featuring the Echo acoustic technology

109 Total Publications


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  • Dual Analysis for Mycobacteria and Propionibacteria in Sarcoidosis BAL
  • Oswald-Richter K A, Beachboard D C, Seeley E H, Abraham S, Shepherd B E, Jenkins C A, Culver D A, Ca
  • Institution: Vanderbilt University
  • Publication: J Clin Immunol. 2012 October; 32(5): 1129–1140.  doi:  10.1007/s10875-012-9700-5
  • 2012

Sarcoidosis is a non-caseating granulomatous disease for which a role for infectious antigens continues to strengthen. Recent studies have reported molecular evidence of mycobacteria or propionibacteria. We assessed for immune responses against mycobacterial and propionibacterial antigens in sarcoidosis bronchoalveolar lavage (BAL) using flow cytometry, and localized signals consistent with microbial antigens with sarcoidosis specimens, using matrix-assisted laser desorption ionization imaging mass spectrometry (MALDI-IMS).

BAL cells from 27 sarcoidosis, 14 PPD- controls, and 9 subjects with nontuberculosis mycobacterial (NTM) infections were analyzed for production of IFN-γ after stimulation with mycobacterial ESAT-6 and Propionibacterium acnes proteins. To complement the immunological data, MALDI-IMS was performed to localize ESAT-6 and Propionibacterium acnes signals within sarcoidosis and control specimens.

CD4+ immunologic analysis for mycobacteria was positive in 17/27 sarcoidosis subjects, compared to 2/14 PPD- subjects, and 5/9 NTM subjects (p = 0.008 and p = 0.71 respectively, Fisher’s exact test). There was no significant difference for recognition of P. acnes, which occurred only in sarcoidosis subjects that also recognized ESAT-6. Similar results were also observed for the CD8+ immunologic analysis. MALDI-IMS localized signals consistent with ESAT-6 only within sites of granulomatous inflammation, whereas P. acnes signals were distributed throughout the specimen.

MALDI-IMS localizes signals consistent with ESAT-6 to sarcoidosis granulomas, whereas no specific localization of P. acnes signals is detected. Immune responses against both mycobacterial and P. acnes are present within sarcoidosis BAL, but only mycobacterial signals are distinct from disease controls. These immunologic and molecular investigations support further investigation of the microbial community within sarcoidosis granulomas.

  • A Cell-Based High-Throughput Screening Assay to Measure Cellular Histone H3 Lys27 Trimethylation with a Modified Dissociation-Enhanced Lanthanide Fluorescent Immunoassay
  • Xie W, Ames R S, Li H
  • Institution: GlaxoSmithKline
  • Publication: J Biomol Screen January 2012 vol. 17 no. 1 99-107
  • 2012

Histone proteins are subject to several modifications, including phosphorylation, acetylation, methylation, sumoylation, and ubiquitination. These posttranslational modifications play critical roles in chromatin structure and gene transcription. Because of their involvement in the progression of a variety of diseases, histone modifications are attracting increased attention. We report herein a high-throughput DELFIA assay to quantify H3K27me3 in the prostate cancer cell line, PC3. Using a high binding MaxiSorp plate, we were able to eliminate the need for the capture antibody. We also developed an effective method, a combination of “freeze-thaw” and 0.2 N HCl, to extract histone proteins in PC3 cells cultured in a 384-well plate. To compensate for cell viability change, we normalized H3K27me3 signal to the total amount of H3 in each sample well. As a result, we show that the assay has a good dynamic range with a robust assay window. Using a methlytransferase inhibitor, DZNep, we show that the change of H3K27me3 signal is target specific. This method simplifies the logistics in screening and profiling and reduces the cost per well to an acceptable level for high-throughput screening. The findings presented here should be applicable to other assays involving binding and extraction of histone proteins.

