The Experts below are selected from a list of 128334 Experts worldwide ranked by ideXlab platform
Leonard I. Zon - One of the best experts on this subject based on the ideXlab platform.
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Current Protocols in Chemical Biology - In situ hybridization assay-based small molecule Screening in zebrafish
Current protocols in chemical biology, 2012Co-Authors: Lili Jing, Ellen M. Durand, Catherine Ezzio, Stephanie M. Pagliuca, Leonard I. ZonAbstract:In vitro biochemical and cell-based small molecule screens have been widely used to identify compounds that target specific signaling pathways. But the identified compounds frequently fail at the animal testing stage, largely due to the in vivo absorption, metabolism and toxicity of chemicals. Zebrafish has recently emerged as a vertebrate whole organism model for small molecule Screening. The in vivo bioactivity and specificity of compounds are examined from the very beginning of zebrafish screens. In addition, zebrafish is suitable for chemical screens at a large scale similar to cellular assays. This protocol describes an approach for in situ hybridization (ISH)-based chemical Screening in zebrafish, which, in principle, can be used to screen any gene product. The described protocol has been used to identify small molecules affecting specific molecular pathways and biological processes. It can also be adapted to zebrafish screens with different readouts.
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Small molecule Screening in zebrafish: swimming in potential drug therapies
Wiley interdisciplinary reviews. Developmental biology, 2012Co-Authors: Owen J. Tamplin, Lili Jing, Richard M. White, Charles K. Kaufman, Scott A. Lacadie, Alison M. Taylor, Leonard I. ZonAbstract:Phenotype-driven chemical genetic screens in zebrafish have become a proven approach for both dissection of developmental mechanisms and discovery of potential therapeutics. A library of small molecules can be arrayed into multiwell plates containing zebrafish embryos. The embryo becomes a whole organism in vivo bioassay that can produce a phenotype upon treatment. Screens have been performed that are based simply on the morphology of the embryo. Other screens have scored complex phenotypes using whole mount in situ hybridization, fluorescent transgenic reporters, and even tracking of embryo movement. The availability of many well-characterized zebrafish mutants has also enabled the discovery of chemical suppressors of genetic phenotypes. Importantly, the application of chemical libraries that already contain FDA-approved drugs has allowed the rapid translation of hits from zebrafish chemical screens to clinical trials.
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Small molecule Screening in the zebrafish.
Methods (San Diego Calif.), 2006Co-Authors: Ryan D. Murphey, Leonard I. ZonAbstract:The zebrafish is an ideal organism for small molecule studies. The ability to use the whole organism allows complex in vivo phenotypes to be assayed and combines animal testing with Screening. Embryos are easily treatable by waterborne exposure. The small size and abundance of embryos make zebrafish suitable for Screening in a high-throughput manner in 96- or 48-well plates. Zebrafish embryos have successfully been used in chemical genetic screens to elucidate biological pathways and find chemical suppressors. Small molecules discovered by Screening zebrafish disease models may also be useful as lead compounds for drug development as there appears to be a high level of conservation of drug activity between mammals and zebrafish. Here we provide the technical aspects of treating embryos with small molecules and performing chemical screens with zebrafish.
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Cancer genetics and drug discovery in the zebrafish.
Nature reviews. Cancer, 2003Co-Authors: Howard M. Stern, Leonard I. ZonAbstract:Fish have a long history of use in cancer toxicology studies, because they develop neoplasms that are histologically similar to human cancers. Because of considerable progress in zebrafish genetics and genomics over the past few years, the zebrafish system has provided many useful tools for studying basic biological processes. These tools include forward genetic screens, transgenic models, specific gene disruptions and Small-Molecule screens. By combining carcinogenesis assays, genetic analyses and Small-Molecule Screening techniques, the zebrafish is emerging as a powerful system for identifying novel cancer genes and for cancer drug discovery.
John D. Brennan - One of the best experts on this subject based on the ideXlab platform.
