Drug Target Strategies
Advances in Cellular Target Engagement
High throughput screening (HTS) cascades have evolved to ensure that high quality hits can be identified from large screening collections. Traditionally, most primary screens focus on the identification of modulators of catalytically active sites, while target engagement assays are placed further down the cascade. Well established technologies like competition-based assays, affinity selection technologies or differential scanning fluorimetry (DSF) depend on availability of protein which is tested in a non-native biochemical setting. Therefore, one of the main concerns when initiating an HTS cascade remains the demonstration of target interaction within a relevant cellular environment. The use of cellular assays during primary screening and the HTS cascade presents an alternative. However, cell-based screens can easily become very complex, risk off-target effects and thus often require time consuming target deconvolution of pathway hitters. To date there has been no single technology that can demonstrate cellular target engagement in a suitable format for HTS primary screening.
The cellular thermal shift assay (CETSA®) can act as an interface between this classic biochemical-cellular screening dichotomy. CETSA® facilitates label free screening in disease relevant cells while approaching the ease of biochemical assays. In an isothermal setup, full assay plates are heated to a setpoint within the target protein melting curve. While most proteins unfold and precipitate upon this heat-shock, a characteristic of protein-ligand interaction is induced thermal stability. The remaining stabilized protein can subsequently be detected with a pair of anti-species antibodies in an AlphaScreen® system. This high throughput (HT) CETSA® format allows large numbers of compounds to be tested in an HTS setting. Here, we report the development of two CETSA®-HT assays along with the application of this technology in HTS for the first time. This has been enabled following the recent agreement between Pelago Bioscience and PerkinElmer to streamline CETSA®-HT into validated kits and to offer support in the assay development. In the Global High Throughput Screening Centre of AstraZeneca we are exploring the potential and the feasibility of CETSA®-HT for large scale HTS campaigns ( >0.5M compounds). These datasets provide an indication of the future impact CETSA®-HT will have in hit identification. This is particularly timely given the expanding interest across drug discovery groups in new target protein classes. With new modalities like PROTACS (proteolysis targeting chimera) non-catalytically active proteins can now therapeutically be targeted. Utilising CETSA®-HT to identify target engagement in cellular environments early during primary screening could shift the paradigm of hit finding.