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- Volume 12, Issue 8, 2009
Combinatorial Chemistry & High Throughput Screening - Volume 12, Issue 8, 2009
Volume 12, Issue 8, 2009
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Editorial [Hot topic: Label-Free Detection Technologies (Guest Editor: John D. McCarter)]
More LessThe use of label-free detection technologies in the pharmaceutical and biotechnology industries continues to grow. In addition, the variety of different label-free detection technologies increases year after year, with many new technologies originally developed both in academic and commercial laboratories now reaching the marketplace. The pharmaceutical and biotechnology sectors, particularly high throughput screening and lead discovery organizations charged with discovering, validating, and developing new drug leads ever more efficiently, are major users and integrators of such technologies. Future innovation in label-free detection technologies should continue to be fueled in part by partnerships with pharmaceutical and biotechnology companies, as well as by environmental and defense applications. At present most large and small pharmaceutical laboratories employ some type of label-free assay technology at one or more stages of the drug discovery process. Label-free cell-based assays for primary screening and functional assays are increasingly popular. Label-free detection technologies are appealing in part because of their potential universality, enabling the quantification of ligand binding to proteins whose function may be unknown, the identification of inhibitors of protein-protein interactions, or the detection of functional events in cellular assays. In this special issue of Combinatorial Chemistry and High Throughput Screening the reader will encounter a sampling of labelfree detection technologies highlighting the variety of applications of such methods in drug discovery, ranging from highthroughput screening to hit validation and lead optimization, as well as the characterization of antigen-antibody, protein-nucleic acid, and protein-small molecule interactions. This issue contains a number of exciting contributions from authors affiliated with research institutes, technology companies, or the biopharmaceutical industry.
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SPR Imaging for High Throughput, Label-Free Interaction Analysis
Authors: Christopher Lausted, Zhiyuan Hu, Leroy Hood and Charles T. CampbellSurface plasmon resonance (SPR) sensors have proven themselves over the last 20 years to be an effective method to study biomolecular binding and kinetics without the use of labeling. More recently, the approach has been adapted to high throughput use with the imaging of SPR-active microarrays. This is an excellent tool for monitoring microarray binding in real-time where the microarray probes and targets can include a wide range of molecules. DNA, RNA, antibodies, enzymes, and a range of other proteins have been arrayed and quantitatively analyzed.
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Mass Spectrometry in High Throughput Screening: A Case Study on Acetyl-Coenzyme A Carboxylase using RapidFire® - Mass Spectrometry (RF-MS)
Authors: Maxine Jonas, William A. LaMarr and Can OzbalIn this review various technologies and approaches for the utilization of mass spectrometry in high throughput analyses are discussed. The use of quadrupole-based mass spectrometry in the screening of chemical libraries against enzymatic targets for the identification of inhibitors and/or activators is highlighted. The RapidFire mass spectrometry system, an integrated on-line solid-phase extraction system interfaced to a triple-quadrupole mass spectrometer is described in detail, and the identification of a series of inhibitors of the acetyl-coenzyme A carboxylase (ACC) assay is described.
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Inhibitors of the Lipid Phosphatase SHIP2 Discovered by High Throughput Affinity Selection-Mass Spectrometry Screening of Combinatorial Libraries
This manuscript describes the discovery and characterization of inhibitors of the lipid phosphatase SHIP2, an important target for the treatment of Type 2 diabetes, using the Automated Ligand Identification System. ALIS is an affinity selection-mass spectrometry platform for label-free, high throughput screening of mixture-based combinatorial libraries. We detail the mass-encoded synthesis of a library that yielded NGD-61338, a pyrazole-based SHIP2 inhibitor. Quantitative ALIS affinity measurements and inhibition of SHIP2 enzymatic activity indicate that this compound has micromolar binding affinity and inhibitory activity for this target. This inhibitor, which does not contain a phosphatase “warhead,” binds the active site of SHIP2 as determined by ALIS-based competition experiments with the enzyme's natural substrate, phosphatidylinositol 3,4,5-triphosphate (PIP3). Structure-activity relationships for NGD-61338 and two other ligand classes discovered by ALIS screening were explored using a combination of combinatorial library synthesis and ALIS-enabled affinity ranking in compound mixtures.
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Recent Developments in Isothermal Titration Calorimetry Label Free Screening
Authors: William B. Peters, Verna Frasca and Richard K. BrownIsothermal titration calorimetry (ITC) is a label free technique used for direct detection of biological interactions by measurement of the heat given off or taken up during the reaction. In this article we will introduce the ITC technique and review two applications of ITC in drug discovery; small molecule/protein interactions and enzyme kinetics. We will also describe the characteristics of a new miniaturized, ultrasensitive calorimeter. This new microcalorimetry system reduces the quantity of protein (or other macromolecule sample) required to obtain a complete thermodynamic profile (n, K, ΔH and ΔS) by up to 7-fold. The reduction in required sample quantities allows ITC to be effectively utilized at earlier stages of the drug discovery and development process.
