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PUBLICATIONS

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103 Total Publications
HOME | RESOURCES | Publications

PUBLICATIONS

103 Total Publications

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TITLES and AUTHORS Year Link PDF + Abstract
  • A High-Throughput Screening Assay for Inhibitors of Bacterial Motility Identifies a Novel Inhibitor of the Na+-Driven Flagellar Motor and Virulence Gene Expression in Vibrio cholerae Rasmussen L, White E L, Pathak A, Ayala J C,Wang H, Wu J H, Benitez J, Silva A J

    Institution: Southern Research Institute, Morehouse School of Medicine

    Publication: Antimicrob Agents Chemother. 2011 September; 55(9): 4134–4143. doi:  10.1128/AAC.00482-11

    2011 abstract

    Numerous bacterial pathogens, particularly those that colonize fast-flow areas in the bladder and gastrointestinal tract, require motility to establish infection and spread beyond the initially colonized tissue. Vibrio cholerae strains of serogroups O1 and O139, the causative agents of the diarrheal illness cholera, express a single polar flagellum powered by sodium motive force and require motility to colonize and spread along the small intestine. Therefore, motility may be an attractive target for small molecules that can prevent and/or block the infective process. In this study, we describe a high-throughput screening (HTS) assay to identify small molecules that selectively inhibit bacterial motility. The HTS assay was used to screen an ∼8,000-compound structurally diverse chemical library for inhibitors of V. cholerae motility. The screen identified a group of quinazoline-2,4-diamino analogs that completely suppressed motility without affecting the growth rate in broth. A further study on the effects of one analog, designated Q24DA, showed that it induces a flagellated but nonmotile (Mot) phenotype and is specific for the Na+-driven flagellar motor of pathogenic Vibrio species. A mutation conferring phenamil-resistant motility did not eliminate inhibition of motility by Q24DA. Q24DA diminished the expression of cholera toxin and toxin-coregulated pilus as well as biofilm formation and fluid secretion in the rabbit ileal loop model. Furthermore, treatment of V. cholerae with Q24DA impacted additional phenotypes linked to Na+ bioenergetics, such as the function of the primary Na+ pump, Nqr, and susceptibility to fluoroquinolones. The above results clearly show that the described HTS assay is capable of identifying small molecules that specifically block bacterial motility. New inhibitors such as Q24DA may be instrumental in probing the molecular architecture of the Na+-driven polar flagellar motor and in studying the role of motility in the expression of other virulence factors.

    Publication / Type:
    Antimicrob Agents Chemother. 2011 September; 55(9): 4134–4143. doi:  10.1128/AAC.00482-11
    Related Subject:
    High-Throughput Screening Assay Na+-Driven Flagellar Motor Virulence in Vibrio cholerae
    Link:
    http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3165335/
  • A Bioluminogenic HDAC Activity Assay: Validation and Screening Halley F, Reinshagen J, Ellinger B, Wolf M, Niles A L, Evans N J, Kirkland T A, Wagner J M, Jung M,

    Institution: European ScreeningPort GmbH, Promega Corporation, Promega Biosciences, Institute of Pharmaceutical S

    Publication: J Biomol Screen December 2011 vol. 16 no. 10 1227-1235

    2011 abstract

    Histone deacetylase (HDAC) enzymes modify the acetylation state of histones and other important proteins. Aberrant HDAC enzyme function has been implicated in many diseases, and the discovery and development of drugs targeting these enzymes is becoming increasingly important. In this article, the authors report the evaluation of homogeneous, single-addition, bioluminogenic HDAC enzyme activity assays that offer less assay interference by compounds in comparison to fluorescence-based formats. The authors assessed the key operational assay properties including sensitivity, scalability, reproducibility, signal stability, robustness (Z′), DMSO tolerance, and pharmacological response to standard inhibitors against HDAC-1, HDAC-3/NcoR2, HDAC-6, and SIRT-1 enzymes. These assays were successfully miniaturized to a 10 µL assay volume, and their suitability for high-throughput screening was tested in validation experiments using 640 drugs approved by the Food and Drug Administration and the Hypha Discovery MycoDiverse natural products library, which is a collection of 10 049 extracts and fractions from fermentations of higher fungi and contains compounds that are of low molecular weight and wide chemical diversity. Both of these screening campaigns confirmed that the bioluminogenic assay was high-throughput screening compatible and yielded acceptable performance in confirmation, counter, and compound/extract and fraction concentration-response assays.