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A Sol−Gel-Derived Acetylcholinesterase Microarray for Nanovolume Small-Molecule Screening
Analytical chemistry, 2010Co-Authors: Maria Rowena N. Monton, Julie M. Lebert, Jessamyn R. L. Little, Jerald J. Nair, James Mcnulty, John D. BrennanAbstract:A fluorimetric acetylcholinesterase (AChE) assay was developed and characterized both in solution and with the enzyme entrapped in sol-gel-derived silica. The assay is based on a disulfide-thiol interchange reaction between the intramolecularly quenched dimeric dye BODIPY FL l-cystine and thiocholine generated by the AChE-catalyzed hydrolysis of acetylthiocholine (ATCh), which results in a brightly fluorescent monomeric product owing to the cleavage of the disulfide-coupled form of the dye. The new assay was validated by comparison with the Ellman assay performed under parallel conditions and was used in both kinetic and end point assays. The assay was extended to the fabrication of functional AChE microarrays using contact pin-printing of sol-gel-derived silica. A total of 392 sol-gel formulations were screened for gelation times and 192 of these were further evaluated for array fabrication on four different surfaces using a factor analysis approach. Of these, 66 sol-gel/surface combinations produced robust microarrays, while 26 sol-gel/surface combinations were identified that could produce highly active AChE microarrays. The Z' factor for the on-array assay using an optimal sol-gel/surface combination, which considers both signal variability and difference in signals between positive and negative controls, was determined to be 0.60, which is above the minimum level required for applicability to Screening. By overprinting nanoliter volumes of solutions containing the dye, ATCh, and potential inhibitors, these microarrays could be used to screen two libraries of small molecules, one composed of newly synthesized alkaloids and another consisting of ∼1000 known bioactive compounds, both as discrete compounds and mixtures thereof, for activity against AChE. IC(50) values were obtained on microarrays for compounds showing significant inhibitory activity, demonstrating the utility of arrays for quantitative inhibition assays.
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Solid-phase assays for small molecule Screening using sol-gel entrapped proteins.
Biochemistry and cell biology = Biochimie et biologie cellulaire, 2008Co-Authors: Julie M. Lebert, Erica M. Forsberg, John D. BrennanAbstract:With compound libraries exceeding one million compounds, the ability to quickly and effectively screen these compounds against relevant pharmaceutical targets has become crucial. Solid-phase assays present several advantages over solution-based methods. For example, a higher degree of miniaturization can be achieved, functional- and affinity-based studies are possible, and a variety of detection methods can be used. Unfortunately, most protein immobilization methods are either too harsh or require recombinant proteins and thus are not amenable to delicate proteins such as kinases and membrane-bound receptors. Sol-gel encapsulation of proteins in an inorganic silica matrix has emerged as a novel solid-phase assay platform. In this minireview, we discuss the development of sol-gel derived protein microarrays and sol-gel based monolithic bioaffinity columns for the high-throughput Screening of small molecule libraries and mixtures.
Eric D. Brown - One of the best experts on this subject based on the ideXlab platform.
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Nutrient Stress Small-Molecule Screening Platform for Escherichia coli.
Methods in molecular biology (Clifton N.J.), 2018Co-Authors: Sara S. El Zahed, Garima Kumar, Madeline Tong, Eric D. BrownAbstract:Clinically approved antibiotics target a narrow spectrum of cellular processes, namely cell wall synthesis, DNA replication, and protein synthesis. Numerous screens have been designed to identify inhibitors that target one of these cellular processes. Indeed, this narrow range of drug mechanisms and a reliance on chemical classes discovered many decades ago are thought to be principally responsible for the current crisis of antibiotic drug resistance. Seeking to expand the target base of antibacterial drug discovery, we developed a nutrient stress Screening platform that identifies inhibitors of the growth of in Escherichia coli under nutrient limitation. Under nutrient stress, bacteria require an expanded biosynthetic capacity that includes the synthesis of amino acids, vitamins, and nucleobases. Growing evidence suggests that these processes may be indispensable to certain pathogens and at particular sites of infection. Indeed, more than 100 biosynthetic enzymes become indispensable to E. coli grown under nutrient stress in vitro. The Screening platform described here puts a focus on these novel targets for new antibiotics and prioritizes growth inhibitory compounds that can be suppressed by individual nutrients and pools thereof.
