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Author Spotlight: Advancing Structural and Biochemical Studies of Proteins Through Thermal Shift Assays
Published on: August 9, 2024
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Homogeneous Assay for Target Engagement Utilizing Bioluminescent Thermal Shift
Melanie L Dart1, Thomas Machleidt1, Emily Jost1
1Promega Corporation, 2800 Woods Hollow Road, Madison, Wisconsin 53711, United States.
ACS Medicinal Chemistry Letters
|June 26, 2018
Summary
We developed a NanoLuc luciferase thermal shift assay (NaLTSA) for drug discovery. This method rapidly assesses target engagement in complex samples without needing purified proteins.
Area of Science:
- Biochemistry
- Drug Discovery
- Assay Development
Background:
- Protein thermal shift assays (TSAs) are crucial for drug discovery but often require purified proteins or antibodies.
- Assessing target engagement in complex biological samples remains challenging.
Purpose of the Study:
- To develop a rapid, sensitive, and broadly applicable assay for target engagement.
- To overcome the limitations of traditional TSAs in drug discovery screening.
Main Methods:
- Appending target proteins with NanoLuc luciferase to couple thermal denaturation with luminescent output.
- Utilizing the NanoLuc luciferase thermal shift assay (NaLTSA) in permeabilized cells.
- Testing NaLTSA on clinically relevant protein families like kinases, bromodomains, and histone deacetylases.
Main Results:
- NanoLuc luciferase has higher intrinsic thermal stability than mammalian proteins.
- The appended NanoLuc does not interfere with the target protein's thermal denaturation.
- NaLTSA successfully assessed target engagement in complex cellular environments.
Conclusions:
- NaLTSA offers a sensitive and rapid method for target engagement assessment in drug discovery.
- This assay is suitable for library screening and compound profiling in complex samples.
- NaLTSA overcomes key operational hurdles of traditional TSAs.
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