Affinity Capillary Electrophoresis in Fragment-based Drug Discovery Projects, A tool for Hit discovery and Binding site Characterization

Introduction
Fragment-based drug discovery is a usual approach to identify innovative lead compounds. This strategy relies on the screening of small-size molecules (‹300 g/mol). These fragments 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.

Experimental Approach
We set an indirect affinity capillary electrophoresis methodology which allows the detection of mM binders, the determination of the dissociation constants, and the characterization of the fragment binding site. Multiple equilibrium theory was used to model the binding events occurring in the capillary.

Results and Discussion
The applicability of our method for fragment hit discovery was evaluated on six references selected across the µM-mM range of affinity. Based on this study, we estimated that the screening concentration has to be set at 0.3-fold the desired lowest detectable dissociation constant. Screening in mixture was also investigated and did not generate false positives or negatives. On the basis of the mathematical models resulting from the multiple equilibrium theory, we determine the dissociation constants of our reference compounds, and we propose an original methodology to establish graphically if two fragments bind the same protein pocket. The applicability of this methodology was demonstrated experimentally on coagulation factor XIIa by evaluating pairs of fragments.

Conclusion
The indirect affinity capillary electrophoresis methodology reported here provides an efficient approach for the early phases of drug discovery projects. This microfluidic technique detects interaction at low occupancy of the protein-binding site (‹30%) without protein tag or immobilization and can be used for binding site mapping, which constitutes a valuable extension to the fragment-based drug discovery analytical toolbox.