  • Publication / Type:J Biomol Screen January 2012 vol. 17 no. 1 99-107
  • Related Subject: Cellular Histone H3 Lys27 Trimethylation Modified Dissociation-Enhanced Lanthanide Fluorescent Immunoassay
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  • Enabling Lead Discovery for Histone Lysine Demethylases by High-Throughput RapidFire Mass Spectrometry
  • Hutchinson S E, Leveridge M V, Heathcote M L, Francis P, Williams L, Gee M, Munoz-Muriedas J, Leaven
  • Institution: GlaxoSmithKline
  • Publication: J Biomol Screen January 2012 vol. 17 no. 1 39-48
  • 2012

A high-throughput RapidFire mass spectrometry assay is described for the JMJD2 family of Fe2+, O2, and α-ketoglutarate-dependent histone lysine demethylases. The assay employs a short amino acid peptide substrate, corresponding to the first 15 amino acid residues of histone H3, but mutated at two positions to increase assay sensitivity. The assay monitors the direct formation of the dimethylated-Lys9 product from the trimethylated-Lys9 peptide substrate. Monitoring the formation of the monomethylated and des-methylated peptide products is also possible. The assay was validated using known inhibitors of the histone lysine demethylases, including 2,4-pyridinedicarboxylic acid and an α-ketoglutarate analogue. With a sampling rate of 7 s per well, the RapidFire technology permitted the single-concentration screening of 101 226 compounds against JMJD2C in 10 days using two instruments, typically giving Z′ values of 0.75 to 0.85. Several compounds were identified of the 8-hydroxyquinoline chemotype, a known series of inhibitors of the Lys9-specific histone demethylases. The peptide also functions as a substrate for JMJD2A, JMJD2D, and JMJD2E, thus enabling the development of assays for all 3 enzymes to monitor progress in compound selectivity. The assay represents the first report of a RapidFire mass spectrometry assay for an epigenetics target.

  • Design of an Automated Enhanced-Throughput Platform for Functional Characterization of Positive Allosteric Modulator-Induced Leftward Shifts in Apparent Agonist Potency In Vitro
  • Hendricson A, Matchett M, Ferrante M, Ferrante C, Hunnicutt E, Westphal R, Kostich W, Huang Y, Masia
  • Institution: Bristol-Myers Squibb Company
  • Publication: Journal of Laboratory Automation April 2012 vol. 17 no. 2 104-115
  • 2012

Prosecution of positive allosteric modulator (PAM) targets demands a specialized assay toolset. Many GPCR or ion channel targets are adaptable to functional assays whereby PAM efficacy can be inferred from left or rightward shifts in the concentration-response curves of orthosteric agonist. The inherent emphasis on throughput and occasional paucity of radioligands for a diverse array of allosteric modulator targets yields a need for an enhanced throughput agonist potency shift assay. Here, we describe a process by which such an assay was automated with robust, reproducible in vitro pharmacology. In direct comparison with a manual CRC shift assay, the enhanced throughput automated platform described here delivered near identical rank orders (r2 = 0.75) at ~4-fold throughput/assay iteration. Correspondingly, average cycle time/plate decreased from 104 to 72 minutes. We also observed reductions in assay interference associated with compounds exhibiting ago-allosterism, which we attribute to preread compound incubation periods which are more precisely time-constrained under automation control. By leveraging automated laboratory technology, we have achieved meaningful throughput with no sacrifice of precision. Rather than to be target-class specific, the present process was specifically designed to serve as a platform template for a variety of cell-based functional allosteric modulation assays.

