Small GTPases are key regulators of cellular activity and represent novel targets for the treatment of human diseases using small molecule inhibitors. We describe a multiplex, flow cytometry bead-based assay for the identification and characterization of inhibitors or activators of small GTPases. Six different GST-tagged small GTPases were bound to glutathione beads each labeled with a different red fluorescence intensity. Subsequently, beads bearing different GTPase were mixed and dispensed into 384-well plates with test compounds, and fluorescent-GTP binding was used as the read-out. This novel multiplex assay allowed us to screen a library of almost 200,000 compounds and identify over 1,200 positive compounds, which were further verified by dose response analyses, using 6 to 8-plex assays. After the elimination of false positive and negative compounds, several small molecule families with opposing effects on GTP-binding activity were identified. Here we detail the characterization of MLS000532223, a general inhibitor that prevents GTP-binding to several GTPases in a dose-dependent manner and is active in biochemical and cell-based secondary assays. Live cell imaging and confocal microscopy studies revealed the inhibitor-induced actin reorganization and cell morphology changes, characteristic of Rho GTPases inhibition. Thus, high throughput screening (HTS) via flow cytometry provides a strategy for identifying novel compounds that are active against small GTPases.

ABCB6 is a member of the adenosine triphosphate (ATP)-binding cassette family of transporter proteins that is increasingly recognized as a relevant physiological and therapeutic target. Evaluation of modulators of ABCB6 activity would pave the way toward a more complete understanding of the significance of this transport process in tumor cell growth, proliferation and therapy-related drug resistance. In addition, this effort would improve our understanding of the function of ABCB6 in normal physiology with respect to heme biosynthesis, and cellular adaptation to metabolic demand and stress responses. To search for modulators of ABCB6, we developed a novel cell-based approach that, in combination with flow cytometric high-throughput screening (HTS), can be used to identify functional modulators of ABCB6. Accumulation of protoporphyrin, a fluorescent molecule, in wild-type ABCB6 expressing K562 cells, forms the basis of the HTS assay. Screening the Prestwick Chemical Library employing the HTS assay identified four compounds, benzethonium chloride, verteporfin, tomatine hydrochloride and piperlongumine, that reduced ABCB6 mediated cellular porphyrin levels. Validation of the identified compounds employing the hemin-agarose affinity chromatography and mitochondrial transport assays demonstrated that three out of the four compounds were capable of inhibiting ABCB6 mediated hemin transport into isolated mitochondria. However, only verteporfin and tomatine hydrochloride inhibited ABCB6’s ability to compete with hemin as an ABCB6 substrate. This assay is therefore sensitive, robust, and suitable for automation in a high-throughput environment as demonstrated by our identification of selective functional modulators of ABCB6. Application of this assay to other libraries of synthetic compounds and natural products is expected to identify novel modulators of ABCB6 activity.

Purpose

Emergency departments (EDs) in the United States are used by patients with cancer for disease or treatment-related problems and unrelated issues. The North Carolina Disease Event Tracking and Epidemiologic Collection Tool (NC DETECT) collects information about ED visits through a statewide database.

Patients and Methods

After approval by the institutional review board, 2008 NC DETECT ED visit data were acquired and cancer-related visits were identified. Descriptive statistics and logistic regressions were performed. Of 4,190,911 ED visits in 2008, there were 37,760 ED visits by 27,644 patients with cancer.

Results

Among patients, 77.2% had only one ED visit in 2008, the mean age was 64 years, and there were slightly more men than women. Among visits, the payor was Medicare for 52.4% and Medicaid for 12.1%. More than half the visits by patients with cancer occurred on weekends or evenings, and 44.9% occurred during normal hours. The top three chief complaints were related to pain, respiratory distress, and GI issues. Lung, breast, prostate, and colorectal cancers were identified in 26.9%, 6.3%, 6%, and 7.7% of visits, respectively, with diagnosis. A total of 63.2% of visits resulted in hospital admittance. When controlling for sex, age, time of day, day of week, insurance, and diagnosis position, patients with lung cancer were more likely to be admitted than patients with other types of cancer.

Conclusion

To the best of our knowledge, this is the first study to provide a population-based snapshot of ED visits by patients with cancer in North Carolina. Efforts that target clinical problems and specific populations may improve delivery of quality cancer care and avoid ED visits.

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.

