Advancing Accurate Quantification of Protein‐Ligand Interactions: Differential Scanning Calorimetry as a Precision Screening Tool Using BCL‐2 as a Model System

Accurate and reliable quantification of protein-ligand energetics at the screening stage is often complicated by ligand aggregation, hydrophobicity-driven artifacts, and the need for cosolvents. Here, differential scanning calorimetry (DSC) as a quantitative, label-free screening method is evaluated using BCL-2 as a model oncogenic target. Nine inhibitors (i.e., venetoclax, navitoclax; and seven previously prioritized BCL-2 hit inhibitors by our research group) are profiled across solvent systems, including neat DMSO, 10% DMSO, and a ternary matrix (S3: 10% DMSO, 90% sulfobutylether-β-cyclodextrin (SBE-β-CD) in saline). DSC yielded thermal transition temperatures and thermodynamic parameters (ΔH, ΔG) that enabled ranking of binding strength. Solubility challenges are addressed by S3, which improved thermal signal quality. Comparisons with time-resolved fluorescence energy transfer (TR-FRET) analysis, in vitro assays, and MM/GBSA binding free energy results confirmed DSC's accuracy in detecting binding energetics. Collectively, these results position DSC as a robust, material-efficient tool for thermodynamic screening of BCL-2 ligands and other poorly soluble compounds, and as a practical complement to isothermal titration calorimetry when solubility or kinetic limitations prevail.