Identification, characterization, and optimization of fragment hits against coagulation factor XIIa supported by affinity capillary electrophoresis and biochemical assay

Coagulation factor XIIa (FXIIa) emerges as a promising target for thromboses induced by blood-contacting devices and several inflammatory diseases, such as hereditary angioedema, Alzheimer’s disease, and multiple sclerosis. FXIIa plays a key role in the initial events that trigger medical device-related thrombosis. Because this protein is dispensable for hemostasis, FXIIa inhibitors have been proposed in this indication as a safe alternative to heparin, which causes major bleeding complications. Growing evidence also indicates the implication of factor XII in neuroinflammatory disorders. The current FXIIa inhibitors are peptides and proteins, including monoclonal antibodies. Today, a potent, selective, small-molecular weight inhibitor is still missing. To design such inhibitors, fragment-based drug discovery (FBDD) has proven to be a reliable approach. This strategy consists of screening small molecules, called fragments, and employing them as building blocks to generate new chemical entities. The advantage over traditional high-throughput screening (HTS) campaigns performed on tens of thousands of molecules is to increase the probability of discovering molecules interacting with the target (hits). This allows the use, in FBDD screening campaigns, of small libraries comprising less than two thousand fragments. However, smaller molecules also mean fewer interactions with the target. Therefore, a FBDD project requires the detection and measurement of weak interactions. Current analytical technologies used in FBDD are confronted with problems of sensitivity, specificity, and/or instrumental costs.
In this context, we investigated the potential of affinity capillary electrophoresis (ACE). Two modes were evaluated. The direct mode proved to be a powerful counter-screening methodology. Indeed, we highlighted its insensitivity to metallic contamination, a common source of false positives that is particularly difficult to detect with current techniques. The indirect mode was able to detect fragment-protein interactions at low occupancy of protein-binding sites (< 30%) and thus proved to be one of the most sensitive techniques that could be used in FBDD. Moreover, we demonstrated that indirect ACE can be designed as a cross-competition assay to determine whether two fragments bind to the same protein pocket using a novel mathematical approach.
Beside the analytical developments, we screened about 1200 fragments on FXIIa by biochemical assay. Thanks to these data, we synthesized analogs of an inhibitor previously identified in our laboratory. We managed to improve a hundredfold the potency of our series while synthesizing less than 15 molecules. The mechanism of inhibition was studied by intact mass protein analysis. In functional ex vivo coagulation assays (aPTT, PT), the best compound showed a potent and selective effect on the contact pathway. Its plasma half-life was determined at 1.9 h. The results of our screening also reveal new scaffolds that could be exploited to design other innovative FXIIa inhibitors.
Altogether, the results presented in this thesis demonstrate that ACE is a powerful analytical technique for FBDD and paves the way for the discovery of small, potent, selective FXIIa inhibitors.