The Cellular Thermal Shift Assay (CETSA) is a scientific method used to understand how potential drug candidates interact with proteins inside living cells. This technique helps researchers investigate if a drug physically binds to its intended protein target. CETSA provides insights into these interactions within a natural biological environment, valuable for fundamental biological research and new medicine development. It offers a direct way to confirm drug-target engagement, moving beyond indirect measures.
The Core Principle of CETSA
The core principle of CETSA relies on changes in a protein’s thermal stability. Proteins maintain specific three-dimensional shapes to function correctly. When heated, proteins lose their shape, a process called denaturation, and then aggregate, becoming insoluble.
If a small molecule binds to a protein, it can increase the protein’s resistance to heat-induced unfolding. This means the protein will remain soluble at higher temperatures than it would without the molecule bound. By exposing cells or tissues to increasing temperatures and then measuring the amount of soluble protein remaining, scientists can detect these shifts in thermal stability.
What CETSA Can Discover
CETSA offers direct evidence of whether a drug physically connects with its target protein within a living cell. This helps confirm if a potential drug candidate reaches and binds its intended target. By assessing this interaction, researchers gain confidence that a compound is acting as hypothesized.
The assay also helps evaluate the strength of this binding, often called target engagement. This allows scientists to understand how effectively a drug interacts with its protein, which is important for optimizing drug properties. Furthermore, CETSA contributes to unraveling a drug’s mechanism of action, providing insights into how it exerts its effects at a molecular level within a biologically relevant setting.
Why CETSA is a Powerful Tool
CETSA measures drug-target interactions directly within intact cells or living organisms, offering data more reflective of physiological conditions compared to traditional in vitro assays. This provides a direct assessment of target engagement without relying on indirect readouts like enzymatic activity.
The method is also label-free, meaning it does not require chemical modifications or reporter tags. This helps preserve the natural behavior of proteins and minimizes assay artifacts. Its versatility allows it to be applied to a wide range of protein classes, including kinases and membrane proteins. CETSA’s ability to validate drug targets in a native cellular context accelerates the drug discovery process by providing reliable and translatable insights into how compounds interact with their biological targets.