Low-affinity binding in cis to P2Y2R mediates force-dependent integrin activation during hantavirus infection.

Pathogenic hantaviruses bind to the plexin-semaphorin-integrin (PSI) domain of inactive, ß3 integrins. Previous studies have implicated a cognate cis interaction between the bent conformation ß5/ß3 integrins and an arginine-glycine-aspartic acid (RGD) sequence in the first extracellular loop of P2Y2R. With single-molecule atomic force microscopy, we show a specific interaction between an atomic force microscopy tip decorated with recombinant αIIbß3 integrins and (RGD)P2Y2R expressed on cell membranes. Mutation of the RGD sequence to RGE in the P2Y2R removes this interaction. Binding of inactivated and fluorescently labeled Sin Nombre virus (SNV) to the integrin PSI domain stimulates higher affinity for (RGD)P2Y2R on cells, as measured by an increase in the unbinding force. In CHO cells, stably expressing αIIbß3 integrins, virus engagement at the integrin PSI domain, recapitulates physiologic activation of the integrin as indicated by staining with the activation-specific mAB PAC1. The data also show that blocking of the Gα13 protein from binding to the cytoplasmic domain of the ß3 integrin prevents outside-in signaling and infection. We propose that the cis interaction with P2Y2R provides allosteric resistance to the membrane-normal motion associated with the switchblade model of integrin activation, where the development of tensile force yields physiological integrin activation.

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).

Quantitative bead-based flow cytometry for assaying Rab7 GTPase interaction with the Rab-interacting lysosomal protein (RILP) effector protein.

Rab7 facilitates vesicular transport and delivery from early endosomes to late endosomes as well as from late endosomes to lysosomes. The role of Rab7 in vesicular transport is dependent on its interactions with effector proteins, among them Rab-interacting lysosomal protein (RILP), which aids in the recruitment of active Rab7 (GTP-bound) onto dynein-dynactin motor complexes to facilitate late endosomal transport on the cytoskeleton. Here we detail a novel bead-based flow cytometry assay to measure Rab7 interaction with the Rab-interacting lysosomal protein (RILP) effector protein and demonstrate its utility for quantitative assessment and studying drug-target interactions. The specific binding of GTP-bound Rab7 to RILP is readily demonstrated and shown to be dose-dependent and saturable enabling K d and B max determinations. Furthermore, binding is nearly instantaneous and temperature-dependent. In a novel application of the assay method, a competitive small molecule inhibitor of Rab7 nucleotide binding (CID 1067700 or ML282) is shown to inhibit the Rab7-RILP interaction. Thus, the assay is able to distinguish that the small molecule, rather than incurring the active conformation, instead 'locks' the GTPase in the inactive conformation. Together, this work demonstrates the utility of using a flow cytometry assay to quantitatively characterize protein-protein interactions involving small GTPases and which has been adapted to high-throughput screening. Further, the method provides a platform for testing small molecule effects on protein-protein interactions, which can be relevant to drug discovery and development.

Discovery of regulators of receptor internalization with high-throughput flow cytometry.

We developed a platform combining 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 facilitates drug discovery for trafficking receptors such as G protein-coupled receptors and was validated with the ß2-adrenergic receptor (ß2AR) system. When a chemical library containing ∼1200 off-patent drugs was screened against cells expressing FAP-tagged ß2ARs, all 33 known ß2AR-active ligands in the library were successfully identified, together with a number of compounds that might regulate receptor internalization in a nontraditional manner. Results indicated that the platform identified ligands of target proteins regardless of the associated signaling pathway; therefore, this approach presents opportunities to search for biased receptor modulators and is suitable for screening of multiplexed targets for improved efficiency. The results revealed that ligands may be biased with respect to the rate or duration of receptor internalization and that receptor internalization may be independent of activation of the mitogen-activated protein kinase pathway.

Simultaneous in vitro molecular screening of protein-peptide interactions by flow cytometry, using six Bcl-2 family proteins as examples.

