A Novel Cellular Thermal Shift Assay Monitors Intracellular Drug Binding

However, optimization of target engagement by drugs within cells is a challenging task, since no methods currently exist for intracellular monitoring of drug binding.

In a potential breakthrough in this field, investigators at the Karolinska Institutet (Stockholm, Sweden) have developed a method for evaluating drug binding to target proteins in cells and tissue samples. Their cellular thermal shift assay (CETSA) was based on the biophysical principle of ligand-induced thermal stabilization of target proteins. In other words, drug binding renders the target protein more resistant to thermal denaturation.

This type of thermal shift assay is a way to monitor the thermal stability of proteins and investigate factors affecting this stability. The technique is used in high-throughput mode to screen optimal buffer conditions, ligands, cofactors, and drugs for their influence on proteins. Two methods to monitor protein denaturation are available: a differential scanning fluorimetry (DSF) method and a differential static light scattering method (DSLS). Changes in the thermal stability of protein-ligand or protein-peptide complexes relative to the stability of the protein alone allow the rapid identification of promising complexes for further structural characterization and to assign functions.

The investigators validated drug binding for a set of important clinical targets and monitored processes of drug transport and activation, off-target effects, and drug resistance in cancer cell lines, as well as drug distribution in tissues and reported these findings in the July 5, 2013, issue of the journal Science.

"We have shown that the method works on a wide variety of target proteins and allows us to directly measure whether the drug molecules reach their targets in cells and animal models," said senior author Dr. Pär Nordlund, professor of medical biochemistry and biophysics at the Karolinska Institutet. "We believe that CETSA will eventually help to improve the efficiency of many drugs and contribute to better drug molecules and more successful treatments."

"We believe that the method can provide an important diagnostic tool in the treatment of cancer, for example, as CETSA can, in principle, enable us to determine which drug is most effective at targeting the proteins in the tumor," said first author Dr. Daniel Martinez Molina, senior lab manager at the Karolinska Institutet. "This also makes it possible for clinicians to ascertain at an early stage of treatment whether the tumor has developed a certain kind of resistance and which type of therapy could then be more suitable for the patient."

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