*2.1.2.1 Binding assays*

Binding assays aim to detect the interaction(s) between two (bio)molecules, such as protein-protein, peptide-protein, nucleic acid-protein, small moleculeprotein, or small molecule-nucleic acid and ideally also evaluate the degree of the interaction [56]. These assays can be used in two ways, in screening approaches to qualitatively identify hits that interact with the target and in a quantitative way to characterize the binding affinity.

There are many examples of different types of qualitative assays that have been used in drug repurposing approaches. Among the most common are immobilization or affinity chromatography, where either the target or the drug are immobilized on a matrix or column followed by exposure to a drug library or potential binding targets [57]. The complexes that have formed can then be eluted and identified using analytic methods. DNA-encoded libraries encompassing wide chemical space have been used in such approaches. After eluting complexes binding compounds are identified by sequencing the DNA-barcode attached to the binding compound. This technique can also be applied to approved drug libraries [58].

The aforementioned assays are aimed at screening large libraries for hits. However, obtaining detailed binding information such as dissociation constants (KD), is crucial in the identification and development of potent drugs. Several biophysical techniques are available to quantify these interactions. Microscale Thermophoresis (MST) can be used to measure binding affinity by detecting changes in molecular motion in a temperature gradient in the presence and absence of different compound concentrations [59]. Differential scanning fluorimetry (DSF) can be used to measure protein unfolding temperature by monitoring in fluorescence of a probe that binds to hydrophobic moieties in a denaturing temperature gradient. Upon binding of drugs to the protein it can stabilize the complex, leading to a shift in unfolding temperature. By using a range of drug concentrations and measuring the effect on the thermal shift the KD can be calculated [60]. Surface plasmon resonance (SPR) is a technique in which the target or drug of interest is immobilized on a thin metal film. A light source is aimed at the other side of the film and the surface Plasmon resonance angle is detected. When a drug or target binds to the immobilized partner the local mass at the sensor surface changes, causing a shift in the angle of reflection proportional to the mass. By measuring these changes in the presence and absence of drug or target, association and dissociation constants can be determined [61]. Isothermal titration calorimetry (ITC), one of the golden standards in KD determination, can directly measure all binding parameters by measuring heat transfer. When binding of drug to target occurs, enthalpy changes (heat absorbed or released) of the system can be measured by a highly sensitive calorimeter. By titrating ligand against the target of interest the Kd, stoichiometry, enthalpy and entropy can be directly measured in the native states of the binding partners since no modifications are required [62].
