discipline. One plausible alternative is the combination of conventional antimicrobial agents/antibiotics with small molecules that block MES known as multidrug efflux pump inhibitors (EPIs). An array of approaches in academic and industrial research settings, varying from high-throughput screening (HTS) ventures to bioassay guided purification and determination, have yielded a number of promising EPIs in a series of pathogenic systems. This synergistic discovery platform has been exploited in translational directions beyond the potentiation of conventional antimicrobial treatments. This venture attempts to highlight different tactical elements of this platform, identifying the need for highly informative and comprehensive EPI-discovery strategies. Advances in assay development genomics, proteomics as well as the accumulation of bioactivity and structural information regarding MES facilitates the basis for a new discovery era. This platform is expanding drastically. A combination of chemogenomics and chemoinformatics approaches will integrate data mining with virtual and physical HTS ventures and populate the chemical-biological interface with a plethora of novel chemotypes. This comprehensive step will expedite the preclinical development of lead EPIs.
PMCID: PMC3717411  PMID: 21470111
Multidrug resistance; efflux pump substrates and inhibitors; natural antimicrobials; high-throughput screening
17.  A Competitive Nucleotide Binding Inhibitor: In vitro Characterization of Rab7 GTPase Inhibition 
ACS chemical biology  2012;7(6):1095-1108.
Mapping the functionality of GTPases through small molecule inhibitors represents an underexplored area in large part due to the lack of suitable compounds. Here we report on the small chemical molecule 2-(benzoylcarbamothioylamino)-5,5-dimethyl-4,7-dihydrothieno[2,3-c]pyran-3-carboxylic acid (PubChem CID 1067700) as an inhibitor of nucleotide binding by Ras-related GTPases. The mechanism of action of this pan-GTPase inhibitor was characterized in the context of the Rab7 GTPase as there are no known inhibitors of Rab GTPases. Bead-based flow cytometry established that CID 1067700 has significant inhibitory potency on Rab7 nucleotide binding with nanomolar inhibitor (Ki) values and an inhibitory response of ≥97% for BODIPY-GTP and BODIPY-GDP binding. Other tested GTPases exhibited significantly lower responses. The compound behaves as a competitive inhibitor of Rab7 nucleotide binding based on both equilibrium binding and dissociation assays. Molecular docking analyses are compatible with CID 1067700 fitting into the nucleotide binding pocket of the GTP-conformer of Rab7. On the GDP-conformer, the molecule has greater solvent exposure and significantly less protein interaction relative to GDP, offering a molecular rationale for the experimental results. Structural features pertinent to CID 1067700 inhibitory activity have been identified through initial structure activity analyses and identified a molecular scaffold that may serve in the generation of more selective probes for Rab7 and other GTPases. Taken together, our study has identified the first competitive GTPase inhibitor and demonstrated the potential utility of the compound for dissecting the enzymology of the Rab7 GTPase as well as serving as a model for other small molecular weight GTPase inhibitors.
doi:10.1021/cb3001099
PMCID: PMC3440014  PMID: 22486388
Rab, Rho, Rac, Cdc42 and Ras GTPases; chemical biology; drug discovery; therapeutics; fluorescent GTP and GDP; enzyme kinetics
18.  Detection of Intracellular Granularity Induction in Prostate Cancer Cell Lines by Small Molecules Using the HyperCyt® High-Throughput Flow Cytometry System 
Journal of biomolecular screening  2009;14(6):596-609.
Prostate cancer is a leading cause of death among men due to the limited number of treatment strategies available for advanced disease. Discovery of effective chemotherapeutics involves the identification of agents that inhibit cancer cell growth. Increases in intracellular granularity have been observed during physiological processes that include senescence, apoptosis, and autophagy, making this phenotypic change a useful marker for identifying small molecules that induce cellular growth arrest or death. In this regard, epithelial-derived cancer cell lines appear uniquely susceptible to increased intracellular granularity following exposure to chemotherapeutics. We have established a novel flow cytometry approach that detects increases in side light scatter in response to morphological changes associated with intracellular granularity in the androgen-sensitive LNCaP and androgen-independent PC3 human prostate cancer cell lines. A cell-based assay was developed to screen for small molecule inducers of intracellular granularity using the HyperCyt® high-throughput flow cytometry platform. Validation was performed using the Prestwick Chemical Library, where known modulators of LNCaP intracellular granularity, such as testosterone, were identified. Nonandrogenic inducers of granularity were also detected. A further screen of ~25,000 small molecules led to the identification of a class of aryl-oxazoles that increased intracellular granularity in both cell lines, often leading to cell death. The most potent agents exhibited submicromolar efficacy in LNCaP and PC3 cells.
doi:10.1177/1087057109335671
PMCID: PMC3666167  PMID: 19470718
HyperCyt® high-throughput flow cytometry; small molecule screening; intracellular granularity; prostate cancer
19.  A Multifunctional Androgen Receptor Screening (MARS) Assay Using the High-Throughput HyperCyt® Flow Cytometry System 
Background
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.