    Publication / Type:
    J Biomol Screen December 2011 vol. 16 no. 10 1227-1235
    Related Subject:
    Bioluminogenic HDAC Activity Assay Screening
    Link:
    http://jbx.sagepub.com/content/16/10/1227.abstract
  • CYP3A Time-Dependent Inhibition Risk Assessment Validated with 400 Reference Drugs Zimmerlin A, Trunzer M, Faller B

    Institution: Novartis Institutes for BioMedical Research

    Publication: DMD June 2011 vol. 39 no. 6 1039-1046, DOI <span class="slug-metadata-note ahead-of-print"><

    2011 abstract

    Although reversible CYP3A inhibition testing is well established for predicting the drug-drug interaction potential of clinical candidates, time-dependent inhibition (TDI) has become the focus of drug designers only recently. Failure of several late-stage clinical candidates has been attributed to TDI, and this mechanism is also suspected to play a role in liver toxicities often observed in preclinical species. Measurement of enzyme inactivation rates (kinact and KI) is technically challenging, and a great deal of variability can be found in the literature. In this article, we have evaluated the TDI potential for 400 registered drugs using a high-throughput assay format based on determination of the inactivation rate (kobs) at a single concentration of test compound (10 M). The advantages of this new assay format are highlighted by comparison with data generated using the IC50 shift assay, a current standard approach for preliminary assessment of TDI. With use of an empirically defined positive/negative kobs bin of 0.02 min1, only 4% of registered drugs were found to be positive. This proportion increased to more than 20% when in-house lead optimization molecules were considered, emphasizing the importance of identifying this property in selection of promising drug candidates. Finally, it is suggested that the data and technology described here may be a good basis for building structure-activity relationships and in silico modeling.

    Publication / Type:
    DMD June 2011 vol. 39 no. 6 1039-1046, DOI <span class="slug-metadata-note ahead-of-print"><
    Related Subject:
    CYP3A Time-Dependent Risk 400 Reference Drugs
    Link:
    http://dmd.aspetjournals.org/content/39/6/1039.full.pdf+html
  • Screening and Identification of a Novel Class of TGF-β Type 1 Receptor Kinase Inhibitor Huynh QK, Wise SJ, Koch KA, Castonguay LA, Reid BG, Pagratis EE, Koditek D, Glascock CB, Pitts KR, T

    Institution: Gilead Sciences

    Publication: J Biomol Screen August 2011 vol. 16 no. 7 724-733, 10.1177/1087057111405846

    2011 abstract

    Transforming growth factor β (TGF-β) type I receptor (activin receptor–like kinase 5, ALK5) has been identified as a promising target for fibrotic diseases. To find a novel inhibitor of ALK5, the authors performed a high-throughput screen of a library of 420 000 compounds using dephosphorylated ALK5. From primary hits of 1521 compounds, 555 compounds were confirmed. In total, 124 compounds were then selected for follow-up based on their unique structures and other properties. Repeated concentration–response testing and final interference assays of the above compounds resulted in the discovery of a structurally novel ALK5 inhibitor (compound 8) (N-(thiophen 2-ylmethyl)-3-(3,4,5 trimethoxyphenyl)imidazo[1,2β]pyridazin 6-amine) with a low IC50 value of 0.7 µM. Compound 8 also inhibited the TGF-β-induced nuclear translocation of SMAD with an EC50 value of 0.8 µM. Kinetic analysis revealed that compound 8 inhibited ALK5 via mixed-type inhibition, suggesting that it may bind to ALK5 differently than other published adenosine triphosphate site inhibitors.  

    Publication / Type:
    J Biomol Screen August 2011 vol. 16 no. 7 724-733, 10.1177/1087057111405846
    Related Subject:
    TGF-β Type 1 Receptor Kinase Inhibitor
    Link:
    http://jbx.sagepub.com/content/16/7/724
  • Acoustically Mounted Microcrystals Yield High-Resolution X-Ray Structures Soares AS, Engle MA, Stearns R, Datwani S, Olechno J, Ellson R, Skinner JM, Allaire M, Orville AM

    Institution: Brookhaven National Laboratory, Labcyte Inc.

    Publication: Biochemistry. 2011 May 31; 50(21): 4399–4401.

    2011 abstract

    We demonstrate a general strategy for determining structures from showers of microcrystals. It uses acoustic droplet ejection to transfer 2.5 nL droplets from the surface of microcrystal slurries, through the air, onto mounting micromesh pins. Individual microcrystals are located by raster-scanning a several-micrometer X-ray beam across the cryocooled micromeshes. X-ray diffraction data sets merged from several micrometer-sized crystals are used to determine 1.8 Ǻ resolution crystal structures.