  • Resorufin Butyrate as a Soluble and Monomeric High-Throughput Substrate for a Triglyceride Lipase
  • Lam V, Henault M, Khougaz K, Fortin L-J, Ouellet M, Melnyk R, Partridge A
  • Institution: Merck Frosst Centre for Therapeutic Research
  • Publication: J Biomol Screen February 2012 vol. 17 no. 2 245-251
  • 2012

Triglyceride lipases such as lipoprotein lipase, endothelial lipase, and hepatic lipase play key roles in controlling the levels of plasma lipoprotein. Accordingly, small-molecule modulation of these species could alter patient lipid profiles with corresponding health effects. Screening of these enzymes for small-molecule therapeutics has historically involved the use of lipid-based particles to mimic native substrates. However, particle-based artifacts can complicate the discovery of therapeutic molecules. As a simplifying solution, the authors sought to develop an approach involving a soluble and monomeric lipase substrate. Using purified bovine lipoprotein lipase as a model system, they show that the hydrolysis of resorufin butyrate can be fluorescently monitored to give a robust assay (Z′ > 0.8). Critically, using parallel approaches, they show that resorufin butyrate is soluble and monomeric under assay conditions. The presented assay should be useful as a simple and inexpensive primary or secondary screen for the discovery of therapeutic lipase modulators.

  • Case Studies of Minimizing Nonspecific Inhibitors in HTS Campaigns That Use Assay-Ready Plates
  • Liu Y, Beresini M H, Johnson A, Mintzer R, Shah K, Clark K, Schmidt S, Lewis C, Liimatta M, Elliott
  • Institution: Genentech, Inc.
  • Publication: J Biomol Screen February 2012 vol. 17 no. 2 225-236
  • 2012

Identifying chemical lead matter by high-throughput screening (HTS) has been a common practice in early stage drug discovery. Evolution of small-molecule library composition to include more drug-like molecules with desirable physical chemical properties combined with improving assay technologies has vastly enhanced the capability of HTS. However, HTS campaigns can still be plagued by false positives arising from nonspecific inhibitors. The generation of assay-ready plates has permitted an incremental advancement to the speed and efficiency of HTS but has the potential to enhance the occurrence of nonspecific inhibitors. A subtle change in the order of reagent addition to the assay-ready plates can greatly alleviate false-positive inhibition. Our case studies with six different kinase and protease targets reveal that this type of inhibition affects targets regardless of enzyme class and is unpredictable based on protein construct or inhibitor chemical scaffold. These case studies support a model where a diversity set of compounds should be tested first for hit rates as a function of order of addition, carrier protein, and relevant mechanistic studies prior to launch of the HTS campaign.

  • A Chemical Genomics Screen to Discover Genes That Modulate Neural Stem Cell Differentiation
  • Kim K J, Wang J, Xu X, Wu S, Zhang W, Qin Z, Wu F, Liu A, Zhao Y, Fang H, Zhu M, Zhao J, Zhong Z
  • Institution: GlaxoSmithKline
  • Publication: J Biomol Screen February 2012 vol. 17 no. 2 129-139
  • 2012

The authors designed a chemical genomics screen with the aim of understanding genes and pathways that modulate neural stem/precursor cell differentiation. Multipotent mouse neural precursor cells isolated from cortices of embryonic day 12 (E12) embryos were subjected to spontaneous differentiation triggered by growth factor withdrawal. A quantitative whole-well immunofluorescence assay was set up to screen tool compound sets to identify small molecules with potent, dose-dependent, and reproducible effects on increasing neural stem cell differentiation toward neuronal lineage. Among the pro-neuronal compounds, kinase inhibitors were shown to exert pro-neuronal effect via a signaling pathway associated with the kinase. The global effect of hit compounds on modulating neuronal differentiation was confirmed by an in vivo mouse study and human neural stem cells culture. This study demonstrates that a phenotypic assay using cell type–specific antibody markers can be used for a large-scale compound screen to discover targets and pathways with impacts on differentiation of lineage-restricted precursor cells toward specific lineages.

