Associate Director HTS Automation Team AstraZeneca Pharmaceuticals, England, United Kingdom
High throughput screening (HTS) is frequently used in drug discovery campaigns to identify high quality hits from large screening collections. Historically, primary screens have focussed on the identification of modulators of catalytically active sites using simple biochemical assays, while target engagement assays have been placed further down the screening cascade, often due to throughput considerations.
Thermal shift assays (TSA) provide measurement of compound-target engagement, measuring protein unfolding by monitoring change in fluorescence as a function of temperature. Differential scanning fluorimetry (DSF) measures the thermal stability of the target protein, binding of a drug causes stabilisation and an increase in protein melting temperature. This simple biophysical technique can be employed for HTS purposes as a primary screening technology. Within the Global High Throughput Screening Centre at AstraZeneca we have successfully deployed an automated DSF screening capability, here we describe the various challenges that we encountered during scale-up and automation to enable screening of up to 400K compound libraries.
The cellular thermal shift assay (CETSA®) has emerged as a technology that allows assessment of interactions between a drug and protein target in a cellular environment, by measuring changes in the thermal stability of the target protein upon ligand binding, and this technology is amenable to testing large numbers of compounds in an HTS setting. We have explored the potential and feasibility of CETSA®-HT for large scale HTS campaigns (up to 0.5M compounds). In 2020 we reported on the development of an HT-CETSA® assay and the application of this technology within the HTS setting. This screening campaign highlighted several practical issues & challenges when automating such assays. Here we report on various improvements & modifications that we have put in place to enable delivery of robust screening assays to deliver high quality hits. These include evaluating different CETSA approaches, and their amenability to automated screening. Combining CETSA with the homogeneous bead-based AlphaLISA® technology requires the development & optimization of target specific antibody pairs, which can be time consuming to develop, and multiple addition steps can make it more complex to automate. Approaches that combine protein-based reporters such as Nano-Glo® HiBiT with CETSA, using luminescence readouts, are often simpler assays with potentially fewer steps making them more amenable to automation. We have also addressed labware issues to improve automation reliability, and investigated alternative thermal cyclers for ease of automation.
Utilising both DSF and HT-CETSA® to identify target engagement in early during primary screening could shift the paradigm of hit finding and the ability to automate these different approaches enlarges the tool box of hit-finding approaches that can be used at scale.