    Publication / Type:
    Biochemistry. 2011 May 31; 50(21): 4399–4401.
    Related Subject:
    Acoustically Mounted Microcrystals High-Resolution X-Ray Structures
    Link:
    http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3144476/
  • Discovery of a Novel Chemical Class of mGlu5 Allosteric Ligands with Distinct Modes of Pharmacology Hammond A S, Rodriguez A L, Townsend S D, Niswender C M, Gregory K J, Lindsley C W, Conn P J

    Institution: Vanderbilt University

    Publication: ACS Chem Neurosci. 2010 October 20; 1(10): 702–716.  doi:  10.1021/cn100051m

    2010 abstract

    We previously discovered a positive allosteric modulator (PAM) of the metabotropic glutamate receptor subtype 5 (mGlu5) termed 4 N-{4-chloro-2-[(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)methyl]phenyl}-2-hydroxybenzamide (CPPHA) that elicits receptor activation through a novel allosteric site on mGlu5, distinct from the classical mGlu5 negative allosteric modulator (NAM) MPEP allosteric site. However, a shallow structure−activity relationship (SAR), poor physiochemical properties, and weak PAM activity at rat mGlu5 limited the utility of CPPHA to explore allosteric activation of mGlu5 at a non-MPEP site. Thus, we performed a functional high-throughput screen (HTS) and identified a novel mGlu5 PAM benzamide scaffold, exemplified by VU0001850 (EC50 = 1.3 μM, 106% Glumax) and VU0040237 (EC50 = 350 nM, 84% Glu Max). An iterative parallel synthesis approach delivered 22 analogues, optimized mGlu5 PAM activity to afford VU0357121 (EC50 = 33 nM, 92% Glumax), and also revealed the first non-MPEP site neutral allosteric ligand (VU0365396). Like CPPHA, PAMs within this class do not appear to bind at the MPEP allosteric site based on radioligand binding studies. Moreover, mutagenesis studies indicate that VU0357121 and related analogues bind to a yet uncharacterized allosteric site on mGlu5, distinct from CPPHA, yet share a functional interaction with the MPEP site.

    Publication / Type:
    ACS Chem Neurosci. 2010 October 20; 1(10): 702–716.  doi:  10.1021/cn100051m
    Related Subject:
    mGlu5 Allosteric Pharmacology
    Link:
    http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2957851/
  • Molecular Target Class Is Predictive of In vitro Response Profile Greshock J, Bachman K E, Degenhardt Y Y, Jing J, Wen Y H, Eastman S, McNeil E, Moy C, Wegrzyn R, Aug

    Institution: GlaxoSmithKline, North Carolina State University

    Publication: Cancer Res; 70(9); 3677–86.

    2010 abstract

    Preclinical cellular response profiling of tumor models has become a cornerstone in the development of novel cancer therapeutics. As efforts to predict clinical efficacy using cohorts of in vitro tumor models have been successful, expansive panels of tumor-derived cell lines can recapitulate an “all comers” efficacy trial, thereby identifying which tumors are most likely to benefit from treatment. The response profile of a therapy is most often studied in isolation; however, drug treatment effect patterns in tumor models across a diverse panel of compounds can help determine the value of unique molecular target classes in specific tumor cohorts. To this end, a panel of 19 compounds was evaluated against a diverse group of cancer cell lines (n = 311). The primary oncogenic targets were a key determinant of concentration-dependent proliferation response, as a total of five of six, four of four, and five of five phosphatidylinositol 3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) pathway, insulin-like growth factor-I receptor (IGF-IR), and mitotic inhibitors, respectively, clustered with others of that common target class. In addition, molecular target class was correlated with increased responsiveness in certain histologies. A cohort of PI3K/AKT/mTOR inhibitors was more efficacious in breast cancers compared with other tumor types, whereas IGF-IR inhibitors more selectively inhibited growth in colon cancer lines. Finally, specific phenotypes play an important role in cellular response profiles. For example, luminal breast cancer cells (nine of nine; 100%) segregated from basal cells (six of seven; 86%). The convergence of a common cellular response profile for different molecules targeting the same oncogenic pathway substantiates a rational clinical path for patient populations most likely to benefit from treatment.