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A Small-Molecule Screening Platform for the Discovery of Inhibitors of Undecaprenyl Diphosphate Synthase
ACS infectious diseases, 2016Co-Authors: Tomasz L. Czarny, Eric D. BrownAbstract:The bacterial cell wall has long been a celebrated target for antibacterial drug discovery due to its critical nature in bacteria and absence in mammalian systems. At the heart of the cell wall biosynthetic pathway lies undecaprenyl phosphate (Und-P), the lipid-linked carrier upon which the bacterial cell wall is built. This study exploits recent insights into the link between late-stage wall teichoic acid inhibition and Und-P production, in Gram-positive organisms, to develop a cell-based Small-Molecule Screening platform that enriches for inhibitors of undecaprenyl diphosphate synthase (UppS). Screening a chemical collection of 142,000 small molecules resulted in the identification of 6 new inhibitors of UppS. To date, inhibitors of UppS have generally shown off-target effects on membrane potential due to their physical-chemical characteristics. We demonstrate that MAC-0547630, one of the six inhibitors identified, exhibits selective, nanomolar inhibition against UppS without off-target effects on membrane potential. Such characteristics make it a unique chemical probe for exploring the inhibition of UppS in bacterial cell systems.
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Screening in Academe: A Perspective on Implementation of University-Based Small Molecule Screening
Journal of biomolecular screening, 2003Co-Authors: Eric D. BrownAbstract:With the emergence ofsmall molecule Screening as a power ful research tool in biology, biomolecular Screening has arrived in the laboratories ofacademe. More and more, researchers in universities and hospital research institutes are recognizing the power ofsmall molecules as probes ofbiochemical and biological systems. At McMaster University in Hamilton, Canada, we have established a state-of-the-art small molecule Screening laboratory that became fully operational a little more than a year ago (http:// hts.mcmaster.ca). It is with considerable enthusiasm, as the founding director of that laboratory, that I offer the following perspectives on Screening in an academic setting and on my own experiences in setting up such a facility. Since the establishment ofthe first academic Screening operations, including, for example, that of the Harvard Institute of Chemistry and Cell Biology in the late 1990s, there has been a groundswell ofinterest among academic researchers in highthroughput Screening (HTS). Today, biological researchers in most research-intensive academic institutions are, at the very least, considering the establishment ofcapabilities and a presence in small molecule Screening to fuel research activities in the emerging field ofchemical biology. Nevertheless, the implementation ofa reason ably capable Screening facility with its associated liquid-handling, instrumentation, compound, and information management systems is a complex, costly, and energetic undertaking. In setting up a state-of-the-art Screening laboratory, it has been instructive for me to reflect on how advances in HTS in the private sector have exerted recent influence on research directions in bio logical research at universities and research institutes. This trend contrasts with the conventional academic view ofinnovation that has emerging technology moving from university to industry. Advances in biological research have, ofcourse, been among the most celebrated university-based innovations. The once purely academic pursuits ofbiological chemistry and cell biology have slowly but firmly established themselves in the research paradigm off ormerly chemistry-centered pharma, beginning with revolu tionary advances in molecular biology in the late 1970s. It is per haps ironic, ifnot remarkable, then, that similarly revolutionary progress made by high-throughput screeners in the pharmaceutical sector has affected the technology now available to chemical biologists in academe. Equipped with robust robotics, information systems, and