Methods
Androgen-independent human prostate cancer-derived PC3 cells were transiently co-transfected 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 co-administered at sub-maximal concentrations with potential AR antagonists. The assay was formatted for high throughput screening using the HyperCyt® flow cytometry system.
Results
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 co-administered with low-dose R1881, which also occurred in a dose-dependent manner.
Conclusions
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. Due to 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.
doi:10.1002/cyto.a.20552
PMCID: PMC3655810  PMID: 18340645
Androgen receptor assay; androgens; antiandrogens; flow cytometry; HyperCyt®; biomolecular screening
20.  Cluster Cytometry for High Capacity Bioanalysis 
Cytometry  2012;81(5):419-429.
Flow cytometry specializes in high content measurements of cells and particles in suspension. Having long excelled in analytical throughput of single cells and particles, only recently with the advent of HyperCyt sampling technology has flow cytometry’s multi-experiment throughput begun to approach the point of practicality for efficiently analyzing hundreds-of-thousands of samples, the realm of high throughput screening (HTS). To extend performance and automation compatibility we built a HyperCyt-linked Cluster Cytometer platform, a network of flow cytometers for analyzing samples displayed in high-density, 1536-well plate format. To assess performance we used cell and microsphere based HTS assays that had been well characterized in previous studies. Experiments addressed important technical issues: challenges of small wells (assay volumes 10 μL or less, reagent mixing, cell and particle suspension), detecting and correcting for differences in performance of individual flow cytometers, and the ability to reanalyze a plate in the event of problems encountered during the primary analysis. Boosting sample throughput an additional four-fold, this platform is uniquely positioned to synergize with expanding suspension array and cell barcoding technologies in which as many as 100 experiments are performed in a single well or sample. As high-performance flow cytometers shrink in cost and size, cluster cytometry promises to become a practical, productive approach for HTS and other large scale investigations of biological complexity.
doi:10.1002/cyto.a.22039
PMCID: PMC3331957  PMID: 22438314
Flow cytometry; suspension array; high content analysis; high throughput screening
21.  Identification of a small molecule yeast TORC1 inhibitor with a flow cytometry-based multiplex screen 
ACS Chemical Biology  2012;7(4):715-722.
TOR (target of rapamycin) is a serine/threonine kinase, evolutionarily conserved from yeast to human, which functions as a fundamental controller of cell growth. The moderate clinical benefit of rapamycin in mTOR-based therapy of many cancers favors the development of new TOR inhibitors. Here we report a high throughput flow cytometry multiplexed screen using five GFP-tagged yeast clones that represent the readouts of four branches of the TORC1 signaling pathway in budding yeast. Each GFP-tagged clone was differentially color-coded and the GFP signal of each clone was measured simultaneously by flow cytometry, which allows rapid prioritization of compounds that likely act through direct modulation of TORC1 or proximal signaling components. A total of 255 compounds were confirmed in dose-response analysis to alter GFP expression in one or more clones. To validate the concept of the high throughput screen, we have characterized CID 3528206, a small molecule most likely to act on TORC1 as it alters GFP expression in all five GFP clones in an analogous manner to rapamycin. We have shown that CID 3528206 inhibited yeast cell growth, and that CID 3528206 inhibited TORC1 activity both in vitro and in vivo with EC50s of 150 nM and 3.9 μM, respectively. The results of microarray analysis and yeast GFP collection screen further support the notion that CID 3528206 and rapamycin modulate similar cellular pathways. Together, these results indicate that the HTS has identified a potentially useful small molecule for further development of TOR inhibitors.
doi:10.1021/cb200452r
PMCID: PMC3331904  PMID: 22260433
22.  A Crowdsourcing Evaluation of the NIH Chemical Probes 
Nature chemical biology  2009;5(7):441-447.
