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In vivo analysis of compound activity and mechanism of action using epistasis in Drosophila

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Journal of Chemical Biology

Abstract

The recent establishment of high-throughput methods for culturing Drosophila provided a unique ability to screen compound libraries against complex disease phenotypes in the context of whole animals. However, as compound studies in Drosophila have been limited so far, the degree of conservation of compound activity between Drosophila and vertebrates or the effectiveness of feeding as a compound delivery system is not well known. Our comprehensive in vivo analysis of 27 small molecules targeting seven signaling pathways in Drosophila revealed a high degree of conservation of compound activity between Drosophila and vertebrates. We also investigated the mechanism of action of AY9944, one of the Hh pathway antagonists that we identified in our compound feeding experiments. Our epistasis analysis of AY9944 provided novel insights into AY9944’s mechanism of action and revealed a novel role for cholesterol transport in Hh signal transduction.

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References

  1. Basler K, Struhl G (1994) Compartment boundaries and the control of Drosophila limb pattern by hedgehog protein. Nature 368:208–214

    Article  CAS  Google Scholar 

  2. Baxter ADA, Boyd Edward Andrew, Guicherit Oivin M, Price Stephen, Rubin Lee D (2003) Mediators of hedgehog signaling pathways, compositions and uses related thereto. Curtis, Inc., Cambridge, MA

  3. Bier E, Bodmer R (2004) Drosophila, an emerging model for cardiac disease. Gene 342:1–11

    Article  CAS  Google Scholar 

  4. Bijlsma MF, Spek CA, Zivkovic D, van de Water S, Rezaee F, Peppelenbosch MP (2006) Repression of smoothened by patched-dependent (pro-)vitamin D3 secretion. PLoS Biol 4:e232

    Article  Google Scholar 

  5. Bilen J, Bonini NM (2005) Drosophila as a model for human neurodegenerative disease. Annu Rev Genet 39:153–171

    Article  CAS  Google Scholar 

  6. Bohni R, Riesgo-Escovar J, Oldham S, Brogiolo W, Stocker H, Andruss BF, Beckingham K, Hafen E (1999) Autonomous control of cell and organ size by CHICO, a Drosophila homolog of vertebrate IRS1-4. Cell 97:865–875

    Article  CAS  Google Scholar 

  7. Brand AH, Perrimon N (1993) Targeted gene expression as a means of altering cell fates and generating dominant phenotypes. Development 118:401–415

    CAS  Google Scholar 

  8. Callejo A, Torroja C, Quijada L, Guerrero I (2006) Hedgehog lipid modifications are required for Hedgehog stabilization in the extracellular matrix. Development 133:471–483

    Article  CAS  Google Scholar 

  9. Chang S, Bray SM, Li Z, Zarnescu DC, He C, Jin P, Warren ST (2008) Identification of small molecules rescuing fragile X syndrome phenotypes in Drosophila. Nat Chem Biol 4:256–263

    Article  CAS  Google Scholar 

  10. Cooper MK, Porter JA, Young KE, Beachy PA (1998) Teratogen-mediated inhibition of target tissue response to Shh signaling. Science 280:1603–1607

    Article  CAS  Google Scholar 

  11. Cooper MK, Wassif CA, Krakowiak PA, Taipale J, Gong R, Kelley RI, Porter FD, Beachy PA (2003) A defective response to Hedgehog signaling in disorders of cholesterol biosynthesis. Nat Genet 33:508–513

    Article  CAS  Google Scholar 

  12. Denef N, Neubuser D, Perez L, Cohen SM (2000) Hedgehog induces opposite changes in turnover and subcellular localization of patched and smoothened. Cell 102:521–531

    Article  CAS  Google Scholar 

  13. Dudley DT, Pang L, Decker SJ, Bridges AJ, Saltiel AR (1995) A synthetic inhibitor of the mitogen-activated protein kinase cascade. Proc Natl Acad Sci USA 92:7686–7689

    Article  CAS  Google Scholar 

  14. Duffy JB (2002) GAL4 system in Drosophila: a fly geneticist’s Swiss army knife. Genesis 34:1–15

    Article  CAS  Google Scholar 

  15. Eggert US, Kiger AA, Richter C, Perlman ZE, Perrimon N, Mitchison TJ, Field CM (2004) Parallel chemical genetic and genome-wide RNAi screens identify cytokinesis inhibitors and targets. PLoS Biol 2:e379

