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The Cellular Thermal Shift Assay (CETSA) has emerged as a powerful tool for evaluating drug-target interactions in live cells, yet its application in ultra-high throughput screening (uHTS) has been limited by technical constraints. In this study, we present significant advancements in CETSA methodology, focusing on the development of an innovative isothermal CETSA platform for primary uHTS screen in 1536 well plates and a Gradient Peltier Device (GPD) for retesting hits in full melting curve CETSA. Our optimized isothermal CETSA allows for the evaluation of adherent cells in their physiological state, enhancing assay performance through a controlled thermal ramp-up instead of traditional heat shock methods and utilizing highly sensitive luminescence detection. The GPD enables all steps of a full melt curve CETSA to be conducted in one single flat bottom microtiter plate, improving data quality by reducing handling and pipetting steps and improving temperature control. We benchmarked both methods against an established fluorescence polarization assay using the androgen receptor as a model target. Results demonstrated a strong correlation between both CETSA methods and the fluorescence polarization assay, indicating the potential for identifying true binders while minimizing false positives. Our findings highlight the utility of this optimized CETSA platform for high throughput drug discovery, paving the way for more effective screening of true binders in live cells.
Hamacher et al. (Sat,) studied this question.