CETSA.. since 2013
Proteins are targets for the majority of drugs and are involved in most cellular process. However, to study proteins and their interactions in live cells and tissues has been very challenging. Recently our group introduced a new method to meet this challenge: the Cellular Thermal Shift Assay (CETSA) (Science 341(6141):84-87). CETSA is a biophysical assay based on the principle of ligand-induced thermal stabilization of target proteins, meaning that a proteins melting temperature will change upon ligand interaction. Thus, by heating samples (lysate, cells or tissue pieces) to different temperatures, and quantifying proteins in the soluble fraction we can detect altered protein interactions after for example drug treatment. This can either be done for selected proteins of interest by using anti-body based assays or proteome wide by using mass spec. CETSA allows direct monitoring of ligand binding to a specific target (target engagement) in lysate, live cells or even tissue pieces. It can also be used to study downstream effects on protein interaction, providing a novel perspective on protein function in situ.
Applications
CETSA in drug discovery
The idea of CETSA was originally conceived for measurement of target engagement in cells and tissues at various stages of drug development. In addition to drug binding, CETSA also reveals biochemical events downstream of drug binding and hence provides a means of establishing mechanistic biomarkers. Since its conception, CETSA has been applied in many animal and patient studies and will be valuable in many stages of pre-clinical and clinical drug development.
cetsa in infectious disease
Apart from target deconvolution in drugs for infectious diseases such as malaria and tuberculosis, CETSA is also able to study other aspects of the disease like the processes in the pathology.
cetsa in cancer cell biology
With the current high resolution MS-CETSA, it is possible to monitor global changes of protein interaction states during specific steps of cancer progression, and in turn identify potentially important molecular processes that can be pharmacological targets. CETSA can also be used gain insights into the drug-target interactions and reveal mechanisms of intrinsic and acquired drug resistance that cannot otherwise be studied in situ.
cetsa to study host-pathogen interactions
In malaria, CETSA has been used to study changes both in the host cell as well as in the parasite after infection. CETSA is also currently being explored to study the infection process in viruses such as HCV.
cetsa to study cellular processes
With the advent of the current format of MS-CETSA, comprehensive studies of protein level changes in addition to stability changes are plausible. The interactions made by proteins with other proteins and biomolecules in the cell determine their protein interaction states (PRINTs), regulate protein activity and serve in recognition of catalytic substrates. Deciphering these PRINTS will provide insights into processes that are elusive with currently available methods.
cetsa in immunology
CETSA can study the temporal effects of cytokines in different cells in the immune system as well as the responses they trigger in each cell type. CETSA can be used to tease out the interactions between the immune cells and the tumor cells.
“Chaos was the law of nature. Order was the dream of man.
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