  • Implementation and Development of an Automated, Ultra-High-Capacity, Acoustic, Flexible Dispensing Platform for Assay-Ready Plate Delivery
  • Griffith D, Northwood R, Owen P, Simkiss E, Brierley A, Cross K, Slaney A, Davis M, Bath C
  • Institution: AstraZeneca
  • Publication: Journal of Laboratory Automation October 2012 vol. 17 no. 5 348-358 10.1177/2211068212457159
  • 2012

Compound management faces the daily challenge of providing high-quality samples to drug discovery. The advent of new screening technologies has seen demand for liquid samples move toward nanoliter ranges, dispensed by contactless acoustic droplet ejection. Within AstraZeneca, a totally integrated assay-ready plate production platform has been created to fully exploit the advantages of this technology. This enables compound management to efficiently deliver large throughputs demanded by high-throughput screening while maintaining regular delivery of smaller numbers of compounds in varying plate formats for cellular or biochemical concentration-response curves in support of hit and lead optimization (structure-activity relationship screening). The automation solution, CODA, has the capability to deliver compounds on demand for single- and multiple-concentration ranges, in batch sizes ranging from 1 sample to 2 million samples, integrating seamlessly into local compound and test management systems. The software handles compound orders intelligently, grouping test requests together dependent on output plate type and serial dilution ranges so that source compound vessels are shared among numerous tests, ensuring conservation of sample, reduced labware and costs, and efficiency of work cell logistics. We describe the development of CODA to address the customer demand, challenges experienced, learning made, and subsequent enhancements.

  • Publication / Type:Journal of Laboratory Automation October 2012 vol. 17 no. 5 348-358 10.1177/2211068212457159
  • Related Subject: Automated Ultra-High-Capacity Acoustic Flexible Dispensing Platform Assay-Ready
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  • Development and Validation of Reagents and Assays for EZH2 Peptide and Nucleosome High-Throughput Screens
  • Diaz E, Machutta C A, Chen S, Jiang Y, Nixon C, Hoffman G, Key D, Sweitzer S, Patel M, Wu Z, Creasy
  • Institution: GlaxoSmithKline
  • Publication: J Biomol Screen. 2012 Dec;17(10):1279-92. doi: 10.1177/1087057112453765
  • 2012

Histone methyltransferases (HMT) catalyze the methylation of histone tail lysines, resulting in changes in gene transcription. Misregulation of these enzymes has been associated with various forms of cancer, making this target class a potential new area for the development of novel chemotherapeutics. EZH2 is the catalytic component of the polycomb group repressive complex (PRC2), which selectively methylates histone H3 lysine 27 (H3K27). EZH2 is overexpressed in prostate, breast, bladder, brain, and other tumor types and is recognized as a molecular marker for cancer progression and aggressiveness. Several new reagents and assays were developed to aid in the identification of EZH2 inhibitors, and these were used to execute two high-throughput screening campaigns. Activity assays using either an H3K27 peptide or nucleosomes as substrates for methylation are described. The strategy to screen EZH2 with either a surrogate peptide or a natural substrate led to the identification of the same tractable series. Compounds from this series are reversible, are [3H]-S-adenosyl-L-methionine competitive, and display biochemical inhibition of H3K27 methylation.

  • Development of a High-Throughput Calcium Flux Assay for Identification of All Ligand Types Including Positive, Negative, and Silent Allosteric Modulators for G Protein-Coupled Receptors
  • Noblin D, Bertekap R L, Burford N T, Hendricson A, Zhang L, Knox R, Banks M, O'Connell J, and Alt A
  • Institution: Yale University, Bristol-Myers Squibb
  • Publication: ASSAY and Drug Development Technologies. October 2012, 10(5): 457-467. doi:10.1089/adt.2011.443
  • 2012

In recent years, the increased use of cell-based fun​ctional assays for G protein-coupled receptors in high-throughput screening has enabled the design of robust assays to identify allosteric modulators (AMs) in addition to the more traditional orthosteric agonists and antagonists. In this article, the authors describe a screening format able to identify all ligand types using a triple-add assay that measures changes in cytosolic calcium concentration with three separate additions and reads in the same assay plate. This triple-add assay captures more small molecule ligand types than previously described assay formats without a significant increase in screening cost. Finally, the customizability of the triple-add assay to suit the needs of various AM screening programs is demonstrated.