Differential scanning calorimetry: a suitable methodology to problem the binding properties of bcl-2 inhibitors

Abstract

AbstractThe Differential Scanning Calorimeter (DSC) technique is commonly used for quantitative analysis of protein stability, and it is one of the methods used to quantify protein-ligand interactions. DSC provides denaturation curves that can be a source of data on binding constants. B-cell leukemia/lymphoma-2 (BCL-2) is an antiapoptotic protein, one of the overexpressed proteins in cancer cells. Here, binding constants for BCL-2 of nine BCL-2 inhibitors were evaluated by utilization of DSC measurements. The binding free energies of suggested inhibitors of BCL-2 were measured by melting temperature, Tm,and heat capacity, Cp. Evaluated BCL-2 binding parameters of candidate molecules agreed with our previously reported biological assays and molecular simulations indicating that the DSC method could be applied for the determination of free energies of enzyme inhibitors with high accuracy. Therefore, our results showed that this fast and relatively cheap technique can be conducted in high throughput drug screening analyses to compare the binding affinity of a set of compounds against a specific target protein. BCL-2 and most of its inhibitors have hydrophobic cavities, and a definite percent of dimethyl sulfoxide (DMSO) was advised to increase the solubility. Our work also suggests that the presence of the recommended high concentration of DMSO as the solvent significantly changes the affinity of the inhibitors to BCL-2, revealing similar DSC curves for all inhibitors.