The B-cell lymphoma-2 (Bcl-2) family contains six antiapoptotic members, each with a hydrophobic pocket in which Bcl-2 homology region 3 (BH3) helices bind. This binding quenches apoptotic signals from activated BH3 family members. Many tumor cells either have increased expression of one of these six proteins or become overexpressed under treatment. Six fusion proteins made up of glutathione-S-transferase and each of the Bcl-2 members are bound individually to six glutathione bead sets, each set being easily distinguished by its different intensity of red fluorescence. The coated bead sets are washed, combined and incubated with green fluorescent Bim-BH3 peptide and a small molecule in 10-µl wells for 1 h. The green fluorescence signal for each bead set is resolved, and selective inhibitors are expected to reduce the signal for individual bead sets. Each 384-well plate is analyzed in 12 min, measuring 200 of 2,000 beads (∼10%) of each type per well.

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.

Drug Repurposing from an Academic Perspective.

Academia and small business research units are poised to play an increasing role in drug discovery, with drug repurposing as one of the major areas of activity. Here we summarize project status for a number of drugs or classes of drugs: raltegravir, cyclobenzaprine, benzbromarone, mometasone furoate, astemizole, R-naproxen, ketorolac, tolfenamic acid, phenothiazines, methylergonovine maleate and beta-adrenergic receptor drugs, respectively. Based on this multi-year, multi-project experience we discuss strengths and weaknesses of academic-based drug repurposing research. Translational, target and disease foci are strategic advantages fostered by close proximity and frequent interactions between basic and clinical scientists, which often result in discovering new modes of action for approved drugs. On the other hand, lack of integration with pharmaceutical sciences and toxicology, lack of appropriate intellectual coverage and issues related to dosing and safety may lead to significant drawbacks. The development of a more streamlined regulatory process world-wide, and the development of pre-competitive knowledge transfer systems such as a global healthcare database focused on regulatory and scientific information for drugs world-wide, are among the ideas proposed to improve the process of academic drug discovery and repurposing, and to overcome the "valley of death" by bridging basic to clinical sciences.

Flow cytometry for real-time measurement of guanine nucleotide binding and exchange by Ras-like GTPases.

Ras-like small GTPases cycle between GTP-bound active and GDP-bound inactive conformational states to regulate diverse cellular processes. Despite their importance, detailed kinetic or comparative studies of family members are rarely undertaken due to the lack of real-time assays measuring nucleotide binding or exchange. Here we report a bead-based flow cytometric assay that quantitatively measures the nucleotide binding properties of glutathione-S-transferase (GST) chimeras for prototypical Ras family members Rab7 and Rho. Measurements are possible in the presence or absence of Mg(2+), with magnesium cations principally increasing affinity and slowing nucleotide dissociation rates 8- to 10-fold. GST-Rab7 exhibited a 3-fold higher affinity for guanosine diphosphate (GDP) relative to guanosine triphosphate (GTP) that is consistent with a 3-fold slower dissociation rate of GDP. Strikingly, GST-Rab7 had a marked preference for GTP with ribose ring-conjugated BODIPY FL. The more commonly used gamma-NH-conjugated BODIPY FL GTP analogue failed to bind to GST-Rab7. In contrast, both BODIPY analogues bound equally well to GST-RhoA and GST-RhoC. Comparisons of the GST-Rab7 and GST-RhoA GTP binding pockets provide a structural basis for the observed binding differences. In sum, the flow cytometric assay can be used to measure nucleotide binding properties of GTPases in real time and to quantitatively assess differences between GTPases.

Estrogen receptors alpha (ESR1) and beta (ESR2) are expressed in circulating human lymphocytes.