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Label-Free Detection of Biomolecular Interactions Using BioLayer Interferometry for Kinetic Characterization
Authors: Joy Concepcion, Krista Witte, Charles Wartchow, Sae Choo, Danfeng Yao, Henrik Persson, Jing Wei, Pu Li, Bettina Heidecker, Weilei Ma, Ram Varma, Lian-She Zhao, Donald Perillat, Greg Carricato, Michael Recknor, Kevin Du, Huddee Ho, Tim Ellis, Juan Gamez, Michael Howes, Janette Phi-Wilson, Scott Lockard, Robert Zuk and Hong TanThe analysis of biomolecular interactions is key in the drug development process. Label-free biosensor methods provide information on binding, kinetics, concentration, and the affinity of an interaction. These techniques provide real-time monitoring of interactions between an immobilized ligand (such as a receptor) to an analyte in solution without the use of labels. Advances in biosensor design and detection using BioLayer Interferometry (BLI) provide a simple platform that enables label-free monitoring of biomolecular interactions without the use of flow cells. We review the applications of BLI in a wide variety of research and development environments for quantifying antibodies and proteins and measuring kinetics parameters.
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A Novel Approach to Label-Free Sensing: Diffractive Optics Technology (dot®)
More LessLabel-free detection methods have played a very significant role in drug design and refinement [1]. They have been used primarily during secondary screening and for in-depth characterization of biomolecular interactions. Misconceptions about the accessibility of these platforms, since they often require specialized training, throughput and robustness in complex media have hampered their adoption in the earliest phases of discovery not to mention their significant and unrealized potential in qualifying reagents for high throughput screening or during novel assay development. A new wave of more cost effective, robust and accessible platforms has made significant inroads, demonstrating that significant information can be derived from these methods all along the drug discovery research continuum. One of these recent entrants, the dotLab® System uses diffractive optics technology (dot®) to detect biomolecular interactions and can be used for a wide variety of applications in the study of a broad spectrum of biological analytes including proteins, DNA and even microorganisms.
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Enhanced Selectivity Screening of GPCR Ligands Using a Label-Free Cell Based Assay Technology
Drug discovery efforts advance in step with advancements in assay technologies, as new technologies provide new lenses through which biology can be viewed. The novel information gathered results in the better understanding of drug-target interactions leading to better decision making during the drug discovery process. One area of rapid development is within label-free technologies. Label-free technologies offer many distinct advantages to the drug discovery workflow. One such novel technology is the CellKey™ System, an impedance-based label-free live cell assay platform. The system is based on impedance technology and is a universal platform for the functional measurement of all classes of G-protein coupled receptors (GPCRs). Data are generated in a kinetic fashion on both endogenously expressed and transfected receptors in a wide variety of cell types. In the studies detailed here, we used the system to perform an enhanced selectivity screen of a small panel of compounds simultaneously against two unrelated GPCR targets signaling through different pathways. Utilizing both the quantitative measures of cellular activation and the qualitative information inherent in the rich output data, we gained knowledge not only about the relative selectivity of each compound across both targets, but also about the character of the interaction of each with the cellular target. In this manner, we successfully demonstrated proof of principal for using an impedance-based technology to perform selectivity analyses and to triage lead compounds in a simplified format.
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MEET THE GUEST EDITOR
More LessJohn McCarter is a Principal Scientist in Lead Discovery at Amgen where he leads a group focusing on the discovery and development of small molecule therapeutics for enzyme and cellular targets. Prior to joining Amgen in 2000, John was a Senior Scientist and Project Leader at Axys Pharmaceuticals where he contributed to the cathepsin K program leading to the clinical development (partnered with Merck) of inhibitors of this enzyme for the treatment of osteoporosis. He was a Medical Research Council of Canada postdoctoral fellow with Prof. Jack Kirsch at the University of California at Berkeley and earned his Ph.D. in Chemistry on mechanisms and inhibitors of glycosidases at the University of British Columbia.
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Volumes & issues
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Volume 27 (2024)
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Volume 26 (2023)
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Volume 25 (2022)
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Volume 24 (2021)
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Volume 23 (2020)
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Volume 22 (2019)
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Volume 21 (2018)
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Volume 20 (2017)
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Volume 19 (2016)
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Volume 18 (2015)
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Volume 17 (2014)
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Volume 16 (2013)
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Volume 15 (2012)
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Volume 14 (2011)
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Volume 13 (2010)
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Volume 12 (2009)
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Volume 11 (2008)
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Volume 10 (2007)
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Volume 9 (2006)
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Volume 8 (2005)
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Volume 7 (2004)
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Volume 6 (2003)
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Volume 5 (2002)
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Volume 4 (2001)
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Volume 3 (2000)
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Label-Free Detection of Biomolecular Interactions Using BioLayer Interferometry for Kinetic Characterization
Authors: Joy Concepcion, Krista Witte, Charles Wartchow, Sae Choo, Danfeng Yao, Henrik Persson, Jing Wei, Pu Li, Bettina Heidecker, Weilei Ma, Ram Varma, Lian-She Zhao, Donald Perillat, Greg Carricato, Michael Recknor, Kevin Du, Huddee Ho, Tim Ellis, Juan Gamez, Michael Howes, Janette Phi-Wilson, Scott Lockard, Robert Zuk and Hong Tan
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