Abstract

AstraZeneca is increasingly pursuing “intractable targets”, such as proteins which are difficult to synthesize, unstable, disordered or don’t have enzymatic activity.  Challenging targets can sometimes be interrogated with reductionist affinity screening methods in solution. However often full-length protein synthesis is not possible and the target may behave differently when tested in isolation from its physiological partners.  The Cellular-Thermal-Shift-Assay (CETSA) can demonstrate target engagement in physiologically-relevant conditions using cells to make their own protein in the right state. 

Previous CETSA workflows have been low-throughput (MS) and high-cost and have required specific antibodies (AlphaLisa). We have worked collaboratively across departments to develop a cost-efficient and scalable HiBit-tagged cell-line approach to quantify target engagement and stabilisation using a luminescence readout. We have developed the first fully automated HiBiT CETSA protocol which allows throughput of up to 35,000 compounds a day in single point screening. We are applying this to high-value novel targets, enabling us to screen up to half a million compounds in under two weeks.By validating this process we have generated a generic protocol that can be applied to a wide range of intractable targets, providing novel chemistry and supporting rapid progression through the portfolio.