Investigating Protein Binding with the Isothermal Ligand‐induced Resolubilization Assay

Target engagement assays typically detect and quantify the direct physical interaction of a protein of interest and its ligand through stability changes upon ligand binding. Commonly used target engagement methods detect ligand‐induced stability by subjecting samples to thermal or proteolytic stress. Here we describe a new variation to these approaches called Isothermal Ligand‐induced Resolubilization Assay (ILIRA), which utilizes lyotropic solubility stress to measure ligand binding through changes in target protein solubility. We identified distinct buffer systems and salt concentrations that compromised protein solubility for four diverse proteins: dihydrofolate reductase (DHFR), nucleoside diphosphate‐linked moiety X motif 5 (NUDT5), poly [ADP‐ribose] polymerase 1 (PARP1), and protein arginine N‐methyltransferase 1 (PRMT1). Ligand‐induced solubility rescue was demonstrated for these proteins, suggesting that ILIRA can be used as an additional target engagement technique. Differences in ligand‐induced protein solubility were assessed by Coomassie blue staining for SDS‐PAGE and dot blot, as well as by NanoOrange, Thioflavin T, and Proteostat fluorescence, thus offering flexibility for readout and assay throughput. Isothermal Ligand‐Induced Resolubilization Assay (ILIRA) is a new screening method for identifying protein ligands that relies on differences in solubility between unliganded and liganded proteins under lyotropic stress conditions. In this proof‐of‐concept study, we demonstrate the application of ILIRA to different protein targets and present alternative assay modalities.