Model Organisms in Drug Discovery

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SCREENING THE GENOME EFFECTIVELY 255 proteins. Indeed, one could argue that all human disease or disease treatment pathways of interest probably contain druggable genes, so that by mutating all the druggable genes in the genome one can interrogate all pathways for points of therapeutic intervention. Demonstrating the scale at which mammalian genes can be mutated, we have industrialized gene knock-out technologies for saturation of the druggable genome within the next 4 years. We have implemented our genome 5000 program to knock out and analyze the resulting phenotypes for 5000 genes from the mammalian genome. The 5000 genes chosen are all members of the currently druggable gene families. Because others have suggested that the druggable genome may be as small as about 3000 genes (Hopkins and Groom, 2002), this scale is sufficient to saturate the mammalian druggable genome in order to identify those genes that have the greatest potential for human disease treatment. 10.3 Screening the genome effectively for novel drug targets Given the possibility of generating knock-out mouse lines at a rate of 1000 per year, the next challenge is to implement a biological evaluation process that has a high probability of identifying potential drug targets, as assessed by the physiological consequences of gene disruption. We have developed a process that maximizes our potential to identify therapeutically significant genes. This process represents the application of increasingly fine filters to genomic information. First, the genome is mined for members of druggable families. Second, knock-out mice are generated for selected genes at an average rate of 20 lines of mutant mice per week. A minimum cohort for initial evaluation is 16 animals; 8 homozygous nulls, 4 heterozygotes and 4 wild-type animals for each gene. This cohort size has produced reliable data from the primary screen upon which decisions for secondary screens can be made. Implementation of this plan has necessitated the integration of bioinformatics, mouse genetics, robotics and high-speed physiological evaluation in a unique and robust infrastructure that has demonstrated already the ability to operate at the required rate. The logistics of generating, maintaining, genotyping and characterizing the required number of animals have been satisfied. Our first biological evaluation of the animals is a comprehensive clinical assessment of all the physiological parameters that we can measure effectively in high-throughput mode. Each test has direct relevance to one or more of our therapeutic areas and is designed to yield information that can be correlated directly with therapeutic intervention. This process includes an extensive battery of behavioral evaluations (neurology), blood pressure and heart rate measurements (cardiology) and a complete hematology survey supplemented
256 SATURATION SCREENING OF DRUGGABLE MAMMALIAN GENOME with fluorescence-activated cell sorting (FACS) scans for immune function (immunology). The animals also are evaluated for body fat content, lean body mass (metabolism), bone mineral density, bone mineral content (endocrinology) and retinal integrity/vascularization (ophthalmology). Effects on cell proliferation and reproductive organ development are studied (oncology) and fertility (reproductive biology) is assessed. This screening phase of biological investigation is called Level 1 analysis. This initial analysis of the physiological consequence of creating null mutations is designed to be unbiased with regard to potential outcome but to encompass phenotypes indicative of utility to our chosen therapeutic areas. All animals in all projects are submitted to the same tests in the same temporal sequence. This means that each test must be self-contained and have minimal impact on the outcome of subsequent tests. The aim of Level 1 analysis is to obtain a comprehensive understanding of gene function within the context of mammalian physiology. Variations from normal in any parameter are detected by comparison with internal cohort controls and, very importantly, with the pooled historical data for all controls. Historical control data are now based on over 2500 animals, giving us a precise quantitative measure of ‘normal’ for each test and the level of background variation. Most of the tests are of primary importance to one particular therapeutic area (e.g. blood pressure and cardiology), but the total picture gained from this type of analysis is critical in identifying possible side-effects of target modulation. This allows the identification of targets with a high potential for success, provided that specific modulators can be developed. Figure 10.1 is a schematic outline of our Level 1 protocol. In addition to therapeutic area-specific tests, multiple general diagnostic tests are performed. Level I pathology examines 52 tissues for the female and 53 tissues for the male. A complete gross necropsy is performed, with collection of tissues and photography of any significant gross lesions. Tissues are immersion-fixed in 10% neutral buffered formalin for 24 h, trimmed, processed to paraffin, embedded, sectioned at 4–5 mm, and stained with hematoxylin and eosin for histopathological examination. A board-certified pathologist examined tissues from one male and one female homozygote for each project (heterozygotes are examined for homozygous lethal projects). The recent introduction of computer-assisted tomography (CAT) scanners, which operate effectively on mice, has enabled non-invasive evaluation of soft-tissue anatomy in addition to very refined skeletal analysis. Application of CAT (MicroCAT, ImTek Inc.) can be used to obtain important morphological information non-invasively. All lesions are recorded and compared with controls in order to facilitate interpretation of phenotypes. The output from all Level 1 tests is reduced to digital data and ported to a relational database. Data acquisition is rapid to the point that no Level 1 test
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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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Page 209 and 210: 8 Lipid Metabolism and Signaling in
Page 211 and 212: FISH AS A MODEL ORGANISM 205 genes
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Page 219 and 220: ZEBRAFISH AS A MODEL SYSTEM 213 Fig
Page 221 and 222: ZEBRAFISH AS A MODEL SYSTEM 215 Reg
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Page 229 and 230: 224 CHEMICAL MUTAGENESIS IN THE MOU
Page 257 and 258: 252 SATURATION SCREENING OF DRUGGAB
Page 259: 254 SATURATION SCREENING OF DRUGGAB
Page 285 and 286: 280 INDEX bupropion 92 busulfan 143
Page 287 and 288: 282 INDEX Dscam 171 dual-energy X-r
Page 289 and 290: 284 INDEX L-685,818 16 lead discove
Page 291 and 292: 286 INDEX protein function 19-22 pr
Page 293: 288 INDEX yeast (continued) functio
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