Between 2004 and 2008, the NIH molecular libraries and imaging initiative (MLI) pilot phase funded ten high-throughput Screening Centers, resulting in the deposition of 691 assays into PubChem and the nomination of 64 chemical probes. We crowdsourced the MLI output to 11 experts, who expressed medium or high levels of confidence in 48 of these 64 probes.
doi:10.1038/nchembio0709-441
PMCID: PMC3596119  PMID: 19536101
23.  A Miniature Couette to Generate Shear for Flow Cytometry: Studying Real-Time Modulation of Intracellular Calcium in Monocytic Cells 
Extracellular hydrodynamic forces may be transmitted to the interior of cells through the alteration of integrin conformation and affinity. Integrin activation regulates leukocyte recruitment, cell activation, and transmigration. The cellular and molecular mechanisms for integrin activation are not precisely known, although intracellular calcium signaling is involved. Flow cytometry offers a versatile way to study intracellular calcium signaling in real-time. We report a novel method to generate defined shear by using a miniature Couette. Testing involved measuring shear induced intracellular calcium signals of human monoblastoid U937 cells in suspension. The Couette was connected externally to a flow cytometer and pressurized at 6 PSI (4.1 N/m2). Cells were subjected to well-defined shear between 0 and 1000 s−1 and delivered continuously within 10 s to a FACScan at 1 μl/s. Intracellular calcium levels and the percentage of cells activated increased as shear increased in duration and intensity.
doi:10.1002/cyto.a.21027
PMCID: PMC3584716  PMID: 22045643
24.  Characterization of a Cdc42 Protein Inhibitor and Its Use as a Molecular Probe* 
The Journal of Biological Chemistry  2013;288(12):8531-8543.
Background: By integrating extracellular signals with actin cytoskeletal changes, Cdc42 plays important roles in cell physiology and has been implicated in human diseases.
Results: A small molecule was found to selectively inhibit Cdc42 in biochemical and cellular assays.
Conclusion: The identified compound is a highly Cdc42-selective inhibitor.
Significance: The described first-in-class Cdc42 GTPase-selective inhibitor will have applications in drug discovery and fundamental research.
Cdc42 plays important roles in cytoskeleton organization, cell cycle progression, signal transduction, and vesicle trafficking. Overactive Cdc42 has been implicated in the pathology of cancers, immune diseases, and neuronal disorders. Therefore, Cdc42 inhibitors would be useful in probing molecular pathways and could have therapeutic potential. Previous inhibitors have lacked selectivity and trended toward toxicity. We report here the characterization of a Cdc42-selective guanine nucleotide binding lead inhibitor that was identified by high throughput screening. A second active analog was identified via structure-activity relationship studies. The compounds demonstrated excellent selectivity with no inhibition toward Rho and Rac in the same GTPase family. Biochemical characterization showed that the compounds act as noncompetitive allosteric inhibitors. When tested in cellular assays, the lead compound inhibited Cdc42-related filopodia formation and cell migration. The lead compound was also used to clarify the involvement of Cdc42 in the Sin Nombre virus internalization and the signaling pathway of integrin VLA-4. Together, these data present the characterization of a novel Cdc42-selective allosteric inhibitor and a related analog, the use of which will facilitate drug development targeting Cdc42-related diseases and molecular pathway studies that involve GTPases.
doi:10.1074/jbc.M112.435941
PMCID: PMC3605667  PMID: 23382385
Cdc42; Cytoskeleton; GTPase; Integrin; Migration
25.  High throughput flow cytometry based yeast two-hybrid array approach for large-scale analysis of protein-protein interactions 
The analysis of protein-protein-interactions is a key focus of proteomics efforts. The yeast two-hybrid system has been the most commonly used method in genome-wide searches for protein interaction partners. However, the throughput of the current yeast two-hybrid array approach is hampered by the involvement of the time-consuming LacZ assay and/or the incompatibility of liquid handling automation due to the requirement for selection of colonies/diploids on agar plates. To facilitate large-scale yeast two-hybrid assays, we report a novel array approach by coupling a GFP reporter based yeast two-hybrid system with high throughput flow cytometry that enables the processing of a 96 well plate in as little as 3 minutes. In this approach, the yEGFP reporter has been established in both AH109 (MATa) and Y187 (MATα) reporter cells. It not only allows the generation of two copies of GFP reporter genes in diploid cells, but also allows the convenient determination of self-activators generated from both bait and prey constructs by flow cytometry. We demonstrate a Y2H array assay procedure that is carried out completely in liquid media in 96-well plates by mating bait and prey cells in liquid YPD media, selecting the diploids containing positive interaction pairs in selective media and analyzing the GFP reporter directly by flow cytometry. We have evaluated this flow cytometry based array procedure by showing that the interaction of the positive control pair P53/T is able to be reproducibly detected at 72 hrs post-mating compared to the negative control pairs. We conclude that our flow cytometry based yeast two-hybrid approach is robust, convenient, quantitative, and is amenable to large-scale analysis using liquid-handling automation.
doi:10.1002/cyto.a.21144
PMCID: PMC3250062  PMID: 21954189
HT flow cytometry; Protein-protein interaction; Yeast two-hybrid system; Array approach

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