wellestablished Screening methodologies, all developed by and large in the biotechnology and pharmaceutical sector, biochemists and cell biologists in academe are turning in earnest to small molecule Screening as a fresh approach to discovering molecular probes of systems under their study. With the freedom to innovate and publish, academic screeners will now surely be in a position to return the favor to their private-sector colleagues with new discoveries and new approaches. It is clearly fitting that the Society for Biomolecular Screening (SBS), founded by private-sector researchers, has taken steps to welcome screeners from academe with the formation of the Academic Outreach Committee in the spring of2003. The committee is composed ofa mix ofindividuals from industry and academe, including myself, who have a keen interest in building bridges among academic screeners and their colleagues in pharmaceutical, biotechnology, and instrumentation companies. The committee also has the mandate offurthering the interests and profile of academic screeners in the SBS and has the potential to function as a nucleus for the exchange of ideas and experiences among academic screeners. Our own HTS lab was founded by 3 principal investigators at McMaster whose research interests include antimicrobial research (Gerry Wright and I) and materials science (John Brennan). The genesis and funding for the laboratory came as part of a provincewide initiative in genomics and included close partnerships with combinatorial chemistry laboratories at York University and the University ofToronto. The thinking was that the chemists and screeners could work together, for example, in lead optimization activities. Indeed, the practicality ofattracting the interest of collaborators in synthetic chemistry for downstream optimization of hits from commercial libraries is frequently a concern among
Per Uhlén - One of the best experts on this subject based on the ideXlab platform.
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Small molecule Screening platform for assessment of cardiovascular toxicity on adult zebrafish heart
BMC Physiology, 2012Co-Authors: Satish Srinivas Kitambi, Erik S Nilsson, Petra Sekyrova, Cristian Ibarra, Gilbert Nyah Tekeoh, Michael Andäng, Patrik Ernfors, Per UhlénAbstract:Background Cardiovascular toxicity is a major limiting factor in drug development and requires multiple cost-effective models to perform toxicological evaluation. Zebrafish is an excellent model for many developmental, toxicological and regenerative studies. Using approaches like morpholino knockdown and electrocardiogram, researchers have demonstrated physiological and functional similarities between zebrafish heart and human heart. The close resemblance of the genetic cascade governing heart development in zebrafish to that of humans has propelled the zebrafish system as a cost-effective model to conduct various genetic and pharmacological screens on developing embryos and larvae. The current report describes a methodology for rapid isolation of adult zebrafish heart, maintenance ex vivo , and a setup to perform quick small molecule throughput Screening, including an in-house implemented analysis script. Results Adult zebrafish were anesthetized and after rapid decapitation the hearts were isolated. The short time required for isolation of hearts allows dissection of multiple fishes, thereby obtaining a large sample size. The simple protocol for ex vivo culture allowed maintaining the beating heart for several days. The in-house developed script and spectral analyses allowed the readouts to be presented either in time domain or in frequency domain. Taken together, the current report offers an efficient platform for performing cardiac drug testing and pharmacological screens. Conclusion The new methodology presents a fast, cost-effective, sensitive and reliable method for performing small molecule Screening. The variety of readouts that can be obtained along with the in-house developed analyses script offers a powerful setup for performing cardiac toxicity evaluation by researchers from both academics and industry.
