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

Abstract

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.