Determining the In Vitro Ligand-Target Interaction by Cellular Thermal Shift Assay and Isothermal Dose-Response Fingerprint Assay.

The cellular thermal shift assay (CETSA) and isothermal dose-response fingerprint assay (ITDRF _CETSA) have been introduced as powerful tools for investigating target engagement by measuring ligand-triggered thermodynamic stabilization of cellular target proteins. Yet, these techniques have rarely been used to evaluate the thermal stability of RNA-binding proteins (RBPs) when exposed to ligands. Here, we present an adjusted approach using CETSA and ITDRF_CETSA to determine the interaction between enasidenib and RBM45. Our assay is sensitive and time-efficient and can potentially be adapted for studying the interactions of RBM45 protein with other potential candidates. Key features • This protocol builds upon the method developed by Molina et al. and extends its application to new protein classes, such as RBPs.