Model Organisms in Drug Discovery

172 MECHANISM OF ACTION IN MODEL ORGANISMS
functionally feasible to test the RNAi of all Drosophila genes by designing cellbased
readouts in 96-well or 384-well formats. Conversely, mammalian cell
experiments use 21–23 length oligonucleotides that are expensive to purchase
and variably mediate an RNAi-like inhibition of gene expression in ‘knockdown’
efficiency. Presently, the expense of RNA oligonucleotides makes
mammalian cell-based ‘genetic’ screens prohibitive, although oligonucleotide
vectors for RNAi are being developed. Also, the smaller, less redundant
genome of Drosophila may be more revealing. Notwithstanding, mammalian
RNAi will be an important resource for rapid validation of Drosophila targets
in vertebrate biology.
6.7 A case study for innate immunity and inflammation drug
discovery
Drosophila S2 cells have macrophage-like properties and therefore should be
informative in understanding cell-mediated innate immunity. Most components
of innate immunity are conserved evolutionarily from Drosophila to
humans, and only higher eukaryotes have acquired immunity (Silverman and
Maniatis, 2001). Insects have a potent and rapid response to a broad spectrum
of pathogens and the response discriminates between types of pathogens.
Fungal and bacterial infections of Drosophila lead to transcriptional
activation of sets of antimicrobial peptide (AMP) genes and eventually S2
cells will phagocytose the microbes. These responses are mediated by nuclear
factor kappa B (NF-kB) family members, which are conserved transcription
factors that also activate the expression of inflammation genes in mammals.
Induction of each AMP gene is regulated by a balance of inputs that are
manifested by combinations of the three Rel/NF-kB proteins Relish, Dorsal
and Dif (Figure 6.6). Activation of Rel/NF-kB pathways is essential for the
Drosophila innate immune response. For example, Drosophila carrying
mutations in the Relish gene do not express certain classes of antimicrobial
peptides, such as Cecropins and Diptercin, and are susceptible to Gramnegative
(Escherichia coli) bacterial infection. Similarly, Dorsal or Dif is
essential for activation of AMPs such as Drosomycin, involved in fungal and
Gram-positive infections (Hedengren et al., 1999). Drosophila Rel proteins,
like mammalian Rels, are sequestered in the cytoplasm as a result of
association with an IkB-like inhibitor protein such as Cactus. When cells are
activated by pathogens, signaling pathways are activated leading to the release
of IkB, nuclear translocation of Rel proteins and Rel-activated transcription.
Cactus is the IkB protein that inhibits Dorsal and Dif. Like NF-kB, Relish is
the mammalian homolog of p105, and contains both a Rel domain and an IkB
inhibitory domain (Silverman and Maniatis, 2001).