The cohesin subunit Rad21 is a negative regulator of hematopoietic self-renewal through epigenetic repression of Hoxa7 and Hoxa9.

Acute myelogenous leukemia (AML) is a high-risk hematopoietic malignancy caused by a variety of mutations, including genes encoding the cohesin complex. Recent studies have demonstrated that reduction in cohesin complex levels leads to enhanced self-renewal in hematopoietic stem and progenitors (HSPCs). We sought to delineate the molecular mechanisms by which cohesin mutations promote enhanced HSPC self-renewal as this represents a critical initial step during leukemic transformation. We verified that RNAi against the cohesin subunit Rad21 causes enhanced self-renewal of HSPCs in vitro through derepression of polycomb repressive complex 2 (PRC2) target genes, including Hoxa7 and Hoxa9. Importantly, knockdown of either Hoxa7 or Hoxa9 suppressed self-renewal, implying that both are critical downstream effectors of reduced cohesin levels. We further demonstrate that the cohesin and PRC2 complexes interact and are bound in close proximity to Hoxa7 and Hoxa9. Rad21 depletion resulted in decreased levels of H3K27me3 at the Hoxa7 and Hoxa9 promoters, consistent with Rad21 being critical to proper gene silencing by recruiting the PRC2 complex. Our data demonstrates that the cohesin complex regulates PRC2 targeting to silence Hoxa7 and Hoxa9 and negatively regulate self-renewal. Our studies identify a novel epigenetic mechanism underlying leukemogenesis in AML patients with cohesin mutations.

Sickle cell disease.

Sickle cell disease (SCD) is an inherited hemoglobinopathy caused by a mutation in the sixth amino acid of the ß-globin gene (HBB). It is the most common serious genetic diseases in childhood, affecting approximately 1 in 2500 births and 100 000 individuals in the USA, in addition to 300 000 new cases globally each year. Central nervous system injury is the most debilitating frequent complication of SCD and includes stroke, silent cerebral infarct (SCI), and cognitive impairment. Among children with sickle cell anemia (HbSS), 11% had a stroke by age 18 years before the implementation of transcranial Doppler screening. SCI is identified in 27% of children with HbSS by their 5th birthday. Children who develop SCI have greater cognitive impairment compared with either children with HbSS without SCI or siblings without SCD. A recent study of adults demonstrated significant cognitive dysfunction, even in participants with apparently mild SCD.

Targeting efflux pumps to overcome antifungal drug resistance.

Resistance to antifungal drugs is an increasingly significant clinical problem. The most common antifungal resistance encountered is efflux pump-mediated resistance of Candida species to azole drugs. One approach to overcome this resistance is to inhibit the pumps and chemosensitize resistant strains to azole drugs. Drug discovery targeting fungal efflux pumps could thus result in the development of azole-enhancing combination therapy. Heterologous expression of fungal efflux pumps in Saccharomyces cerevisiae provides a versatile system for screening for pump inhibitors. Fungal efflux pumps transport a range of xenobiotics including fluorescent compounds. This enables the use of fluorescence-based detection, as well as growth inhibition assays, in screens to discover compounds targeting efflux-mediated antifungal drug resistance. A variety of medium- and high-throughput screens have been used to identify a number of chemical entities that inhibit fungal efflux pumps.

The Association between Educational Attainment and Patterns of Emergency Department Utilization among Adults with Sickle Cell Disease.

PURPOSE: Patients with low educational attainment may be at increased risk for unplanned health care utilization. This study aimed to determine what factors are related to emergency department (ED) visits in hopes of guiding treatments and early interventions. METHODS: At two medical centers in the Mid-Atlantic United States, 258 adults with sickle cell disease aged 19-70 years participated in a retrospective study where we examined whether education level is independently associated with ED visits after accounting for other socioeconomic status (SES) variables, such as pain and disease severity and psychosocial functioning. RESULTS: The data showed that patients without a high school education visited the ED three times as frequently as patients with post secondary education. Controlling for poverty and employment status decreased the effect of education on ED visits by 33.24 %. Further controlling for disease severity and/or psychosocial functioning could not account for the remaining association between education and ED visits, suggesting that education is independently associated with potentially avoidable emergency care. CONCLUSIONS: Early interventions addressing disparities in academic performance, especially for those children most at risk, may lead to improved long-term health outcomes in this population.

