Affinity Capillary Electrophoresis for Fragment-based Drug Discovery Projects: Hit Discovery and Binding Site Characterization

peer reviewedFragment-based drug discovery is a usual approach to identify innovative lead compounds. This strategy relies on the screening of small-size compounds (< 300 g/mol). These fragments have a weak affinity for the target but explore more efficiently the protein chemical space. For successful applications, this strategy requires the detection of weak binders (µM–mM range) and characterization of their binding site. NMR and X-ray crystallography can address this challenge and, in consequence, are commonly used in fragment-based drug discovery. However, their accessibility is limited due to their large protein consumption and the cost of equipment. Here, we present an affinity capillary electrophoresis methodology that detects mM binders, determines dissociation constants, and characterizes the fragment binding site. We use the multiple equilibrium theory to model the binding events occurring in the capillary. On the basis of the resulting mathematical models, we determine dissociation constants in the μM–mM range, and we propose an original methodology to establish graphically if two fragments bind the same protein pocket [1]. [1] C. Davoine, A. Pardo, L. Pochet, M. Fillet, Analytical Chemistry, 2021, 10.1021/acs.analchem.1c03611Development of new compounds targeting coagulation factor XIIa using innovative microfluidic assays in the context of fragment-based drug discover