Model Organisms in Drug Discovery

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. Olfactory discrimination test (olfaction and social recognition) . Trace and delay aversive conditioning . Social interaction and social recognition tests . Zero maze (anxiety) Oncology The targets of current oncology therapeutics fall into three major categories: cytotoxic agents such as DNA damaging agents or inhibitors of tubulin or topoisomerase, tissue-specific growth regulators such as estrogen receptor blockers and leutinizing hormone blockers, and disease-specific antitumor agents such as Gleevec, Herceptin and Rituxan. The oncology Level 1 screen is based on the hypothesis that targets for the next generation of cancer drugs are likely to fall into the same categories operating through control points in mammalian cell cycle, apoptosis or response to DNA damage. Level 1 tests Embyronic lethality and reduced viability Targets for future cytotoxic agents are likely to be identified first by embryonic lethality or reduced viability. These phenotypes are examined further to detemine effects on cell cycle, apoptosis and angiogensis. Tissue-specific growth regulation Targets affecting growth, differentiation and function of reproductive organs are examined through histopathologic survey of males, virgin females and lactating female mice. Cell proliferation HIGH-THROUGHPUT BIOLOGY 271 Oncogene targets that have a direct effect on cell cycle, DNA repair or apoptosis can manifest their function through changes in adult skin fibroblast proliferation. Punch biopsies are taken of skin samples from the backs of mutant mice and cohort controls. These are developed into primary fibroblast cultures and the fibroblast proliferation rates are measured in a strictly controlled protocol. The ability of this assay to detect hyperproliferative and hypoproliferative phenotypes has been demonstrated with p53 and Ku80 (unpublished results).
272 SATURATION SCREENING OF DRUGGABLE MAMMALIAN GENOME Level 2 oncology tests Targets identified from Level 1 are characterized further for their potential role in human tumorigenesis. Focus is placed on targets that are highly expressed in human tumor cell lines and capable of driving the tumor phenotype as demonstrated by gene knock-down studies or overexpressiondriven tumorigenesis models in nude mice. Quantitative polymerase chain reaction for analysis of expression in cancerous and normal cell lines and tissues Quantitative polymerase chain reaction of candidate genes is done using cDNA prepared from 66 cancer and nine normal cell lines from ATCC, seven primary cell strains from Clonetics, about three cancer lines and matched adjacent normal tissue controls from Ambion, MCF-7 breast cancer cells +/ 17b-estradiol and LNCaP prostate cancer cells +/ dihydrotestosterone. This is done to identify targets that are overexpressed in cancerous cell lines relative to normal cell and tissue controls. Gene knock-down studies with short interfering RNA Cancer cell lines determined to be overexpressing a target of interest are cotransfected with 3–6 short hairpin RNA vectors and blasticidin resistance vectors or synthetic short interfering RNAs to knock down the expression of specific targets. Assessment is made of the effects of RNA interference on in vitro proliferation, anchorage-dependent and anchorage-independent colony formation and the ability of cell lines to form tumors in nude mice. Overexpression studies for putative oncogenes Potential oncology targets are tested to determine whether they can drive tumor formation. Full-length genes of interest are cloned into a mammalian expression vector and co-transfected into NIH3T3 and RK3E cells with a blasticidin-resistance vector. The resulting blasticidin-resistant polyclonal pools are tested in vitro for acquisition of anchorage independence, reduced serum dependence and increased focus-forming ability. Stably transfected cell lines expressing exogenous cDNAs of interest are then analyzed for their ability to form tumors in athymic nude mice.
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Model Organisms in Drug Discovery M
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Copyright u 2003 John Wiley & Sons
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Contents List of contributors .....
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CONTENTS ix 7 Genetics and Genomics
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List of Contributors Hector Beltran
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LIST OF CONTRIBUTORS xiii Stefan Sc
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1 Introduction to Model Systems in
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Organism INTEGRATING MODEL ORGANISM
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INTEGRATING MODEL ORGANISM RESEARCH
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INTEGRATING MODEL ORGANISM RESEARCH
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10 GROWING YEAST FOR FUN AND PROFIT
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3 Caenorhabditis elegans Functional
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THE DRUG DISCOVERY PROCESS 43 thoug
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multivulva phenotype of Ras gain-of
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disease. These molecular targets ar
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FROM DISEASE TO TARGET 49 Figure 3.
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FROM DISEASE TO TARGET 51 Figure 3.
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signaling pathway. The third catego
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FROM DISEASE TO TARGET 55 resistant
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phenomenon was first observed in C.
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profiles. The C. elegans gene expre
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emerging technologies. Forward gene
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The compound library LEAD DISCOVERY
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LEAD DISCOVERY 65 previous section,
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LEAD DISCOVERY 67 Figure 3.5 Distri
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Compound learning set for assay val
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10 000-15 000 human drug targets ar
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transporter itself. The SSRIs that
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REFERENCES 75 de Montigny, C., Blie
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REFERENCES 77 Lewis, J. A., Wu, C.
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REFERENCES 79 Tissenbaum, H. A. and
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82 DROSOPHILA AS A TOOL FOR DRUG DI
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100 DROSOPHILA AS A TOOL FOR DRUG D
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5 Drosophila - a Model System for T
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EVOLUTIONARY CONSERVATION OF DISEAS
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TARGET IDENTIFICATION/TARGET VALIDA
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CHEMICAL GENETICS 143 acid identity
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3. A hit identifies a biologically
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about their function in a multicell
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REFERENCES 149 Han, Z. S., Enslen,
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REFERENCES 151 Rintelen, F., Stocke
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6 Mechanism of Action in Model Orga
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INTRODUCTION TO COMPOUND DEVELOPMEN
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MODEL ORGANISMS ARRIVE ON THE SCENE
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ELUCIDATING THE MECHANISM OF COMPOU
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ELUCIDATING THE MECHANISM OF COMPOU
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A CASE STUDY FOR ALZHEIMER’S DISE
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NEW CHEMICAL GENETIC STRATEGIES 171
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A CASE STUDY FOR INNATE IMMUNITY 17
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GLOBAL GENE EXPRESSION STUDIES IN M
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As described above, the extensive i
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REFERENCES 179 Austin, J. and Kimbl
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REFERENCES 181 Hughes, T. R., Marto
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REFERENCES 183 Sin, N., Meng, L., W
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186 GENETICS AND GENOMICS IN THE ZE
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8 Lipid Metabolism and Signaling in
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FISH AS A MODEL ORGANISM 205 genes
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LIPID METABOLISM SCREEN 207 transpo
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LIPID METABOLISM SCREEN 209 Figure
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the embryo media containing radioac
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ZEBRAFISH AS A MODEL SYSTEM 213 Fig
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ZEBRAFISH AS A MODEL SYSTEM 215 Reg
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aspirin, which has potent inhibitor
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Page 285 and 286: 280 INDEX bupropion 92 busulfan 143
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Page 289 and 290: 284 INDEX L-685,818 16 lead discove
Page 291 and 292: 286 INDEX protein function 19-22 pr
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