(1169-B) Measuring protein ligand binding with an endogenous HiBiT CETSA test system
Monday, February 5, 2024
2:00 PM – 3:00 PM EST
Location: Exhibit Halls AB
Abstract: One of the critical steps in drug development is the assessment of binary complex formation between compounds and their target proteins of interest (POI). Usually, binary complex formation is investigated by testing purified proteins in vitro using fluorescence polarization, isothermal titration calorimetry (ITC), surface plasmon resonance (SPR) or differential scanning fluorimetry (DSF) methods. However, most proteins need to be truncated for soluble expression and assays like ITC require high protein concentrations or immobilisation of the protein as found in SPR. Fluorescence polarization on the other hand is a tracer-based method requiring a binder excluding the discovery of novel binders. While DSF does not require a tracer it highly interferes with coloured compounds (especially red-yellow) severely restricting the amount of compatible test samples. Accordingly, there is a need for experimental setups that can improve the screening for new ligands.
One possible approach is the Cellular Thermal Shift Assay (CETSA), which allows the measurement of endogenous full-length protein in a tracer-free manner, however normally requires subsequent proteomics or antibody-based detection which drastically decreases the throughput. Herein, the authors describe a HiBiT CETSA which addresses this problem. For this, the POI´s gene locus is modified to express a HiBiT fusion tag, circumventing the needs for specific antibodies or to measure proteomics which allows to run HiBiT CETSA in a target-focused system.
The HiBiT CETSA enables the qualities of DSF, which allows novel binder identification with low amounts of protein, without the downsides in form of protein truncations and restrictions in compound selection. HiBiT CETSA therefore allows screening efforts including proteins which are not available in vitro. Furthermore, due to sensitivity of detection it was possible to miniaturize this approach for use in 1536 well plates.