    Article  Google Scholar 

  16. Fang N, Casida JE (1998) Anticancer action of cube insecticide: correlation for rotenoid constituents between inhibition of NADH:ubiquinone oxidoreductase and induced ornithine decarboxylase activities. Proc Natl Acad Sci USA 95:3380–3384

    Article  CAS  Google Scholar 

  17. Gallet A, Ruel L, Staccini-Lavenant L, Therond PP (2006) Cholesterol modification is necessary for controlled planar long-range activity of Hedgehog in Drosophila epithelia. Development 133:407–418

    Article  CAS  Google Scholar 

  18. Gazit A, Osherov N, Gilon C, Levitzki A (1996) Tyrphostins. 6. Dimeric benzylidenemalononitrile tyrophostins: potent inhibitors of EGF receptor tyrosine kinase in vitro. J Med Chem 39:4905–4911

    Article  CAS  Google Scholar 

  19. Gazit A, Osherov N, Posner I, Yaish P, Poradosu E, Gilon C, Levitzki A (1991) Tyrphostins. 2. Heterocyclic and alpha-substituted benzylidenemalononitrile tyrphostins as potent inhibitors of EGF receptor and ErbB2/neu tyrosine kinases. J Med Chem 34:1896–1907

    Article  CAS  Google Scholar 

  20. Guizzetti M, Costa LG (2008) Sonic hedgehog in Smith–Lemli–Opitz syndrome and tumor development. J Pediatr Hematol Oncol 30:641–642

    Article  Google Scholar 

  21. Incardona JP, Eaton S (2000) Cholesterol in signal transduction. Curr Opin Cell Biol 12:193–203

    Article  CAS  Google Scholar 

  22. Incardona JP, Gaffield W, Kapur RP, Roelink H (1998) The teratogenic Veratrum alkaloid cyclopamine inhibits sonic hedgehog signal transduction. Development 125:3553–3562

    CAS  Google Scholar 

  23. Jacobs HT, Fernandez-Ayala DJ, Manjiry S, Kemppainen E, Toivonen JM, O’Dell KM (2004) Mitochondrial disease in flies. Biochim Biophys Acta 1659:190–196

    Article  CAS  Google Scholar 

  24. Johnson RL, Milenkovic L, Scott MP (2000) In vivo functions of the patched protein: requirement of the C terminus for target gene inactivation but not Hedgehog sequestration. Mol Cell 6:467–478

    Article  CAS  Google Scholar 

  25. Kango-Singh M, Halder G (2004) Drosophila as an emerging model to study metastasis. Genome Biol 5:216

    Article  Google Scholar 

  26. Karim FD, Rubin GM (1998) Ectopic expression of activated Ras1 induces hyperplastic growth and increased cell death in Drosophila imaginal tissues. Development 125:1–9

    CAS  Google Scholar 

  27. Karten B, Peake KB, Vance JE (2009) Mechanisms and consequences of impaired lipid trafficking in Niemann–Pick type C1-deficient mammalian cells. Biochim Biophys Acta 1791:659–670

    CAS  Google Scholar 

  28. Lange Y, Steck TL (1994) Cholesterol homeostasis. Modulation by amphiphiles. J Biol Chem 269:29371–29374

    CAS  Google Scholar 

  29. Lasko P (2002) Diabetic flies? Using Drosophila melanogaster to understand the causes of monogenic and genetically complex diseases. Clin Genet 62:358–367

    Article  CAS  Google Scholar 

  30. Lawrence PA, Morata G (1976) Compartments in the wing of Drosophila: a study of the engrailed gene. Dev Biol 50:321–337

    Article  CAS  Google Scholar 

  31. Li W, Ohlmeyer JT, Lane ME, Kalderon D (1995) Function of protein kinase A in hedgehog signal transduction and Drosophila imaginal disc development. Cell 80:553–562

    Article  CAS  Google Scholar 

  32. Liscum L, Munn NJ (1999) Intracellular cholesterol transport. Biochim Biophys Acta 1438:19–37

    CAS  Google Scholar 

  33. McGuire SE, Mao Z, Davis RL (2004) Spatiotemporal gene expression targeting with the TARGET and gene-switch systems in Drosophila. Sci STKE 2004, pl6

  34. Micchelli CA, Esler WP, Kimberly WT, Jack C, Berezovska O, Kornilova A, Hyman BT, Perrimon N, Wolfe MS (2003) Gamma-secretase/presenilin inhibitors for Alzheimer’s disease phenocopy Notch mutations in Drosophila. FASEB J 17:79–81

