Isothermal Titration Calorimetry (ITC)

Isothermal Titration Calorimetry (ITC) directly measures the thermodynamics of molecular interactions by monitoring heat released or absorbed during binding. The technique provides a comprehensive interaction profile, including binding affinity (K_d), stoichiometry (n), enthalpy (ΔH), and entropy (ΔS).

As a label-free, solution-based method, ITC is suitable for protein–ligand, protein–protein, and nucleic-acid interactions. It supports detailed characterization of binding mechanisms in drug discovery, lead optimization, and biomolecular research.

Direct measurement of binding thermodynamics

In an ITC assay, a solution containing the ligand is gradually titrated into a solution containing the target molecule under isothermal conditions. The heat released or absorbed during each binding event is measured with high sensitivity, enabling calculation of binding affinity (K_d), stoichiometry (n), enthalpy (ΔH), and entropy (ΔS).

Key Features

ITC characterization of AK-1690 binding to STAT6

AK-1690, a STAT6 SH2-domain-targeted PROTAC reported by Wang et al. (2024), was titrated into the non-phosphorylated STAT6 core domain using PEAQ-ITC™. The ITC data were analyzed with the “One Set of Sites” model.

The binding affinity measured by ITC was comparable to the value obtained by SPR (K_D = 33.7 nM). The interaction was driven by both enthalpy (ΔH) and entropy (TΔS) changes. The ITC n value of 1.18 confirmed an approximately 1:1 molar ratio of STAT6 to AK-1690 binding.

ITC binding isotherm of AK-1690 to STAT6.

Binding energetics of AK-1690 and STAT6.

ITC-derived binding parameters for the AK-1690–STAT6 interaction are summarized below.