PUBLICATIONS

featuring the Echo acoustic technology

108 Total Publications

TITLES and AUTHORS

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  • 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
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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.

  • 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
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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.

  • 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
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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.

  • 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
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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.

  • 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
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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.

  • Insulin crystallization depends on zinc transporter ZnT8 expression, but is not required for normal glucose homeostasis in mice
  • Lemaire K, Ravier M A, Schraenen A, Creemers J W M, Van de Plas R, Granvik M, Van Lommel L, Waelkens
  • Institution: Katholieke Universiteit Leuven
  • Publication: PNAS vol. 106 no. 35: 14872–14877, doi: 10.1073/pnas.0906587106
  • 2009
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Zinc co-crystallizes with insulin in dense core secretory granules, but its role in insulin biosynthesis, storage and secretion is unknown. In this study we assessed the role of the zinc transporter ZnT8 using ZnT8-knockout (ZnT8−/−) mice. Absence of ZnT8 expression caused loss of zinc release upon stimulation of exocytosis, but normal rates of insulin biosynthesis, normal insulin content and preserved glucose-induced insulin release. Ultrastructurally, mature dense core insulin granules were rare in ZnT8−/− beta cells and were replaced by immature, pale insulin “progranules,” which were larger than in ZnT8+/+ islets. When mice were fed a control diet, glucose tolerance and insulin sensitivity were normal. However, after high-fat diet feeding, the ZnT8−/− mice became glucose intolerant or diabetic, and islets became less responsive to glucose. Our data show that the ZnT8 transporter is essential for the formation of insulin crystals in beta cells, contributing to the packaging efficiency of stored insulin. Interaction between the ZnT8−/− genotype and diet to induce diabetes is a model for further studies of the mechanism of disease of human ZNT8 gene mutations.

  • Identification of Novel Non-Hydroxamate Anthrax Toxin Lethal Factor Inhibitors by Topomeric Searching, Docking and Scoring, and In Vitro Screening
  • Chiu T-L, Solberg J, Patil S, Geders T W, Zhang X, Rangarajan S, Francis R, Finzel B C, Walters M A,
  • Institution: University of Minnesota
  • Publication: J Chem Inf Model. 2009 December; 49(12): 2726–2734.  doi:  10.1021/ci900186w
  • 2009
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Anthrax is an infectious disease caused by Bacillus anthracis, a Gram-positive, rod-shaped, anaerobic bacterium. The lethal factor (LF) enzyme is secreted by B. anthracis as part of a tripartite exotoxin and is chiefly responsible for anthrax-related cytotoxicity. As LF can remain in the system long after antibiotics have eradicated B. anthracis from the body, the preferred therapeutic modality would be the administration of antibiotics together with an effective LF inhibitor. Although LF has garnered a great deal of attention as an attractive target for rational drug design, relatively few published inhibitors have demonstrated activity in cell-based assays and, to date, no LF inhibitor is available as a therapeutic or preventive agent. Here we present a novel in silico high-throughput virtual screening protocol that successfully identified 5 non-hydroxamic acid small molecules as new, preliminary LF inhibitor scaffolds with low micromolar inhibition against that target, resulting in a 12.8% experimental hit rate. This protocol screened approximately thirty-five million non-redundant compounds for potential activity against LF and comprised topomeric searching, docking and scoring, and drug-like filtering. Among these 5 hit compounds, none of which has previously been identified as a LF inhibitor, three exhibited experimental IC50 values less than 100 µM. These three preliminary hits may potentially serve as scaffolds for lead optimization, as well as templates for probe compounds to be used in mechanistic studies. Notably, our docking simulations predicted that these novel hits are likely to engage in critical ligand-receptor interactions with nearby residues in at least two of the three (S1’, S1–S2 and S2’) subsites in the LF substrate binding area. Further experimental characterization of these compounds is in process. We found that micromolar-level LF inhibition can be attained by compounds with non-hydroxamate zinc-binding groups that exhibit monodentate zinc chelation, as long as key hydrophobic interactions with at least two LF subsites are retained.