Ten years ago, we introduced a fluorescent probe that shed light on the inside-out regulation of one of the major leukocyte integrins, very late antigen-4 (VLA-4, CD49d/CD29). Here we describe the regulation of another leukocyte integrin, lymphocyte function-associated antigen-1 (LFA-1, CD11a/CD18) using a novel small fluorescent probe in real time on live cells. We found that multiple signaling mechanisms regulate LFA-1 conformation in a manner analogous to VLA-4. LFA-1 can be rapidly activated by Gαi-coupled G protein-coupled receptors (GPCRs) and deactivated by Gαs-coupled GPCRs. The effects of Gαs-coupled GPCR agonists can be reversed in real time by receptor-specific antagonists. The specificity of the fluorescent probe binding has been assessed in a competition assay using the natural LFA-1 ligand ICAM-1 and the LFA-1-specific α I allosteric antagonist BIRT0377. Similar to VLA-4 integrin, modulation of the ligand dissociation rate can be observed for different LFA-1 affinity states. However, we also found a striking difference in the binding of the small fluorescent ligand. In the absence of inside-out activation ligand, binding to LFA-1 is extremely slow, at least 10 times slower than expected for diffusion-limited binding. This implies that an additional structural mechanism prevents ligand binding to inactive LFA-1. We propose that such a mechanism explains the inability of LFA-1 to support cell rolling, where the absence of its rapid engagement by a counterstructure in the inactive state leads to a requirement for a selectin-mediated rolling step.

Background

More needs to be known about the prevalence of risk and protective factors for fires, burns, and carbon monoxide poisoning in U.S. households.

Methods

A random-digit-dial survey was conducted about home safety with 1003 respondents representing households in the continental United States. Descriptive statistics assess the prevalence of risk and protective factors for fires, burns, and carbon monoxide overall, and by demographic characteristics, household structure, region, and residential tenure. The data were weighted to adjust for nonresponse and to reflect the U.S. population.

Results

Although most respondents reported having a smoke alarm (97%), and 80% reported having one on each level of their home, <20% reported checking the alarm at least every 3 months. Seventy-one percent reported having a fire extinguisher, 29% had a carbon monoxide detector, and 51% of those living with at least one other person had a fire escape plan. Few could report the temperature of their hot water at the tap (9%), or the setting on the hot water heater (25%). Only 6% had an antiscald device.

Conclusions

Results suggest that there is much room for improvement regarding adoption of measures to prevent fires, burns, and carbon monoxide poisoning. Further investigations of the efficacy of carbon monoxide detectors, fire extinguishers, and escape plans, as well as effectiveness studies of fire and burn-prevention efforts are needed.

Fluorescence intensity of the pH-sensitive carboxyfluorescein derivative 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 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-term 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 in order to identify V-ATPase inhibitors. One compound, disulfiram, enhanced BCECF fluorescence intensity (although to a degree beyond anticipated for pH changes alone in the mutant cells). Once confirmed by dose response assays (EC50=26 μM), we verified V-ATPase inhibition by disulfiram in secondary assays which 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.

α4β1-Integrin (very late antigen-4 (VLA-4)) mediates cell adhesion to cell surface ligands (VCAM-1). Binding of VLA-4 to VCAM-1 initiates rolling and firm adhesion of leukocytes to vascular endothelium followed by the extravasation into the tissue. VLA-4-dependent adhesion plays a key role in controlling leukocyte adhesive events. Small molecules that bind to the integrin ligand-binding site and block its interaction with natural ligands represent promising candidates for treatment of several diseases. Following a flow cytometric screen for small molecule discovery, we took advantage of a conformationally sensitive anti-β1-integrin antibody (HUTS-21) and a small LDV-containing ligand (LDV-FITC) with known affinity to study binding affinities of several known and recently discovered integrin ligands. We found that binding of the LDV-containing small molecule induced exposure of HUTS-21 epitope and that the EC50 for antibody binding was equal to previously reported Kd for fluorescent LDV (LDV-FITC). Thus, binding of HUTS-21 can be used to report ligand-binding site occupancy. We studied binding of two known integrin ligands (YLDV and TR14035), as well as of two novel compounds. EC50 values for HUTS-21 binding showed good correlation with Kis determined in the competition assay with LDV-FITC for all ligands. A docking model suggests a common mode of binding for the small molecule VLA-4 ligands. This novel approach described here can be used to determine ligand-binding affinities for unlabeled integrin ligands, and can be adapted to a high-throughput screening format for identification of unknown integrin ligands.

Integrins are heterodimeric adhesion receptors that regulate immune cell adhesion. Integrin-dependent adhesion is controlled by multiple conformational states that include states with different affinity to the ligand, states with various degrees of molecule unbending, and others. Affinity change and molecule unbending play major roles in the regulation of cell adhesion. The relationship between different conformational states of the integrin is unclear. Here we have used conformationally sensitive antibodies and a small LDV-containing ligand to study the role of the inside-out signaling through formyl peptide receptor and CXCR4 in the regulation of α4β1 integrin conformation. We found that in the absence of ligand, activation by formyl peptide or SDF-1 did not result in a significant exposure of HUTS-21 epitope. Occupancy of the ligand binding pocket without cell activation was sufficient to induce epitope exposure. EC50 for HUTS-21 binding in the presence of LDV was identical to a previously reported ligand equilibrium dissociation constant at rest and after activation. Furthermore, the rate of HUTS-21 binding was also related to the VLA-4 activation state even at saturating ligand concentration. We propose that the unbending of the integrin molecule after guanine nucleotide-binding protein-coupled receptor-induced signaling accounts for the enhanced rate of HUTS-21 binding. Taken together, current results support the existence of multiple conformational states independently regulated by both inside-out signaling and ligand binding. Our data suggest that VLA-4 integrin hybrid domain movement does not depend on the affinity state of the ligand binding pocket.

