*3.1.2.6 Thermal shift or calorimetry-based method*

Isothermal titration calorimetry (ITC) is the only technique which is currently available for the direct determination of enthalpy, **Δ***H*, of a ligand binding to a protein [44]. Thermodynamic evaluation might be useful to provide information about specificity, agonist versus antagonist effects of ligands, and other important properties [45]. Fragment-based drug discovery (FBDD) is an approach of particular interest and relevance here. Fragments are molecules smaller than typical drugs, and they generally bind with lower affinity than conventional drug screening hits [46]. Measuring the contributions of enthalpy and entropy to the free energy of binding provides information that can be useful in fragment elaboration and subsequent medicinal chemistry work [47]. ITC is a uniquely powerful tool for characterization of the thermodynamics of test compounds binding to target proteins. Interaction between the compound and protein leads to release or uptake of small amounts of heat, while the mixture is held at a close approximation to


#### **Figure 4.**

*Comparison between the advantages and disadvantages of target-based and phenotypic screening based upon the different features such as molecular target of disease, its mechanism of action, confirmation methods, SAR optimization methods, and hypothesis limitation.*

constant temperature [48]. Thermal shift screening methods has allowed to identify compounds that interact with *Trypanosoma brucei* choline kinase (TBCK) and inhibit TBCK, a validated drug target against African sleeping sickness [49].
