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

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.

ERK signaling couples nutrient status to antiviral defense in the insect gut

“…Using an orthogonal approach, we took advantage of the ERK pathway inhibitor U0126 that blocks MEK activation across diverse species (29,30), including Drosophila (31,32). Using an antibody that recognizes activated Drosophila Erk, we found that vertebrate insulin induces Erk activation within 15 min (SI Appendix, Fig.…”

“…S30). U0126 (500 μM) was used as described (31,32). Flies were fed 10 μL of the following concentrations of virus: VSV (1 × 10 8 pfu/mL), SINV (1 × 10 9 pfu/mL), and DCV (1 × 10 12 IU/mL), unless otherwise indicated.…”

Cancer Drug Development Using Drosophila as an in vivo Tool: From Bedside to Bench and Back

“…Inhibited the differentiation of stem cells into enterocytes [47] Acivicin Inhibited tumor formation in Drosophila by inhibiting CTP synthase [22] Gefitinib and erlotinib Suppressed eye and wing phenotypes induced by EGFR in Drosophila; inhibited dp-ERK1/2 in the eye and wing imaginal discs of wild-type larvae [56] Artemisinin, curcumin Exhibited anticancer activities against brain cancer through generation of reactive oxygen species [60] TAE684, crizotinib Ameliorated the rough-eye phenotype caused by overexpression of hALK F1174L and hALK R1275Q in Drosophila; the effect of crizotinib was less than that of TAE684 [70,71] Imipramine Inhibited fascin pathway in Drosophila; known mediator of tumor invasion and metastasis [76] Triptolide Induced apoptosis in third-instar larval wing discs of Drosophila by inhibiting the ATPase activity of the XPB subunit of TFIIH [84] F14512 Exhibited antiproliferative properties in Drosophila cells; stabilized ternary Topo II-DNA-cleavable complexes at unique sites located in moderately repeated sequences [87] AY9944 Inhibited Hh-induced internalization of the transmembrane protein Ptc as well as the expression of the Hh target gene en; depleted cholesterol from the plasma membrane and its intracellular accumulation in Drosophila tissues [89] Bouvardin Enhanced the effects of radiation in Drosophila larvae through inhibition of the elongation step of protein synthesis [92] Trametinib and fluvastatin Improved tracheal development and reduced over-proliferation and whole-animal toxicity in a Ras-PTEN Drosophila lung cancer model [94]. several aspects of tumor development [23], small molecules that disrupt the function of STAT, such as sunitinib and dasatinib, are now being developed for cancer therapy [24].…”

“…High-throughput screening is an extremely powerful assay that allows quick screening of drugs in vivo against a predetermined target at large scale in a costeffective manner. In brief, cells, embryos, larvae, or adult Drosophila with cancer-causing TFIIH Not defined Larval wing disc [84] Topo II Not defined Adult eye cells [87] Hh Not defined Tissues [89] genes tagged with a luciferase or GFP reporter can be cultured with food containing drugs. After a specific period of time, the effect of the drug on the cancer phenotype can be quantified by various means (Figure 4).…”

Drosophila and the Hallmarks of Cancer

“…Twenty of these compounds can modify specific signalling pathways and the concomitantly induced phenotypes such as the Hh antagonist that linked cholesterol transport to Hh signaling [176]. Latest research deployed Drosophila as a whole organism for investigating the targets and antitargets for cancer polypharmacology [177].…”