Thermal Shift as an Entropy-Driven Effect

Redhead M, et al., 56(47):6187-6199, Biochemistry, 2017

The goal of this study was to provide more clarity and quantitative interpretation to the data obtained from Thermal Shift Assays (TSAs). The models and methodologies were designed to determine the ligand affinity, enthalpy, and molecular mechanism of ligand pairs. Duplex DNA as well as two protein models, carbonic anhydrase II (CAII) and glutathione S-transferase (GST) were chosen as model systems. Surface Plasmon Resonance (SPR) and Isothermal Calorimetry (ITC) were chosen as orthogonal biophysical techniques to verify TSA data. The binding of furosemide to CAII was investigated by SPR using a Pall ForteBio Pioneer FE instrument. CAII was immobilized onto the sensor chip surface using standard amine coupling chemistry. Ethanolamine was injected to cap any residual active sites. Subsequently, furosemide was injected using a OneStep injection protocol in the presence or absence of guanidine. SPR data obtained were analyzed using the QDat software. The kinetic rate constants (ka and kd) and and the KD values were determined. Overall results obtained from assay systems ranging from protein?small molecule interactions to duplex DNA stability, demonstrate the utility of TSAs in all stages of drug discovery.

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