Discovery of Small-Molecule Nonfluorescent Inhibitors of Fluorogen-Fluorogen Activating Protein Binding Pair.

A new class of biosensors, fluorogen activating proteins (FAPs), has been successfully used to track receptor trafficking in live cells. Unlike the traditional fluorescent proteins (FPs), FAPs do not fluoresce unless bound to their specific small-molecule fluorogens, and thus FAP-based assays are highly sensitive. Application of the FAP-based assay for protein trafficking in high-throughput flow cytometry resulted in the discovery of a new class of compounds that interferes with the binding between fluorogens and FAP, thus blocking the fluorescence signal. These compounds are high-affinity, nonfluorescent analogs of fluorogens with little or no toxicity to the tested cells and no apparent interference with the normal function of FAP-tagged receptors. The most potent compound among these, N,4-dimethyl-N-(2-oxo-2-(4-(pyridin-2-yl)piperazin-1-yl)ethyl)benzenesulfonamide (ML342), has been investigated in detail. X-ray crystallographic analysis revealed that ML342 competes with the fluorogen, sulfonated thiazole orange coupled to diethylene glycol diamine (TO1-2p), for the same binding site on a FAP, AM2.2. Kinetic analysis shows that the FAP-fluorogen interaction is more complex than a homogeneous one-site binding process, with multiple conformational states of the fluorogen and/or the FAP, and possible dimerization of the FAP moiety involved in the process.

A high throughput flow cytometric assay platform targeting transporter inhibition.

This review highlights the concepts, recent applications and limitations of High Throughput Screening (HTS) flow cytometry-based efflux inhibitory assays. This platform has been employed in mammalian and yeast efflux systems leading to the identification of small molecules with transporter inhibitory capabilities. This technology offers the possibility of substrate multiplexing and may promote novel strategies targeting microbial efflux systems. This platform can generate a comprehensive dataset that may support efforts to map the interface between chemistry and transporter biology in a variety of pathogenic systems.

Fluorescent substrates for flow cytometric evaluation of efflux inhibition in ABCB1, ABCC1, and ABCG2 transporters.

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 anticancer 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.

Characterization of a Cdc42 protein inhibitor and its use as a molecular probe.

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.

A selective ATP-binding cassette subfamily 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 structure-activity relationship (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-overexpressing tumor model. At least two analogues 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.

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.

A competitive nucleotide binding inhibitor: in vitro characterization of Rab7 GTPase inhibition.

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 (K(i)) 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.

Identification of a small molecule yeast TORC1 inhibitor with a multiplex screen based on flow cytometry.

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 a manner analogous to that of 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 EC(50)'s 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.

Microbial efflux pump inhibition: tactics and strategies.

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.

Synthesis and anti-Hantaan virus activity of N(1)-3-fluorophenyl-inosine.

As part of an ongoing effort to develop new antiviral nucleoside analogs, our interest was drawn to N(1)-aryl purines as a novel structural class and potential scaffold for drug discovery. Herein, we describe the synthesis of N(1)-3-fluorophenyl-inosine (FPI) and N(1)-3-fluorophenyl-hypoxanthine (FP-Hx) and their antiviral activity against hantaviruses. The EC(50) for FPI and FP-Hx were 94 and 234microM, respectively, against Hantaan virus. FPI was not toxic to mammalian cells at concentrations that exhibited antiviral activity. Analysis of its metabolism revealed a low conversion of FPI in Vero E6 or human cells to a 5'-triphosphate, and it was a poor substrate for human purine nucleoside phosphorylase. Further, the compound did not alter GTP levels indicating FPI does not inhibit inosine monophosphate dehydrogenase. With respect to the virus, FPI did not decrease viral RNA levels or increase the mutation frequency of the viral RNA. This suggests that the antiviral activity of FPI might be solely due to the interaction of FPI or its metabolites with viral or host proteins involved in post-replication events that would affect the levels of infectious virus released. Synthesis of other compounds structurally similar to FPI is warranted to identify more potent agents that selectively abrogate production of infectious virus.

Detection of intracellular granularity induction in prostate cancer cell lines by small molecules using the HyperCyt high-throughput flow cytometry system.

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 approximately 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.

A novel fluorescent cross-reactive formylpeptide receptor/formylpeptide receptor-like 1 hexapeptide ligand.

