We have developed a high throughput technology for functional discovery and validation drug targets associated with specific signaling pathway. A lentiviral siRNA library with siRNA sequences for all known genes and fluorescent transcriptional reporter vector are transduced into target cells and cells with modulation in specific pathway are isolated by FACS. Lentiviral siRNA inserts and corresponding target genes associated with specific pathway are then identified by hybridization with microarrays designed with probe sequences that are complementary to every siRNA. Data will be presented that demonstrates the application of genome-wide functional analysis based on the combined use of siRNA libraries and reporter vectors for the discovery drug targets controlling p53-dependent pathway and apoptosis in several cancer cell models.

One of the major challenges facing pharmaceutical research today is selection of the most important drug targets for any given disease indication. Gene screening on a genomic scale, using oligonucleotides, is quickly developing into a major source of novel molecular targets. The design, construction and application of a genomic set of siRNAs is described. An example of application of the set to link novel genes to a pathway is given.

Although kinases and phosphatases are prominent drug targets, only a small number has been associated with particular diseases and therefore been addressed by drug development. The majority of kinases and phosphatases has not been characterized yet. However, the availability of RNA interference for efficient and reproducible silencing of individual genes in human cells now provides the opportunity to gain functional information on each individual target. We applied kinase and phosphatase siRNA libraries to multi-parametric high-content assays using the automated high-throughput microscope OPERA. First, we characterized the libraries in a time-course for the impact of target mRNA down-regulation on cell viability and apoptosis to define a screening window. Subsequently, the libraries were applied in primary screens for viral infection; in secondary assays the pathways were dissected to characterize novel targets in regulating endocytosis. The complex phenotypic patterns were compared with those of natural phosphatase inhibitors and their derivates.

GPCRs and ion channels represent the most attractive target classes for the drug discovery industry. In order to acquire more knowledge about these important target classes, a large scale expression profiling approach was applied using QPCR. A high quality human tissue collection, containing 50 different tissues mirroring the human body, was established for that purpose. Several software tools, for the analysis and visualization of the obtained data were additionally developed. Currently the expression profiles for all human rhodopsin-like GPCRs and a substantial number of the ion channels were obtained and analyzed. Several expression profiles will be shown with an emphasis on those obtained from: a.) different splice variants of the same gene; b.) human genes and their rat orthologue showing quite substantial species differences; and c.) known targets, which will explain the observed side effects of the corresponding drugs.