Bone marrow thymocytes in part mediate the bone-preserving effects of estrogen by decreasing their production of osteoclast growth factors such as interleukin-1 and -6 and tumor necrosis factor alpha in the presence of physiological amounts of estradiol. Although several in vitro studies implicate the T-lymphocyte as a candidate mediator of estrogen signaling in the skeleton, whether these cells or any lymphocytes ordinarily express one or both nuclear estrogen receptors was previously unresolved. The purpose of our investigation was therefore to ascertain, by using real-time PCR, immmunoblotting, and cytometric techniques, if any of the nuclear estrogen receptors could be detected in normal peripheral blood mononuclear cells (PBMNC) collected from healthy volunteers. The results of immunoblotting experiments revealed that both estrogen receptor alpha (ESR1) and beta (ESR2) proteins are expressed in nuclei, but not in the cytoplasm of PBMNC harvested from all of the 15 healthy male and female volunteers (aged 23-50 years) we tested. PBMNCs contained mRNA coding for the two major full-length isoforms of ESR2 and the expression of ESR2 protein was localized within a lymphocyte subpopulation by cytometric analysis. Our data provide further evidence that lymphocytes and monocytes are responsive to estrogen and underscore its importance in modulating the immune response, as well as the vascular and skeletal health of men and women.

Rapid-mix flow cytometry measurements of subsecond regulation of G protein-coupled receptor ternary complex dynamics by guanine nucleotides.

We have used rapid-mix flow cytometry to analyze the early subsecond dynamics of the disassembly of ternary complexes of G protein-coupled receptors (GPCRs) immobilized on beads to examine individual steps associated with guanine nucleotide activation. Our earlier studies suggested that the slow dissociation of Galpha and Gbetagamma subunits was unlikely to be an essential component of cell activation. However, these studies did not have adequate time resolution to define precisely the disassembly kinetics. Ternary complexes were assembled using three formyl peptide receptor constructs (wild type, formyl peptide receptor-Galpha(i2) fusion, and formyl peptide receptor-green fluorescent protein fusion) and two isotypes of the alpha subunit (alpha(i2) and alpha(i3)) and betagamma dimer (beta(1)gamma(2) and beta(4)gamma(2)). At saturating nucleotide levels, the disassembly of a significant fraction of ternary complexes occurred on a subsecond time frame for alpha(i2) complexes and tau(1/2)< or =4s for alpha(i3) complexes, time scales that are compatible with cell activation. beta(1)gamma(2) isotype complexes were generally more stable than beta(4)gamma(2)-associated complexes. The comparison of the three constructs, however, proved that the fast step was associated with the separation of receptor and G protein and that the dissociation of the ligand or of the alpha and betagamma subunits was slower. These results are compatible with a cell activation model involving G protein conformational changes rather than disassembly of Galphabetagamma heterotrimer.

Some mechanistic insights into GPCR activation from detergent-solubilized ternary complexes on beads.

The binding of full and partial agonist ligands (L) to G protein-coupled receptors (GPCRs) initiates the formation of ternary complexes with G proteins [ligand-receptor-G protein (LRG) complexes]. Cyclic ternary complex models are required to account for the thermodynamically plausible complexes. It has recently become possible to assemble solubilized formyl peptide receptor (FPR) and beta(2)-adrenergic receptor (beta(2)AR) ternary complexes for flow cytometric bead-based assays. In these systems, soluble ternary complex formation of the receptors with G proteins allows direct quantitative measurements which can be analyzed in terms of three-dimensional concentrations (molarity). In contrast to the difficulty of analyzing comparable measurements in two-dimensional membrane systems, the output of these flow cytometric experiments can be analyzed via ternary complex simulations in which all of the parameters can be estimated. An outcome from such analysis yielded lower affinity for soluble ternary complex assembly by partial agonists compared with full agonists for the beta(2)AR. In the four-sided ternary complex model, this behavior is consistent with distinct ligand-induced conformational states for full and partial agonists. Rapid mix flow cytometry is used to analyze the subsecond dynamics of guanine nucleotide-mediated ternary complex disassembly. The modular breakup of ternary complex components is highlighted by the finding that the fastest step involves the departure of the ligand-activated GPCR from the intact G protein heterotrimer. The data also show that, under these experimental conditions, G protein subunit dissociation does not occur within the time frame relevant to signaling. The data and concepts are discussed in the context of a review of current literature on signaling mechanism based on structural and spectroscopic (FRET) studies of ternary complex components.

