featuring the Echo acoustic technology

109 Total Publications


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  • Target Inhibition Networks: Predicting Selective Combinations of Druggable Targets to Block Cancer Survival Pathways
  • Tang, J, Karhinen L, Xu T, Szwajda A, Yadav B, Wennerberg K, Aittokallio T
  • Institution: Institute for Molecular Medicine Finland (FIMM), University of Helsinki, Helsinki, Finland
  • Publication: PLoS Comput Biol 9(9): e1003226. doi:10.1371/journal.pcbi.1003226
  • 2013

A recent trend in drug development is to identify drug combinations or multi-target agents that effectively modify multiple nodes of disease-associated networks. Such polypharmacological effects may reduce the risk of emerging drug resistance by means of attacking the disease networks through synergistic and synthetic lethal interactions. However, due to the exponentially increasing number of potential drug and target combinations, systematic approaches are needed for prioritizing the most potent multi-target alternatives on a global network level. We took a functional systems pharmacology approach toward the identification of selective target combinations for specific cancer cells by combining large-scale screening data on drug treatment efficacies and drug-target binding affinities. Our model-based prediction approach, named TIMMA, takes advantage of the polypharmacological effects of drugs and infers combinatorial drug efficacies through system-level target inhibition networks. Case studies in MCF-7 and MDA-MB-231 breast cancer and BxPC-3 pancreatic cancer cells demonstrated how the target inhibition modeling allows systematic exploration of functional interactions between drugs and their targets to maximally inhibit multiple survival pathways in a given cancer type. The TIMMA prediction results were experimentally validated by means of systematic siRNA-mediated silencing of the selected targets and their pairwise combinations, showing increased ability to identify not only such druggable kinase targets that are essential for cancer survival either individually or in combination, but also synergistic interactions indicative of nonadditive drug efficacies. These system-level analyses were enabled by a novel model construction method utilizing maximization and minimization rules, as well as a model selection algorithm based on sequential forward floating search. Compared with an existing computational solution, TIMMA showed both enhanced prediction accuracies in cross validation as well as significant reduction in computation times. Such cost-effective computational-experimental design strategies have the potential to greatly speed-up the drug testing efforts by prioritizing those interventions and interactions warranting further study in individual cancer cases.

  • An HTS-Compatible 3D Colony Formation Assay to Identify Tumor-Specific Chemotherapeutics
  • Horman S, To J, Orth A
  • Institution: Genomics Institute of the Novartis Research Foundation (GNF)
  • Publication: J Biomol Sc high-content screening, HTS, colon, colony formation, soft agar, co-culture, 3D assay de
  • 2013

There has been increasing interest in the development of cellular behavior models that take advantage of three-dimensional (3D) cell culture. To enable assessment of differential perturbagen impacts on cell growth in 2D and 3D, we have miniaturized and adapted for high-throughput screening (HTS) the soft agar colony formation assay, employing a laserscanning cytometer to image and quantify multiple cell types simultaneously. The assay is HTS compatible, providing high-quality, image-based, replicable data for multiple, co-cultured cell types. As proof of concept, we subjected colorectal carcinoma colonies in 3D soft agar to a mini screen of 1528 natural product compounds. Hit compounds from the primary screen were rescreened in an HTS 3D co-culture matrix containing colon stromal cells and cancer cells. By combining tumor cells and normal, nontransformed colon epithelial cells in one primary screening assay, we were able to obtain differential IC50 data, thereby distinguishing tumor-specific compounds from general cytotoxic compounds. Moreover, we were able to identify compounds that antagonized tumor colony formation in 3D only, highlighting the importance of this assay in identifying agents that interfere with 3D tumor structural growth. This screening platform provides a fast, simple, and robust method for identification of tumor-specific agents in a biologically relevant microenvironment.  

  • Is Your Liquid Handling Misleading You?
  • Olechno J
  • Institution: Labcyte Inc., Institute for Molecular Medicine Finland, FIMM, University of Helsinki
  • Publication: Innovations in Pharmaceutical Technology June 2013 Issue 45 31-35 
  • 2013

Selecting the most suitable liquid handling method can have a huge influence over your final results, particularly with miniaturised volumes. Acoustic liquid handling using the relatively new direct dilution technique may offer scientists greater accuracy.

