Model Organisms in Drug Discovery

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68 C. ELEGANS FUNCTIONAL GENOMICS IN DRUG DISCOVERY Figure 3.6 Hit filtering with C. elegans mutants. The two compounds SER1 (&), a putative serotonin reuptake inhibitor, and DOP1 (^), a putative dopamine receptor antagonist, enhance drinking in the ‘drinking assay’. The compound concentration (in mM) is given on the x-axis and the relative fluorescence (in %) is given on the y-axis; 100% corresponds to no effect on drinking for a given strain. The control strain has only minimal levels of the neurotransmitters serotonin and dopamine. Any compound that increases serotonergic signaling via the serotonin reuptake transporter cannot enhance drinking in this strain. Similarly, dopamine receptor antagonists cannot enhance drinking because the dopaminergic signaling is already reduced in this strain. The strain nodop-1 lacks only dopaminergic signaling. A serotonergic compound can enhance drinking in this strain whereas a dopaminergic compound cannot. The results of the two compounds on the strain nodop-1 suggest that only SER1 is a serotonergic compound. For the strain noser-1 the situation is reversed. This strain lacks serotonergic signaling, hence SER1 cannot enhance drinking. Compound DOP1 enhances drinking and is therefore unlikely to be a serotonergic compound screens with C. elegans only offer real advantages if data on complex phenotypes such as cell migration in a living animal, movement pattern and morphology can be generated. A special type of C. elegans automation technology is COPAS TM from Union Biometrica, USA. This C. elegans animal sorter works like a conventional fluorescence-activated cell sorter (FACS). Different from conventional cell sorting, COPAS TM is able to sort tube-shaped living animals of lengths varying from 70 to 1300 mm. COPAS TM records four parameters per object passing the sorting chamber: the time of flight to measure length, the extinction to discriminate transparent larvae from darker adults and two fluorescent parameters (www.unionbio.com). Sophisticated software, the COPAS Profiler TM , has been developed to sort fluorescencelabeled animals by the position of the fluorescence signal along the body axis. The COPAS TM platform is equipped with a plate handling system and operates quickly enough to be plugged into a high-throughput screening process.
Compound learning set for assay validation In addition to confirming the quality of a C. elegans assay, the assay must also be pharmacologically validated. Devgen has used a learning set of about 250 CNS drugs to include drugs with mode of actions that modulate drinking and drugs that should not affect drinking. These classes of drugs have been used to validate the C. elegans ‘drinking assay’. Serotonin reuptake inhibition should increase drinking and consequently 485% of the tested SSRIs and 475% of all 5-HT reuptake inhibitors have been shown to enhance drinking rates in the assay. Because dopamine negatively regulates drinking, inhibitors of dopaminergic signaling should also enhance drinking, but indirectly. Fortyfive percent of the dopamine antagonists tested have been shown to influence drinking rates. The third important neurotransmitter that increases drinking rates is acetylcholine. Consequently, none of the tested antagonists were detected in the screen. A range of unrelated CNS compounds have been tested, including adrenergic antagonists, opioids and histaminergics. Fifteen percent of the adrenergic modulators were shown to be enhancers of drinking rates and must be considered as false positives. The conclusion is that the drinking screen for enhancers is highly sensitive because it identifies most of the SSRIs for which the assay has been configured. It should be noted that this C. elegans assay is able to identify reliable human drugs for a specific target and that the assay is another example of the high level of conservation between C. elegans and human pharmacology. Another question is the number of false positives that can be expected. Thirty-three percent of all hits obtained in the ‘drinking assay’ are the desired SSRIs (Figure 3.5). In addition, 51% of the hits act specifically on the biology under investigation because a serotonin receptor agonist also increases drinking. Although these hits may be still of interest, we will show later how to filter out dopaminergics and serotonin receptor modulators. The remaining 16% of the hits are false positives, which need to be filtered out as well. The data validate the drinking assay as highly robust, sensitive and selective. We will describe the C. elegans screening platform and a screening campaign with the drinking assay. The C. elegans screening platform LEAD DISCOVERY 69 The HTS equipment used in a C. elegans screening unit includes the same robotics and technology found in standard HTS laboratories but with one important difference (Seethala, 2001): instead of targets or cells being present in the wells for screening, the wells are filled with living animals swimming through the medium. The growth and handling requirements associated with the use of living C. elegans animals limits the time available for screening to 3
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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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Page 27 and 28: 16 GROWING YEAST FOR FUN AND PROFIT
Page 51 and 52: 3 Caenorhabditis elegans Functional
Page 53 and 54: THE DRUG DISCOVERY PROCESS 43 thoug
Page 55 and 56: multivulva phenotype of Ras gain-of
Page 57 and 58: disease. These molecular targets ar
Page 59 and 60: FROM DISEASE TO TARGET 49 Figure 3.
Page 61 and 62: FROM DISEASE TO TARGET 51 Figure 3.
Page 63 and 64: signaling pathway. The third catego
Page 65 and 66: FROM DISEASE TO TARGET 55 resistant
Page 67 and 68: phenomenon was first observed in C.
Page 69 and 70: profiles. The C. elegans gene expre
Page 71 and 72: emerging technologies. Forward gene
Page 73 and 74: The compound library LEAD DISCOVERY
Page 75 and 76: LEAD DISCOVERY 65 previous section,
Page 77: LEAD DISCOVERY 67 Figure 3.5 Distri
Page 81 and 82: 10 000-15 000 human drug targets ar
Page 83 and 84: transporter itself. The SSRIs that
Page 85 and 86: REFERENCES 75 de Montigny, C., Blie
Page 87 and 88: REFERENCES 77 Lewis, J. A., Wu, C.
Page 89 and 90: REFERENCES 79 Tissenbaum, H. A. and
Page 91 and 92: 82 DROSOPHILA AS A TOOL FOR DRUG DI
Page 109 and 110: 100 DROSOPHILA AS A TOOL FOR DRUG D
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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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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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