Duplexed, bead-based competitive assay for inhibitors of protein kinases.

BACKGROUND: Many cellular signal transduction cascades have protein kinases as critical components. Small molecule protein kinase inhibitors can be effective as laboratory probes and drugs. Methods that allow two or more kinases to be evaluated simultaneously for inhibition by a small molecule would allow unequivocal tests of specificity and selectivity of action of the small molecule. METHODS: Two hexahistidine-tagged activin receptor-like kinases were expressed in E. coli, purified, and bound to nickel beads. A fluorescent kinase ligand (F-KL) that binds to the ATP-binding site of these kinases with nanomolar affinity was developed. Binding of F-KL with kinase on the bead made the beads bright, and inhibitors decreased the brightness. RESULTS: A test panel of 17 nonfluorescent kinase inhibitors, spanning two orders of magnitude affinity for the kinases, gave K(d) values for the kinases that correlated well with a fluorescence polarization assay. Results were obtained for the kinases in duplex, using an autosampler to send beads from a 96-well plate to a flow cytometer in a format suitable for high throughput screening. CONCLUSIONS: Inhibitors of kinases can be measured in duplex in a high throughput format by flow cytometry, if a suitable fluorescent ligand is available.

Glutathione-S-transferase-green fluorescent protein fusion protein reveals slow dissociation from high site density beads and measures free GSH.

BACKGROUND: Glutathione, a ubiquitous tripeptide, is an important cellular constituent, and measurement of reduced and oxidized glutathione is a measure of the redox state of cells. Glutathione-S-transferase (GST) fusion proteins bind naturally to beads derivatized with glutathione, and elution of such bead-bound fusion proteins with buffer containing millimolar glutathione is a commonly used method of protein purification. Many protein-protein interactions have been established by using GST fusion proteins and measuring binding of fusion protein binding partners by GST pulldown assays, usually monitored by Western blot methodology. METHODS: Dextran beads suitable for flow cytometry were derivatized with glutathione. A fusion protein of GST and green fluorescent protein was used to define kinetic and equilibrium binding characteristics of GST fusion proteins to glutathione beads. Free glutathione competes with this binding, and this competition was used to measure free glutathione concentration. RESULTS: A 10 microl assay can measure 5 microl of 20 microM glutathione (100 pmol glutathione) in 2 h by flow cytometry. This concentration is two orders of magnitude lower than cellular glutathione concentrations, and three orders of magnitude lower than affinity chromatography eluates. One important result is that by generating high site density, the GST fusion proteins can be constrained to the surface of one bead without hopping to the next bead in multiplex assays. CONCLUSIONS: Glutathione in cellular lysates and GST-fusion protein affinity chromatography eluates can be measured by flow cytometry. Many interactions between GST fusion proteins and their fluorescent binding partners should be quantifiable by flow cytometry. Although a system may have the disadvantage that it has a low affinity and a correspondingly quick off-rate in solution, it may remain on beads if the site density can be increased to offer a slow apparent off rate.

Dynamics of fluorescence dequenching of ostrich-quenched fluorescein biotin: a multifunctional quantitative assay for biotin.

We describe a simple and rapid quantitative assay for biotin and biotin conjugates. The assay is based on the kinetic analysis of the enhancement of fluorescence of streptavidin/fluorescein biotin complexes in the presence of biotin. The kinetic response of fluorescence enhancement is proportional to the concentration of biotin. Standard calibration curves based on the kinetic response are obtained and detection limits of approximately 10(-9)M are established. Because the assay is amenable for use in small volumes of 5-50 microL or bead-based assays, the detection limits can be extended to the femtomole range. Since the assay depends on kinetic analysis, routine quantitation can be achieved without reference to standard curves. The dynamic aspects allow the assay to be extended to a broader range of applications including its use as an indicator of reagent mixing in laminar-flow assays carried out in microfluidic devices.

