Advanced Isothermal Titration Calorimetry for Medicinal Chemists with ITCcalc

Abstract

Isothermal titration calorimetry (ITC) is the method of choice for thermodynamic binding profiling. As a label-free in-solution technique, ITC experiments do not require modified or immobilized proteins or ligands and hence can be performed under close-to-native environmental conditions. It directly follows the released heat of the molecular interaction and does not need competing reporter molecules. However, advanced experimental ITC setups are frequently necessary to derive thermodynamic parameters for medicinal chemistry applications. Beyond 1:1 protein−ligand binding, complex systems include displacement titrations to determine thermodynamics for ligands with affinities either too high or too low to be derived from direct titrations, the characterization of protonation changes upon binding, or the elucidation of heat capacities. We present hands-on experiments for graduate medicinal chemists and students familiar with standard ITC methods, reliably highlighting these issues and their respective solutions. The setup can be either a biochemical trypsin−ligand system or a more cost-efficient EDTA-based variant. This demonstration of best practice protocols comes together with the freely accessible ITC web calculator ITCcalc (https://itccalc.uni-mainz.de), guiding through the underlying evaluation of the obtained experimental results.

Assessment of Nucleobase Protomeric and Tautomeric States in Nucleic Acid Structures for Interaction Analysis and Structure-Based Ligand Design

“…To our knowledge, the specific targeting of modified nucleobases or locally altered protomeric/tautomeric states via small molecules to achieve selective ligands is a largely unexplored field. However, examples where ligand or target protomers or tautomers change upon ligand binding are well known for proteins − and might occur for RNA as well. Previous studies demonstrated the need for correct ligand protomer and tautomer annotation not only for structure-based protein-ligand design − but also for RNA-ligand design. ,,, To elucidate the ability of Protonate3D to annotate the correct protomeric and tautomeric states, RNA-ligand complex structures for which specific ligand protomers and tautomers are reported were investigated.…”