Background

Activation of integrins in response to inside-out signaling serves as a basis for leukocyte arrest on endothelium, and migration of immune cells. Integrin-dependent adhesion is controlled by the conformational state of the molecule (i.e. change in the affinity for the ligand and molecular unbending (extension)), which is regulated by seven-transmembrane Guanine nucleotide binding Protein-Coupled Receptors (GPCRs). α4β1-integrin (CD49d/CD29, Very Late Antigen-4, VLA-4) is expressed on leukocytes, hematopoietic stem cells, hematopoietic cancer cells, and others. Affinity and extension of VLA-4 are both rapidly up-regulated by inside-out signaling through several Gαi-coupled GPCRs. The goal of the current report was to study the effect of Gαs-coupled GPCRs upon integrin activation.

Results

Using real-time fluorescent ligand binding to assess affinity and a FRET based assay to probe α4β1-integrin unbending, we show that two Gαs-coupled GPCRs (H2-histamine receptor and β2-adrenergic receptor) as well as several cAMP agonists can rapidly down modulate the affinity of VLA-4 activated through two Gαi-coupled receptors (CXCR4 and FPR) in U937 cells and primary human peripheral blood monocytes. This down-modulation can be blocked by receptor-specific antagonists. The Gαs-induced responses were not associated with changes in the expression level of the Gαi-coupled receptors. In contrast, the molecular unbending of VLA-4 was not significantly affected by Gαs-coupled GPCR signaling. In a VLA-4/VCAM-1-specific myeloid cell adhesion system, inhibition of the VLA-4 affinity change by Gαs-coupled GPCR had a statistically significant effect upon cell aggregation.

Conclusion

We conclude that Gαs-coupled GPCRs can rapidly down modulate the affinity state of VLA-4 binding pocket through a cAMP dependent pathway. This plays an essential role in the regulation of cell adhesion. We discuss several possible implications of this described phenomenon.

Emergency Department (ED) chief complaint (CC) data are key components of syndromic surveillance systems. However, it is difficult to use CC data because they are not standardized and contain varying semantic and lexical forms for the same concept. The purpose of this project was to revise a previously-developed text processor for preprocessing CC data specifically for syndromic surveillance and then evaluate it for acute respiratory illness surveillance to support decisions by public health epidemiologists. We evaluated the text processor accuracy and used the results to customize it for respiratory surveillance. We sampled 3,699 ED records from a population-based public health surveillance system. We found equal sensitivity, specificity, and positive and negative predictive value of syndrome queries of data processed through the text processor compared to a standard keyword method on raw, unprocessed data.

The sensitivity and specificity of syndrome definitions used in early event detection (EED) systems affect the usefulness of the system for end-users. The ability to calculate these values aids system designers in the refinement of syndrome definitions to better meet public health needs. Utilizing a stratified sampling method and expert review to create a gold standard dataset for the calculation of sensitivity and specificity, we describe how varying syndrome structure impacts these statistical parameters and discuss the relevance of this to outbreak detection and investigation.

The purpose of this poster is to describe plans for improving the functionality of the North Carolina’s early event detection system by allowing users to document and track alerts within the system. This unique approach to syndromic surveillance follow up aims to improve communication among the local, regional and state level users of the system, as well as reduce investigation time and eliminate duplication of effort.

Emergency Department (ED) data are key components of syndromic surveillance systems. While diagnosis data are widely available in electronic form from EDs and often used as a source of clinical data for syndromic surveillance, our previous survey of North Carolina EDs found that the data were not available in a timely manner for early detection. The purpose of this study was to measure the time of availability of participating EDs’ diagnosis data in a state-based syndromic surveillance system. We found that a majority of the ED visits transmitted to the state surveillance system for 12/1/05 did not have a diagnosis until more than a week after the visit. Reasons for the lack of timely transmission of diagnoses included coding problems, logistical issues and the lack of IT personnel at smaller hospitals.

Emergency Department (ED) data are a key component of bioterrorism surveillance systems. Little research has been done to examine differences in ED data capture and entry across hospitals, regions and states. The purpose of this study was to describe the current state of ED data for use in bioterrorism surveillance in 2 regions of the country. We found that chief complaint (CC) data are available electronically in 54% of the North Carolina EDs surveyed, and in 100% of the Seattle area EDs. Over half of all EDs reported that CCs are recorded in free text form. Though all EDs have electronic diagnosis data, less than half report that diagnoses are coded within 24 hours of the ED visit.