Model Organisms in Drug Discovery

NEW CHEMICAL GENETIC STRATEGIES 171
RNAi as a genetic screening tool in C. elegans is covered in Chapter 3.
Another approach is to use RNAi technologies in MOA studies as a cell-based
gene knock-out system in Drosophila-cultured cells to analyze systematically
the function of the 14 000 predicted genes in the Drosophila genome. The
simple addition of dsRNA to Drosophila cells in culture ablates the protein
expression of target genes by RNAi mechanisms, thereby efficiently
‘phenocopying’ loss-of-function mutations (Caplen et al., 2000, 2001; Clemens
et al., 2000). For example, the insulin signaling pathway was studied for RNAi
efficiency in Drosophila S2 cells (Clemens et al., 2000). As expected from
knowledge of the insulin pathway, inhibiting the expression of MAPKK by
dsRNA prevents human insulin-stimulated phosphorylation of MAPK. In
another branch of the insulin pathway, dsRNA directed against PTEN (a
negative regulator of insulin signaling), leads to constitutive activation of the
insulin-responsive PI3K pathway. Therefore, RNAi combined with established
biochemical reagents allows deeper characterization of complex
signaling pathways.
In a drug discovery setting, RNAi in cell-based systems can be used to
identify novel targets in compound-validated pathways. For example,
modulation by an antagonist should ‘phenocopy’ cells treated with RNAi to
the compound’s target. Cell-based screening in Drosophila cells will be useful
when compound activity can be correlated with phenotypic detection
methods, such as using markers, functional assays or microscopic imaging
of cells. In cell-based genetic screens RNAi is a rapid method for identifying
MOA pathways but not all disease pathways can be represented in the limited
cell lines available in Drosophila. Reasonable expectations can be made that
S2 cells will be relevant in conserved cell-based functions such as apoptosis,
cell division, cytoskeletal morphology and metabolism, or molecular readouts
such as specific phosphorylation or gene expression changes.
The RNAi technologies for use in mammalian cell-based system are rapidly
evolving but the ease, cost, efficiency and reproducibility using RNAi in S2
cells will allow for routine genome-wide functional analysis (Elbashir et al.,
2001; Tuschl, 2002). For example, RNAi was used to identify a cellular
tyrosine kinase that acts upstream of the phosphorylation of Dscam, a protein
found to be important in axonal pathfinding (Muda et al., 2002). Only one of
six RNAi treatments (Src42A) directed at suspected kinases was able to
decrease tyrosine phosphorylation on Dscam in S2 cells. This suggests that
Src42A acts upstream of Dscam and may be a candidate Src42A substrate.
This approach could be scaled up in S2 cells to test all 200+ Drosophila
kinases for changes in a phosphorylation event.
The RNAi in Drosophila is most effectively induced by dsRNAs of more
than 80 nucleotides in length, which are easy to generate by polymerase chain
reaction (PCR) (Clemens et al., 2000). We routinely generate dsRNA to
complementary (c)DNA clones using generic primers. This makes it