New Microcalorimetric Methods for Measuring Ultratight Protein-Ligand Interactions

Isothermal titration calorimetry (ITC) is the gold standard for the quantitative characterization of biomolecular interactions. However, reliable determination of the dissociation constant (KD) is typically limited to the range 100 μM > KD > 1 nM. Nevertheless, interactions characterized by a higher KD can be assessed indirectly, provided that a suitable competitive ligand is available whose KD falls within the directly accessible window. Unfortunately, the established competitive ITC displacement method has two major limitations. First, it requires the high-affinity ligand be soluble at high concentrations in aqueous solution (typically >100 μM). This poses serious problems in quantifying high-affinity interactions involving poorly water-soluble small-molecule ligands taken from compound libraries, as is usually the case in many drug-discovery projects. A second limitation of the displacement method is that the protocol necessitates at least two titrations to characterize one high-affinity inhibitor. This limits the great potential of microcalorimetry both in drug discovery efforts, where high sample throughput is required, as well as in basic research, where precious biological samples are often available only in limited amounts.Here, we present two novel competition assays for measuring ultratight protein-ligand interactions by ITC that overcome these limitations. The first assay allows for a precise thermodynamic description of high-affinity protein-ligand interactions involving poorly water-soluble compounds (Krainer et al. Anal. Chem.2012, 84, 10715), while the second assay provides a simultaneous quantification of both the competitive, moderate-affinity and the high-affinity ligand in a single experiment with minimal sample requirement. We discuss the theoretical background of the approach and demonstrate some practical applications using examples of high-affinity protein-ligand interactions.