DiscussionHuman whole genome libraries and sub-libraries are commercially available for high throughput loss of function screens, with RNAi now a recognised and widely-used technique in functional genomics, pathway analysis and target validation. Whilst RNAi platforms have been used across AstraZeneca in low throughput format, RNAi screening had not previously been utilised for target deconvolution within the high throughput screening (HTS) centre. Here we report the use of a kinase targeted siRNA sub-library to help deconvolve the kinases involved in driving Nrf2 pathway activity in lung cancer cell lines.
A 384-well siRNA assay was developed and optimised to align with a recent Nrf2 pathway phenotypic screen. The primary screening campaign had identified active compounds which selectively inhibited the transcriptional activity of Nrf2 in lung cancer cells; however the specific target(s) remained unknown. As many of the active chemical clusters contained kinase cores, the Dharmacon siRNA kinase sub-library was purchased to help deconvolve the kinases involved and an automated process to generate assay-ready siRNA plates was developed.
Application of the above processes to Nrf2 pathway assays identified numerous kinase ‘hits’ from the siRNA screen for follow-up studies. One kinase in particular correlated strongly with existing compound data and was subsequently validated for effects downstream of Nrf2. Further work is ongoing to understand the role of this kinase in the Nrf2 signalling pathway, whilst other kinases identified in the cascade are being explored as potential new targets.
Overall this project has demonstrated the feasibility of RNAi screening in HTS, and has shown that the early identification of genes involved in pathway activity can help direct the screening cascade; thus increasing project efficiency and reducing time spent on downstream target de-convolution.
Jessica Taylor, Undergraduate Industrial Placement Student.
Supervisor: Dr Simon Woodcock
Global High Throughput Screening Centre. AstraZeneca