Denaturation-sensitive BRET probes enable intracellular thermal shift measurements across diverse protein targets and biomolecular complexes

Vasta, J. D.; Michaud, A.; Beck, M. T.; Hwang, I.; Schwarzenstein, A.; Corona, C. R.; Sequeira, P.; Happe, C.; Schwalm, M. P.; Gehrke, B.; Nealey, K.; Russell, S.; Mikulsky, B.; Dunn-Hoffman, K. K.; Damgaard, C.; Zhou, M.; Berry, S.; Usselmann, L.; Forbes, C.; Vazquez-Chantada, M.; Lentz, T. B.; Johnson, R. L.; Slater, M. R.; Ames, C.; Szewczyk, M. M.; Barsyte-Lovejoy, D.; Zhou, W.; Cong, M.; Meisenheimer, P. L.; Shaw, J.; Robers, M. B.

doi:10.64898/2026.11.10.765695

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bioRxiv · 10.64898/2026.11.10.765695

Denaturation-sensitive BRET probes enable intracellular thermal shift measurements across diverse protein targets and biomolecular complexes

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Abstract

Thermal shift assays have revolutionized drug discovery, but can be limited in sensitivity and scalability. Combining the unique attributes of differential scanning fluorimetry (DSF) and cellular thermal shift (CETSA), we report the methodological framework for the bioluminescence resonance energy transfer shift assay (BRETSA). BRETSA utilizes denaturation sensitive BRET probes, in combination with luciferase-tagged target proteins, to track and longitudinally record thermal shift in living cells. Denaturation profiles were successfully measured for over 500 globular proteins, spanning 24 target families in diverse cellular compartments. Among these, target engagement was confirmed for over 200 proteins; including validated drug targets as well as understudied members of the kinase subfamily. Leveraging orthosteric, allosteric, and covalent ligands in potency studies, this method offers improved sensitivity and scalability compared to other thermal shift methods. We extend the utility of BRETSA to interrogate target engagement at biomolecular complexes and query cooperative binding for molecular glues and emerging tricomplex drug modalities.

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Vasta, J. D., Michaud, A., Beck, M. T., Hwang, I., Schwarzenstein, A., Corona, C. R., Sequeira, P., Happe, C., Schwalm, M. P., Gehrke, B., Nealey, K., Russell, S., Mikulsky, B., Dunn-Hoffman, K. K., Damgaard, C., Zhou, M., Berry, S., Usselmann, L., Forbes, C., Vazquez-Chantada, M., Lentz, T. B., Johnson, R. L., Slater, M. R., Ames, C., Szewczyk, M. M., Barsyte-Lovejoy, D., Zhou, W., Cong, M., Meisenheimer, P. L., Shaw, J., Robers, M. B.. 2026-11-11. Denaturation-sensitive BRET probes enable intracellular thermal shift measurements across diverse protein targets and biomolecular complexes. https://doi.org/10.64898/2026.11.10.765695

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