    CAS  Google Scholar 

  35. Porter JA, Ekker SC, Park WJ, von Kessler DP, Young KE, Chen CH, Ma Y, Woods AS, Cotter RJ, Koonin EV et al (1996) Hedgehog patterning activity: role of a lipophilic modification mediated by the carboxy-terminal autoprocessing domain. Cell 86:21–34

    Article  CAS  Google Scholar 

  36. Sakamoto N, Terai M, Takenaka T, Maeno H (1978) Inhibition of cyclic AMP phosphodiesterase by 2,6-dimethyl-4-(3-nitrophenyl)-1,4-dihydropyridine-3,5-dicarboxylic acid 3-[2-(N-benzyl-N-methylamino)] ethyl ester 5-methyl ester hydrochloride (YC-93), a potent vasodilator. Biochem Pharmacol 27:1269–1274

    Article  CAS  Google Scholar 

  37. Santos AC, Lehmann R (2004) Isoprenoids control germ cell migration downstream of HMGCoA reductase. Dev Cell 6:283–293

    Article  CAS  Google Scholar 

  38. Seamon KB, Daly JW, Metzger H, de Souza NJ, Reden J (1983) Structure–activity relationships for activation of adenylate cyclase by the diterpene forskolin and its derivatives. J Med Chem 26:436–439

    Article  CAS  Google Scholar 

  39. Segalat L (2007) Invertebrate animal models of diseases as screening tools in drug discovery. ACS Chem Biol 2:231–236

    Article  CAS  Google Scholar 

  40. Selcher JC, Atkins CM, Trzaskos JM, Paylor R, Sweatt JD (1999) A necessity for MAP kinase activation in mammalian spatial learning. Learn Mem 6:478–490

    Article  CAS  Google Scholar 

  41. Strigini M, Cohen SM (1997) A Hedgehog activity gradient contributes to AP axial patterning of the Drosophila wing. Development 124:4697–4705

    CAS  Google Scholar 

  42. Vidal M, Wells S, Ryan A, Cagan R (2005) ZD6474 suppresses oncogenic RET isoforms in a Drosophila model for type 2 multiple endocrine neoplasia syndromes and papillary thyroid carcinoma. Cancer Res 65:3538–3541

    Article  CAS  Google Scholar 

  43. Witte HT, Jeibmann A, Klambt C, Paulus W (2009) Modeling glioma growth and invasion in Drosophila melanogaster. Neoplasia 11:882–888

    CAS  Google Scholar 

  44. Xu T, Rubin GM (1993) Analysis of genetic mosaics in developing and adult Drosophila tissues. Development 117:1223–1237

    CAS  Google Scholar 

  45. Yoshikawa H (1991) Effects of drugs on cholesterol esterification in normal and Niemann–Pick type C fibroblasts: AY-9944, other cationic amphiphilic drugs and DMSO. Brain Dev 13:115–120

    CAS  Google Scholar 

  46. Zecca M, Basler K, Struhl G (1995) Sequential organizing activities of engrailed, hedgehog and decapentaplegic in the Drosophila wing. Development 121:2265–2278

    CAS  Google Scholar 

  47. Zhu AJ, Zheng L, Suyama K, Scott MP (2003) Altered localization of Drosophila Smoothened protein activates Hedgehog signal transduction. Genes Dev 17:1240–1252

    Article  CAS  Google Scholar 

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Author notes

  1. Dan Garza

    Present address: Proteostasis Therapeutics, Cambridge, MA, 02139, USA

  2. Erdem Bangi

    Present address: Developmental and Regenerative Biology, Mount Sinai Medical Center, New York, NY, 10029, USA

Authors and Affiliations

  1. Developmental and Molecular Pathways, Novartis Institutes for Biomedical Research, Cambridge, MA, 02139, USA

    Marc Hild

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  1. Erdem Bangi
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  2. Dan Garza
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  3. Marc Hild
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Correspondence to Erdem Bangi.

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Bangi, E., Garza, D. & Hild, M. In vivo analysis of compound activity and mechanism of action using epistasis in Drosophila . J Chem Biol 4, 55–68 (2011). https://doi.org/10.1007/s12154-010-0051-5

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  • DOI: https://doi.org/10.1007/s12154-010-0051-5

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