  • Publication / Type:J Chem Inf Model. 2009 December; 49(12): 2726–2734.  doi:  10.1021/ci900186w
  • Related Subject:Identification Novel Non-Hydroxamate Anthrax Toxin Lethal Factor Inhibitor Topomeric Docking Scoring In Vitro
  • Link:http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2805240/
  • Validation of Low-Volume 1536-Well Assay-Ready Compound Plates
  • Eriksson H, Brengdahl J, Sandström P, Rohman M, Becker B
  • Institution: AstraZeneca and Tessela plc.
  • Publication: J Biomol Screen June 2009 vol. 14 no. 5 468-475
  • 2009
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Assay-ready compound plates (ARPs) are sealed assay plates that contain DMSO solutions of screening compounds predispensed for particular assays. Assays are started by adding assay buffer and reagents to the ARPs. This concept offers important logistical advantages for screening such as decoupling of the plate preparation from the screening process and exchange of assay plates between different geographical locations. Compound solutions can be accurately and precisely dispensed by acoustic droplet ejection technology in the small volumes required for screening in the 1536-well format. At such low volumes, however, potential effects such as solvent evaporation, compound degradation, precipitation, or adsorption are reasons for concern with regard to assay reproducibility, performance, and shelf life of ARPs. To address these concerns, the authors screened freshly prepared ARPs using several types of assays. The results were compared to results obtained from plates stored for up to 13 days under 2 storage conditions (22 °C, —18 °C). Tight correlations between results were found that indicated that temperature and time had very little influence on the assay performance for up to about 1 week storage time of the plates. In addition, using a time series of microphotographs of DMSO droplets, the authors visually confirmed that the sizes of the droplets in ARPs apparently do not change over 13 days under certain storage conditions.

  • Innovation Leading the Way: Application of Lean Manufacturing to Sample Management
  • Allen M, Wigglesworth M J
  • Institution: GlaxoSmithKline
  • Publication: Journal of Biomolecular Screening. 2009:515-522
  • 2009
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Historically, sample management successfully focused on providing compound quality and tracking distribution within a diverse geographic. However, if a competitive advantage is to be delivered in a changing environment of outsourcing, efficiency and customer service must now improve or face reconstruction. The authors have used discrete event simulation to model the compound process from chemistry to assay and applied lean manufacturing techniques to analyze and improve these processes. In doing so, they identified a value-adding process time of just 11 min within a procedure that took days. Modeling also allowed the analysis of equipment and human resources necessary to complete the expected demand in an acceptable cycle time. Layout and location of sample management and screening departments are key in allowing process integration, creating rapid flow of work, and delivering these efficiencies. Following this analysis and minor process changes, the authors have demonstrated for 2 programs that solid compounds can be converted to assay-ready plates in less than 4 h. In addition, it is now possible to deliver assay data from these compounds within the same working day, allowing chemistry teams more flexibility and more time to execute the next chemistry round. Additional application of lean manufacturing principles has the potential to further decrease cycle times while using fewer resources.

  • Applications of Adaptive Focused Acoustics to Compound Management
  • Nixon E, Holland-Crimmin S, Lupotsky B, Chan J, Curtis J, Dobbs K, Blaxill Z
  • Institution: GlaxoSmithKline
  • Publication: J Biomol Screen June 2009 vol. 14 no. 5 460-467
  • 2009
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Since the introduction of lithotripsy kidney stone therapy, Focused Acoustics and its properties have been thoroughly utilized in medicine and exploration. More recently, Compound Management is exploring its applications and benefits to sample integrity. There are 2 forms of Focused Acoustics: Acoustic Droplet Ejection and Adaptive Focused Acoustics, which work by emitting high-powered acoustic waves through water toward a focused point. This focused power results in noncontact plate-to-plate sample transfer or sample dissolution, respectively. For the purposes of this article, only Adaptive Focused Acoustics will be addressed. Adaptive Focused Acoustics uses high-powered acoustic waves to mix, homogenize, dissolve, and thaw samples. It facilitates transferable samples through noncontact, closed-container, isothermal mixing. Experimental results show significantly reduced mixing times, limited degradation, and ideal use for heat-sensitive compounds. Upon implementation, acoustic dissolution has reduced the number of samples requiring longer mixing times as well as reducing the number impacted by incomplete compound dissolution. It has also helped in increasing the overall sample concentration from 6 to 8 mM to 8 to 10 mM by ensuring complete compound solubilization. The application of Adaptive Focused Acoustics, however, cannot be applied to all Compound Management processes, such as sample thawing and low-volume sample reconstitution. This article will go on to describe the areas where Adaptive Focused Acoustics adds value as well as areas in which it has shown no clear benefit.