Techniques: GPCR assembly, pharmacology and screening by flow cytometry.

Flow cytometers are well known for their ability to analyze and sort cells at high rates based on physiological responses and expression of protein markers. The potential for flow cytometry in G-protein-coupled receptor (GPCR) research, however, is less well appreciated. Potential applications include: (i) the homogenous discrimination of free and bound ligands or proteins in both cellular and microsphere-based assays; and (ii) multiplexed ('suspension array') analysis of cell responses and protein-protein interactions. Innovative sample-handling systems also provide sub-second resolution of interaction kinetics and 1 second per well throughput of microliter-sized samples from multiwell plates. Flow cytometric methods using microspheres for analysis of GPCRs that interact with intracellular and extracellular binding partners such as ligands, G proteins and kinases have been established. These analyses can produce quantitative pharmacological data analogous to radioligand assays, and, in some cases, the probes can be integrated into the assembly as fluorescent fusion proteins.

Real-time analysis of ternary complex on particles: direct evidence for partial agonism at the agonist-receptor-G protein complex assembly step of signal transduction.

We developed a novel and generalized approach to investigate G protein-coupled receptor molecular assemblies. We solubilized a fusion protein consisting of the beta(2)-adrenergic receptor and green fluorescent protein (GFP) for bead-based flow cytometric analysis. beta(2)-Adrenergic receptor GFP bound to dihydroalprenolol-conjugated beads, providing a K(d) for the fusion protein and, in competition with beta(2)-adrenergic receptor ligands, K(d) values for agonists and antagonists. Beads displaying chelated nickel bound purified hexahistidine-tagged G protein heterotrimers and, subsequently, the binary complex of agonist with beta(2)-adrenergic receptor GFP. The dose-response curves of ternary complex formation revealed maximal assembly for ligands previously classified as full agonists and reduced assembly for ligands previously classified as partial agonists. Guanosine 5'-3-O-(thio)triphosphate-induced dissociation rates of the ternary complex were the same for full and partial agonists. Soluble G protein, competing with ternary complexes on beads provided an affinity estimate of agonist-receptor complexes to G protein. When performed simultaneously, the two assemblies discriminated between agonist, antagonist or inactive molecule in a manner appropriate for high throughput, small volume drug discovery. The assemblies can be further generalized to other G protein coupled receptor protein-protein interactions.

Ligand-receptor-G-protein molecular assemblies on beads for mechanistic studies and screening by flow cytometry.

G protein-coupled receptors form a ternary complex of ligand, receptor, and G protein heterotrimer (LRG) during signal transduction from the outside to the inside of a cell. Our goal was to develop a homogeneous, small-volume, bead-based approach compatible with high-throughput flow cytometry that would allow evaluation of G protein coupled receptor molecular assemblies. Dextran beads were derivatized to carry chelated nickel to bind hexahistidine-tagged green fluorescent protein (GFP) and hexahistidine-tagged G proteins. Ternary complexes were assembled on these beads using fluorescent ligand with wild-type receptor or a receptor-Gialpha2 fusion protein, and with a nonfluorescent ligand and receptor-GFP fusion protein. Streptavidin-coated polystyrene beads used biotinylated anti-FLAG antibodies to bind FLAG-tagged G proteins for ternary complex assembly. Validation was achieved by showing time and concentration dependence of ternary complex formation. Affinity measurements of ligand for receptor on particles, of the ligand-receptor complex for G protein on the particles, and receptor-Gialpha2 fusion protein for Gbetagamma, were consistent with comparable assemblies in detergent suspension. Performance was assessed in applications representing the potential of these assemblies for ternary complex mechanisms. We showed the relationship for a family of ligands between LR and LRG affinity and characterized the affinity of both the wild-type and GFP fusion receptors with G protein. We also showed the potential of kinetic measurements to allow observation of individual steps of GTP-induced ternary complex disassembly and discriminated a fast step caused by RG disassembly compared with the slower step of Galphabetagamma disassembly.