Formylpeptide receptors (FPRs) are implicated in a variety of immunological and inflammatory response cascades. Further understanding of FPR-family ligand interactions could play an integral role in biological and therapeutic discovery. Fluorescent reporter ligands for the family are desirable experimental tools for increased understanding of ligand/receptor interactions. The ligand binding affinity and fluorescent reporting activity of the peptide WK(FL)YMVm was explored though use of the high throughput HyperCyt flow cytometric platform. Relative binding affinities of several known FPR and FPRL1 peptide ligands were compared in a duplex assay format. The fluorescent W-peptide ligand, WK(FL)YMVm, proved to be a high-affinity, cross-reactive reporter ligand for the FPR/FPRL1 duplex assay. Ligand specificity was demonstrated for each receptor, with known, selective peptide ligands. The binding site specificity of the reporter ligand was further verified by a fluorescent confocal microscopy internalization experiment. The fluorescent peptide ligand WK(FL)YMVm binds with high affinity to both FPR and FPRL1. The differential affinities of known peptide ligands were observed with the use of this fluorescent probe in high throughput screening flow cytometry.

Distinct reactions catalyzed by bacterial and yeast trans-aconitate methyltransferases.

The trans-aconitate methyltransferase from the bacterium Escherichia coli catalyzes the monomethyl esterification of trans-aconitate and related compounds. Using two-dimensional (1)H/(13)C nuclear magnetic resonance spectroscopy, we show that the methylation is specific to one of the three carboxyl groups and further demonstrate that the product is the 6-methyl ester of trans-aconitate (E-3-carboxy-2-pentenedioate 6-methyl ester). A similar enzymatic activity is present in the yeast Saccharomyces cerevisiae. Although we find that yeast trans-aconitate methyltransferase also catalyzes the monomethyl esterification of trans-aconitate, we identify that the methylation product of yeast is the 5-methyl ester (E-3-carboxyl-2-pentenedioate 5-methyl ester). The difference in the reaction catalyzed by the two enzymes may explain why a close homologue of the E. coli methyltransferase gene is not found in the yeast genome and furthermore suggests that these two enzymes may play distinct roles. However, we demonstrate here that the conversion of trans-aconitate to each of these products can mitigate its inhibitory effect on aconitase, a key enzyme of the citric acid cycle, suggesting that these methyltransferases may achieve the same physiological function with distinct chemistries.

Gender and family as moderators of the relationship between music video exposure and adolescent sexual permissiveness.

This study examined family environment and gender as moderators of an hypothesized relationship between exposure to rock music videos and premarital sexual attitudes and behavior. Results of a survey of 214 adolescents revealed a stronger association between permissive sexual attitudes and behavior and reported exposure to music videos for females than for males. As predicted, the association for females was much stronger for those from unsatisfactory family environments.

Sexual harassment: early adolescents' self-reports of experiences and acceptance.

Considerable attention has been focused on sexual harassment experiences and attitudes of older adolescents and adults. Recently, educational and judicial institutions have recognized that harassment also occurs among junior and senior high school students. The primary aim of this project was to gather information regarding early adolescents' experiences with and acceptance of sexual harassment behaviors. Results indicate a considerable proportion of females (50%) and males (37%) have been victims of sexual harassment perpetrated by their peers, even though their acceptance of these behaviors is quite low. Suggestions for a sexual harassment educational program for early adolescents are presented.

Inhibitors of human renin with C-termini derived from amides and esters of alpha-mercaptoalkanoic acids.

New transition-state analogues bearing C-termini derived from alpha-mercaptoalkanoic acids, esters, and amides were prepared and evaluated as inhibitors of human renin. Addition of alpha-mercaptoalkanoate esters to a chiral Boc-amino epoxide intermediate led ultimately to the target [(2R,3S)-3-(BocPheHis-amino)-4-cyclohexyl-2-hydroxy-1-butyl]thio derivatives. The corresponding sulfoxide and sulfone analogues were also investigated. Some of these derivatives, including one with a stable BocPhe replacement, were relatively potent inhibitors of human plasma renin, having IC50 values below 10 nM. When selected compounds were administered intravenously to sodium-deficient rhesus monkeys (Macaca mulatta) at 0.06-1 mg/kg, they reduced plasma renin activity by 87-94%. However, the accompanying drop in blood pressure was of short duration.