Background

RNA-binding proteins (RBPs) contribute considerably to post-transcriptional regulation; thus, their dysregulation has been linked to a variety of illnesses, including malignancies [1]. Thereafter, targeting RBPs with specific ligands to perturb cancer development has attracted much attention [2]. Recent studies, including ours, have unraveled the role of RNA-binding motif protein 45 (RBM45) in promoting hepatocellular carcinoma (HCC) progression [3,4], highlighting its importance in cancer development. More crucially, our previous work, which combines a molecular docking approach as well as molecular and cellular techniques, identified enasidenib, a previously considered mutant isocitrate dehydrogenase 2 (mIDH2) inhibitor that binds to RBM45. Therefore, a better clarification of the binding affinity between enasidenib and RBM45 would be beneficial for the design of RBM45-targeted therapeutics.

Recently, several label-free techniques have been developed to determine ligand-target engagement using intact cells or cell lysates. These techniques include drug affinity responsive target stability (DARTS) [5], stability of proteins from rates of oxidation (SPROX) [6], and cellular thermal shift assay (CETSA) [7]. Among these, CETSA is more widely applicable since it can determine the specific ligand-induced target thermodynamic stabilization in biological settings such as whole cell lysates, intact cells, or even tissues [7]. Typically, the shift in thermal stability is assessed by quantifying the density of the remaining soluble target proteins using available antibodies via immunoblots analysis. As a result, thermal melt curves are generated, in which the target protein of interest, with or without ligands, is exposed to a panel of temperatures in the CETSA experiment. Alternatively, an isothermal dose-response curve is also generated, in which the target protein is subjected to increasing concentrations of compounds under a constant temperature (a checkpoint at which the unliganded target protein is degraded as determined by the CETSA experiment), referred to as the isothermal dose-response fingerprint assay (ITDRF_CETSA) [8]. Undoubtedly, CETSA and ITDRF_CETSA techniques offer the opportunity to investigate whether ligands engage their intended targets in complicated environments and determine at what concentration they exert their effects.

Moreover, the CETSA approach has been demonstrated to be effective in determining the thermal stability of enzymes such as dihydrofolate reductase (DHFR) and thymidylate synthase (TS) [7], kinases including cyclin-dependent kinases CDK4 and CDK6 [7], mitogen‐activated protein kinases p38α and ERK1/2 [8]), as well as membrane proteins like solute carriers (SLCs) [9], in the absence and presence of potential ligands. However, the CETSA technique has rarely been applied to assess the thermodynamic stabilization of RBPs when subjected to compounds. Furthermore, a cell-based CETSA decreases the sensitivity to the effects of low-affinity ligands due to drug dissociation from the target after cell lysis, making lysate-based CETSA a preferred choice [9]. Here, we report the modified cell lysate CETSA and ITDRF_CETSA techniques for determining the interaction between enasidenib and RBM45. Our assay is sensitive and time saving and can potentially be adapted to investigate RBM45 protein with other candidate ligands.