featuring the Echo acoustic technology

111 Total Publications


  • Year
  • Link
  • PDF
  • + 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
  • 2011

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.

  • 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

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:
  • 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

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.

  • 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

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

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

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

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

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

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

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: