Model Organisms in Drug Discovery

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132 DROSOPHILA – A MODEL SYSTEM gene, for example, is required for early patterning of the embryo and was found in the famous Nu¨sslein–Volhard screen. Its later function in imaginal disc growth and patterning could not be detected by examining homozygous mutant animals. 3. Genes whose proteins possess overlapping (redundant) functions cannot be detected. 4. The screens for recessive mutations are laborious because it requires the establishment of independent lines in which the homozygous phenotype is detected only in the third generation (see Figure 5.2A). Screens for recessive mutations in tissue-specific mosaic animals To circumvent some of these problems, a method to identify genes in tissuespecific genetic mosaics was developed (Newsome et al., 2000): the aim of the method is to generate flies that are homozygous for mutations on a chromosome arm in a tissue such as the adult eye. Because each fly receives a different set of mutagenized chromosomes from the mutagen-treated father, each individual fly will manifest the phenotype of a recessive mutation in this particular tissue. In the remaining tissues and in the germ-line, the fly is heterozygous for the same mutation. In contrast to the classical recessive screens over three generations, tissue-specific screens reveal the results in the first generation (see Figure 5.2B). Individual flies exhibiting the desired phenotype can be selected and established in a line. Furthermore, in many cases the mutant tissue survives to adulthood while homozygosity of the same mutation in the entire animal is lethal. The principle of the method is depicted in Figure 5.2B. Homozygous mutant clones are produced by the Flp/FRT system (Xu and Rubin, 1993): Flp recombinase from yeast mediates site-specific recombination between its target sites, called FRT (Flp recombinase target). When these FRT sequences are integrated at identical positions on the homologous chromosomes, the Flp recombinase will mediate sister chromatid exchange and thereby induce sitespecific mitotic recombination. When the Flp recombinase is induced in a cell heterozygous for a newly induced mutation on an FRT-bearing chromosome, mitotic recombination will generate two unequal daughter cells, one homozygous for the mutation and the other homozygous for the nonmutagenized chromosome. In subsequent divisions, each of these cells will develop into a clone of cells. To eliminate the clone of cells carrying the nonmutagenized chromosome, this chromosome is made to carry a recessive cell lethal mutation. Homozygosity for this mutation after mitotic recombination will eliminate this cell. Expression of Flp recombinase under the control of a tissue-specific promoter expressed in the early progenitor cells permits mitotic recombination to be restricted to a single tissue (see Figure 5.2B).
TARGET IDENTIFICATION/TARGET VALIDATION STRATEGIES 133 We will demonstrate the principle and efficiency of this type of screen with the example of the so-called ‘pinhead screen’: a screen to search for genes involved in cell growth and cell proliferation (Oldham et al., 2000). In the pinhead screen, Flp recombinase was induced by an eye-specific enhancer (ey-Flp). This limits clone induction to the head capsule and prevents deleterious effects of the mutations in other tissues. Such flies were analyzed for mutations that affect cell growth and cell size. So-called pin- or bigheads were recovered when growthpromoting or growth-inhibiting genes were hit, respectively (see Figure 5.3D and 5.3E). In this screen, already known and novel components of the insulin pathway (chico), oncogenes (PI3K, Akt, Tor) and the tumor suppressors PTEN, TSC1 and TSC2 were identified (Oldham et al., 2000; H. Stocker, S. Breuer and E. Hafen, unpublished results). In addition, we identified some 20 novel loci that either promote or inhibit growth. The corresponding genes are in the process of being characterized. Given their central role in the control of cellular growth, the novel growth-promoting genes are promising targets for anticancer therapy. The growth-inhibiting genes are potential tumor suppressor genes in humans and may serve as diagnostic markers. The close link between cell growth and basic metabolism manifested by components of the insulin signaling pathways in mammals and Drosophila may also offer the opportunity to validate the products of the genes identified in this screen as target of metabolic disorders such as type 2 diabetes. Dominant modifier screens Most LOF mutations are recessive, which means that 50% of the wild-type protein is sufficient for normal function. When a particular process is already partially disrupted by another mutation, however, the amount of components in the same pathway may become rate-limiting. A sensitized genetic background therefore can be used to screen for dominant enhancers or suppressors of a particular mutation. The advantage of such a screen is its simplicity, because only one of the two alleles has to be mutant (F1 screen). Even more importantly, such dosage-sensitive genetic interactions are usually indicative of a specific association of the newly identified components with the sensitized signaling pathway. Examples of such screens have been discussed above in the context of the WNT and Ras pathways. For illustration of a dominant modifier screen, see Figures 5.2C and 5.3A–C. EP overexpression screens Traditionally, genes are characterized based on LOF phenotypes. However, it is estimated that two-thirds of all Drosophila genes have no obvious LOF phenotype (Miklos and Rubin, 1996). This is at least in part due to functional
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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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Page 91 and 92: 82 DROSOPHILA AS A TOOL FOR DRUG DI
Page 109 and 110: 100 DROSOPHILA AS A TOOL FOR DRUG D
Page 127 and 128: 5 Drosophila - a Model System for T
Page 129 and 130: EVOLUTIONARY CONSERVATION OF DISEAS
Page 137 and 138: TARGET IDENTIFICATION/TARGET VALIDA
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Page 151 and 152: CHEMICAL GENETICS 143 acid identity
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Page 157 and 158: REFERENCES 149 Han, Z. S., Enslen,
Page 159 and 160: REFERENCES 151 Rintelen, F., Stocke
Page 161 and 162: 6 Mechanism of Action in Model Orga
Page 163 and 164: INTRODUCTION TO COMPOUND DEVELOPMEN
Page 165 and 166: MODEL ORGANISMS ARRIVE ON THE SCENE
Page 167 and 168: ELUCIDATING THE MECHANISM OF COMPOU
Page 169 and 170: ELUCIDATING THE MECHANISM OF COMPOU
Page 171 and 172: A CASE STUDY FOR ALZHEIMER’S DISE
Page 179 and 180: NEW CHEMICAL GENETIC STRATEGIES 171
Page 181 and 182: A CASE STUDY FOR INNATE IMMUNITY 17
Page 183 and 184: GLOBAL GENE EXPRESSION STUDIES IN M
Page 185 and 186: As described above, the extensive i
Page 187 and 188: REFERENCES 179 Austin, J. and Kimbl
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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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REFERENCES 219 Chau, I. and Cunning
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REFERENCES 221 Patrono, C., Patrign
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224 CHEMICAL MUTAGENESIS IN THE MOU
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252 SATURATION SCREENING OF DRUGGAB
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280 INDEX bupropion 92 busulfan 143
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282 INDEX Dscam 171 dual-energy X-r
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284 INDEX L-685,818 16 lead discove
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286 INDEX protein function 19-22 pr
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288 INDEX yeast (continued) functio
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