  • Dispensing Processes Impact Apparent Biological Activity as Determined by Computational and Statistical Analyses
  • Ekins S, Olechno J, Williams AJ
  • Institution: Collaborations in Chemistry, Labcyte Inc., Royal Society of Chemistry
  • Publication: PLoS ONE 8(5): e62325, 2013. http://doi:10.1371/journal.pone.0062325
  • 2013

Dispensing and dilution processes may profoundly influence estimates of biological activity of compounds. Published data show Ephrin type-B receptor 4 IC50 values obtained via tip-based serial dilution and dispensing versus acoustic dispensing with direct dilution differ by orders of magnitude with no correlation or ranking of datasets. We generated computational 3D pharmacophores based on data derived by both acoustic and tip-based transfer. The computed pharmacophores differ significantly depending upon dispensing and dilution methods. The acoustic dispensing-derived pharmacophore correctly identified active compounds in a subsequent test set where the tip-based method failed. Data from acoustic dispensing generates a pharmacophore containing two hydrophobic features, one hydrogen bond donor and one hydrogen bond acceptor. This is consistent with X-ray crystallography studies of ligand-protein interactions and automatically generated pharmacophores derived from this structural data. In contrast, the tip-based data suggest a pharmacophore with two hydrogen bond acceptors, one hydrogen bond donor and no hydrophobic features. This pharmacophore is inconsistent with the X-ray crystallographic studies and automatically generated pharmacophores. In short, traditional dispensing processes are another important source of error in high-throughput screening that impacts computational and statistical analyses. These findings have far-reaching implications in biological research.

  • High-Throughput RNA Interference Screening: Tricks of the Trade
  • Nebane MN, Coric T, Whig K, McKellip S, Woods L, Sosa M, Sheppard R, Rasmussen L, Bjornsti M, White
  • Institution: Southern Research Institute, University of Alabama at Birmingham
  • Publication: Journal of Laboratory Automation April 24, 2013 2211068213486786
  • 2013

The process of validating an assay for high-throughput screening (HTS) involves identifying sources of variability and developing procedures that minimize the variability at each step in the protocol. The goal is to produce a robust and reproducible assay with good metrics. In all good cell-based assays, this means coefficient of variation (CV) values of less than 10% and a signal window of fivefold or greater. HTS assays are usually evaluated using Z′ factor, which incorporates both standard deviation and signal window. A Z′ factor value of 0.5 or higher is acceptable for HTS. We used a standard HTS validation procedure in developing small interfering RNA (siRNA) screening technology at the HTS center at Southern Research. Initially, our assay performance was similar to published screens, with CV values greater than 10% and Z′ factor values of 0.51 ± 0.16 (average ± standard deviation). After optimizing the siRNA assay, we got CV values averaging 7.2% and a robust Z′ factor value of 0.78 ± 0.06 (average ± standard deviation). We present an overview of the problems encountered in developing this whole-genome siRNA screening program at Southern Research and how equipment optimization led to improved data quality.

  • Structural Basis of Signaling Blockade by Anti-IL-13 Antibody Lebrikizumab
  • Ultsch M, Bevers J, Nakamura G, Vandlen R, Kelley RF, Wu LC, Eigenbrot C.
  • Institution: Genentech Inc.
  • Publication: J Molecular Biology, 425 (8) 1330–1339, 2013.
  • 2013

The cytokine interleukin 13 (IL-13) is a major effector molecule for T-helper type 2 inflammation and is pathogenic in allergic diseases such as asthma. The effects of IL-13 are mediated via a pathway that is initiated by binding to a heterodimeric receptor consisting of IL-13Rα1 and IL-4Rα. Antibodies raised against IL-13 can block its inflammatory effects by interfering with binding to either of the two receptor polypeptides. Lebrikizumab is a monoclonal anti-IL-13 antibody that has shown clinical benefit in a phase II study for the treatment of moderate-to-severe uncontrolled asthma. Here we report the molecular structure of IL-13 in complex with the Fab from lebrikizumab by X-ray crystallography at 1.9 Å resolution. We show that lebrikizumab inhibits IL-13 signaling by binding to IL-13 with very high affinity and blocking IL-13 binding to IL-4Rα. In addition, we use site-directed mutations to identify the most important antibody contributors to binding. Our studies define key features of lebrikizumab binding and its mechanism of action that may contribute to its clinical effects.