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Small molecule Screening platform for assessment of cardiovascular toxicity on adult zebrafish heart
BMC physiology, 2012Co-Authors: Satish Srinivas Kitambi, Petra Sekyrova, Cristian Ibarra, Gilbert Nyah Tekeoh, Michael Andäng, Patrik Ernfors, Erik Nilsson, Per UhlénAbstract:Cardiovascular toxicity is a major limiting factor in drug development and requires multiple cost-effective models to perform toxicological evaluation. Zebrafish is an excellent model for many developmental, toxicological and regenerative studies. Using approaches like morpholino knockdown and electrocardiogram, researchers have demonstrated physiological and functional similarities between zebrafish heart and human heart. The close resemblance of the genetic cascade governing heart development in zebrafish to that of humans has propelled the zebrafish system as a cost-effective model to conduct various genetic and pharmacological screens on developing embryos and larvae. The current report describes a methodology for rapid isolation of adult zebrafish heart, maintenance ex vivo, and a setup to perform quick small molecule throughput Screening, including an in-house implemented analysis script. Adult zebrafish were anesthetized and after rapid decapitation the hearts were isolated. The short time required for isolation of hearts allows dissection of multiple fishes, thereby obtaining a large sample size. The simple protocol for ex vivo culture allowed maintaining the beating heart for several days. The in-house developed script and spectral analyses allowed the readouts to be presented either in time domain or in frequency domain. Taken together, the current report offers an efficient platform for performing cardiac drug testing and pharmacological screens. The new methodology presents a fast, cost-effective, sensitive and reliable method for performing small molecule Screening. The variety of readouts that can be obtained along with the in-house developed analyses script offers a powerful setup for performing cardiac toxicity evaluation by researchers from both academics and industry.
Julie M. Lebert - One of the best experts on this subject based on the ideXlab platform.
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A Sol−Gel-Derived Acetylcholinesterase Microarray for Nanovolume Small-Molecule Screening
Analytical chemistry, 2010Co-Authors: Maria Rowena N. Monton, Julie M. Lebert, Jessamyn R. L. Little, Jerald J. Nair, James Mcnulty, John D. BrennanAbstract:A fluorimetric acetylcholinesterase (AChE) assay was developed and characterized both in solution and with the enzyme entrapped in sol-gel-derived silica. The assay is based on a disulfide-thiol interchange reaction between the intramolecularly quenched dimeric dye BODIPY FL l-cystine and thiocholine generated by the AChE-catalyzed hydrolysis of acetylthiocholine (ATCh), which results in a brightly fluorescent monomeric product owing to the cleavage of the disulfide-coupled form of the dye. The new assay was validated by comparison with the Ellman assay performed under parallel conditions and was used in both kinetic and end point assays. The assay was extended to the fabrication of functional AChE microarrays using contact pin-printing of sol-gel-derived silica. A total of 392 sol-gel formulations were screened for gelation times and 192 of these were further evaluated for array fabrication on four different surfaces using a factor analysis approach. Of these, 66 sol-gel/surface combinations produced robust microarrays, while 26 sol-gel/surface combinations were identified that could produce highly active AChE microarrays. The Z' factor for the on-array assay using an optimal sol-gel/surface combination, which considers both signal variability and difference in signals between positive and negative controls, was determined to be 0.60, which is above the minimum level required for applicability to Screening. By overprinting nanoliter volumes of solutions containing the dye, ATCh, and potential inhibitors, these microarrays could be used to screen two libraries of small molecules, one composed of newly synthesized alkaloids and another consisting of ∼1000 known bioactive compounds, both as discrete compounds and mixtures thereof, for activity against AChE. IC(50) values were obtained on microarrays for compounds showing significant inhibitory activity, demonstrating the utility of arrays for quantitative inhibition assays.
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Solid-phase assays for small molecule Screening using sol-gel entrapped proteins.
Biochemistry and cell biology = Biochimie et biologie cellulaire, 2008Co-Authors: Julie M. Lebert, Erica M. Forsberg, John D. BrennanAbstract:With compound libraries exceeding one million compounds, the ability to quickly and effectively screen these compounds against relevant pharmaceutical targets has become crucial. Solid-phase assays present several advantages over solution-based methods. For example, a higher degree of miniaturization can be achieved, functional- and affinity-based studies are possible, and a variety of detection methods can be used. Unfortunately, most protein immobilization methods are either too harsh or require recombinant proteins and thus are not amenable to delicate proteins such as kinases and membrane-bound receptors. Sol-gel encapsulation of proteins in an inorganic silica matrix has emerged as a novel solid-phase assay platform. In this minireview, we discuss the development of sol-gel derived protein microarrays and sol-gel based monolithic bioaffinity columns for the high-throughput Screening of small molecule libraries and mixtures.