    Publication / Type:
    Cancer Res; 70(9); 3677–86.
    Related Subject:
    Molecular Target Predictive of In vitro Response
    Link:
    http://cancerres.aacrjournals.org/content/70/9/3677.long
  • Novel HTS Strategy Identifies TRAIL-Sensitizing Compounds Acting Specifically Through the Caspase-8 Apoptotic Axis Finlay D, Richardson R D, Landberg L K, Howes A L, Vuori K

    Institution: Sanford-Burnham Medical Research Institute, La Jolla, California

    Publication: PLoS One. 2010; 5(10): e13375. doi:  10.1371/journal.pone.0013375

    2010 abstract

    Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand (TRAIL) is potentially a very important therapeutic as it shows selectivity for inducing apoptosis in cancer cells whilst normal cells are refractory. TRAIL binding to its cognate receptors, Death Receptors-4 and -5, leads to recruitment of caspase-8 and classical activation of downstream effector caspases, leading to apoptosis. As with many drugs however, TRAIL's usefulness is limited by resistance, either innate or acquired. We describe here the development of a novel 384-well high-throughput screening (HTS) strategy for identifying potential TRAIL-sensitizing agents that act solely in a caspase-8 dependent manner. By utilizing a TRAIL resistant cell line lacking caspase-8 (NB7) compared to the same cells reconstituted with the wild-type protein, or with a catalytically inactive point mutant of caspase-8, we are able to identify compounds that act specifically through the caspase-8 axis, rather than through general toxicity. In addition, false positive hits can easily be “weeded out” in this assay due to their activity in cells lacking caspase-8-inducible activity. Screening of the library of pharmacologically active compounds (LOPAC) was performed as both proof-of-concept and to discover potential unknown TRAIL sensitizers whose mechanism is caspase-8 mediated. We identified known TRAIL sensitizers from the library and identified new compounds that appear to sensitize specifically through caspase-8. In sum, we demonstrate proof-of-concept and discovery of novel compounds with a screening strategy optimized for the detection of caspase-8 pathway-specific TRAIL sensitizers. This screen was performed in the 384-well format, but could easily be further miniaturized, allows easy identification of artifactual false positives, and is highly scalable to accommodate diverse libraries.

    Publication / Type:
    PLoS One. 2010; 5(10): e13375. doi:  10.1371/journal.pone.0013375
    Related Subject:
    HTS STRAIL-Sensitizing Caspase-8 Apoptotic Axis
    Link:
    http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2953515/
  • An Innovative Way to Create Assay Ready Plates for Concentration Response Testing using Acoustic Technology Turmel M, Itkin Z, Liu D, Nie D

    Institution: AstraZeneca

    Publication: JALA 2010;15:297–305 

    2010 abstract

    A totally integrated serial dilution assay plate preparation system that fully uses the high precision nanoliter dispensing capabilities of acoustic liquid handlers has been developed and implemented. The application uses a hybrid of a serial dilution method and a direct dilution method, achieving a wide concentration range for the dilution series, while avoiding additive errors inherent to traditional serial dilution methods. The method allows assay miniaturization, which greatly reduces reagent and consumable costs to the customers. The system is in production at AstraZeneca and has generated high-quality assay ready plates for high-throughput screening and secondary screening since 2005. Further development in recent years has expanded the flexibility of the assay ready plate creation process to meet varied screening requirements.
    We will discuss the requirements for assay ready plates for concentration response testing and describe the novel plate creation method in detail with the rigorous validation procedures. Along with method validation data, some real-life screening results will be presented to compare an experiment conducted on compounds prepared using the novel hybrid method and those prepared using a more traditional serial dilution method, which endorses the application of the novel method.

    Publication / Type:
    JALA 2010;15:297–305 
    Related Subject:
    Assay Ready Concentration Response Acoustic
    Link:
    http://intl-jla.sagepub.com/content/15/4/297.full.pdf+html
  • Gradient, Contact-Free Volume Transfers Minimize Compound Loss in Dose-Response Experiments Harris D, Olechno J, Datwani S and Ellson R

    Institution: Labcyte Inc.

    Publication: J Biomol Screen January 2010 vol. 15 no. 1 86-94, <span class=

    2009 abstract

    More accurate dose-response curves can be constructed by eliminating aqueous serial dilution of compounds. Traditional serial dilutions that use aqueous diluents can result in errors in dose-response values of up to 4 orders of magnitude for a significant percentage of a compound library. When DMSO is used as the diluent, the errors are reduced but not eliminated. The authors use acoustic drop ejection (ADE) to transfer different volumes of model library compounds, directly creating a concentration gradient series in the receiver assay plate. Sample losses and contamination associated with compound handling are therefore avoided or minimized, particularly in the case of less water-soluble compounds. ADE is particularly well suited for assay miniaturization, but gradient volume dispensing is not limited to miniaturized applications.

    Publication / Type:
    J Biomol Screen January 2010 vol. 15 no. 1 86-94, <span class=
    Related Subject:
    Gradient, Contact-Free Volume Transfers Minimize Compound Loss Dose-Response
    Link:
    http://jbx.sagepub.com/content/15/1/86.abstract
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