  • Identification and Characterization of Novel Human Glucose-6-Phosphate Dehydrogenase Inhibitors
  • Preuss J, Richardson A D, Pinkerton A, Hedrick M, Sergienko E, Rahlfs S, Becker K, Bode L
  • Institution: University of California, Sanford-Burnham Medical Research Institute, Justus Liebig University
  • Publication: Journal of Biomedical Screening, September 2012
  • 2013

Glucose-6-phosphate dehydrogenase (G6PD) is the key enzyme of the pentose phosphate pathway, converting glucose-6-phosphate to 6-phosphoglucono-δ-lactone with parallel reduction of NADP+. Several human diseases, including cancer, are associated with increased G6PD activity. To date, only a few G6PD inhibitors have been available. However, adverse side effects and high IC50 values hamper their use as therapeutics and basic research probes. In this study, we developed a high-throughput screening assay to identify novel human G6PD (hG6PD) inhibitors. Screening the LOPAC (Sigma-Aldrich; 1280 compounds), Spectrum (Microsource Discovery System; 1969 compounds), and DIVERSet (ChemBridge; 49 971 compounds) small-molecule compound collections revealed 139 compounds that presented ≥50% hG6PD inhibition. Hit compounds were further included in a secondary and orthogonal assay in order to identify false-positives and to determine IC50 values. The most potent hG6PD inhibitors presented IC50 values of <4 µM. Compared with the known hG6PD inhibitors dehydroepiandrosterone and 6-aminonicotinamide, the inhibitors identified in this study were 100- to 1000-fold more potent and showed different mechanisms of enzyme inhibition. One of the newly identified hG6PD inhibitors reduced viability of the mammary carcinoma cell line MCF10-AT1 (IC50 ~25 µM) more strongly than that of normal MCF10-A cells (IC50 >50 µM).

  • Analysis of Compound Weighing Precision in Drug Discovery
  • Popa-Burke I, Novick S, Mantilla M, McGrath K
  • Institution: GlaxoSmithKline, North Carolina State University
  • Publication: Journal of Biomedical Screening, September 2012
  • 2013

Early drug discovery laboratories often call for the precise weighing of 1- to 5-mg solids into 4- to 5-g glass vials. For the balance used in this study (Mettler Toledo XP205), the manufacturer rates its accuracy at ±0.01 mg over the working range of 1 mg to 220 g and its precision or repeatability at 0.015 mg for 10-g weights. The manufacturer ratings were confirmed using standard steel weights, but these calibrators do not well represent the weighing precision of drug compound. For example, when pre-taring a 4- to 5-g vial on the balance and then weighing 1- to 5-mg calibration weights, although no bias was observed, precision dropped appreciably. When measuring solid sample in the range of 1 to 5 mg, deviation of the measured weight from the actual (true) weight was even worse, in the range of ±20% to 50%. Balance settings and environmental factors exert a strong influence on weighing precision. Although most environmental factors, such as air draughts, temperature, vibrations, and levelness, can be optimized to the extent practical in laboratory settings, problems due to static electricity are often overlooked. By controlling static electricity, we demonstrate how we optimized the process to where measurements were within ±10% of actual weight when weighing solid sample in the range of 2 to 5 mg and ±20% when weighing 1 mg into a 4- to 5-g vial. Our weighing process and method to calculate actual weight are given in detail.

  • High-Precision, Room Temperature Screening Assay for Inhibitors of Microsomal Prostaglandin E Synthase-I
  • Anderson S, Norman M, Olsson R, Smith R, Liu G, Nord J
  • Institution: AstraZeneca
  • Publication: Journal of Biomolecular Screening 17(10) 1372–1378 © 2012 SLAS
  • 2012

Microsomal prostaglandin E synthase-1 (mPGES-1) is the major enzyme catalyzing the isomerization of prostaglandin (PG) H2 to PGE2. Here we report the development of a robust and practical automated assay in a 384-well format for room temperature screening of mPGES-1 inhibitors with high precision and low reagent consumption. The assay should enable precise structure-activity relationship development. It uses acetonitrile as solvent for PGH2, FeCl2/citrate as stop reagent, and a short reaction time. Combined with high-precision liquid transfer and extensive mixing after addition of reactants, these properties let the assay reach Z' > 0.7 and high reproducibility of inhibitor IC50 values. Thorough investigation of the quality of mixing in all liquid transfer steps proved crucial for reaching high-precision performance.

  • Publication / Type:Journal of Biomolecular Screening 17(10) 1372–1378 © 2012 SLAS
  • Related Subject:automation or robotics, liquid handling, enzyme assays or enzyme kinetics, fluorescence methods, inflammation, autoimmune diseases, Room Temperature Microsomal Prostaglandin E Synthase-I
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  • 3D Imaging by Mass Spectrometry: A New Frontier
  • Seeley E H, Caprioli R M
  • Institution: Vanderbilt University
  • Publication: Anal Chem. 2012 Mar 6;84(5):2105-10. doi: 10.1021/ac2032707
  • 2012

Imaging mass spectrometry can generate three-dimensional volumes showing molecular distributions in an entire organ or animal through registration and stacking of serial tissue sections. Here, we review the current state of 3D imaging mass spectrometry as well as provide insights and perspectives on the process of generating 3D mass spectral data along with a discussion of the process necessary to generate a 3D image volume.