Coumarins as factor XIIa inhibitors: Potency and selectivity improvements using a fragment-based strategy.

Previously, we described weak coumarin inhibitors of factor XIIa, a promising target for artificial surface-induced thrombosis and various inflammatory diseases. In this work, we used fragment-based drug discovery approach to improve our coumarin series. First, we screened about 200 fragments for the S1 pocket. The S1 pocket of trypsin-like serine proteases, such as factor XIIa, is highly conserved and is known to drive a major part of the association energy. From the screening, we selected fragments displaying a micromolar activity and studied their selectivity on other serine proteases. Then, these fragments were merged to our coumarin templates, leading to the generation of nanomolar inhibitors. The mechanism of inhibition was further studied by mass spectrometry demonstrating the covalent binding through the formation of an acyl enzyme complex. The most potent compound was tested in plasma to evaluate its stability and efficacy on coagulation assays. It exhibited a plasmatic half-life of 1.9 h and a good selectivity for the intrinsic coagulation pathway over the extrinsic one.

Fragment Hit Discovery and Binding Site Characterization by Indirect Affinity Capillary Electrophoresis: Application to Factor XIIa.

Fragment-based lead discovery is a usual strategy in drug discovery to identify innovative lead compounds. The success of this approach strongly relies on the capacity to detect weak binders and characterize their binding site. NMR and X-ray crystallography are the conventional technologies used to tackle this challenge. However, their large protein consumption and the cost of equipment reduce their accessibility. Here, an affinity capillary electrophoresis methodology was developed that enables the detection of mM binders, the determination of dissociation constants, and the characterization of the fragment binding site. On the basis of multiple equilibrium theory, dissociation constants in the µM-mM range were determined, and a new methodology is proposed 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 with expected behavior. This study reinforces the significance of using affinity capillary electrophoresis to gather valuable information for medicinal chemistry projects.

Factor XII/XIIa inhibitors: Their discovery, development, and potential indications.

Coagulation factor XII (FXII), a S1A serine protease, was discovered more than fifty years ago. However, its in vivo functions and its three-dimensional structure started to be disclosed in the last decade. FXII was found at the crosstalk of several physiological pathways including the intrinsic coagulation pathway, the kallikrein-kinin system, and the immune response. The FXII inhibition emerges as a therapeutic strategy for the safe prevention of artificial surface-induced thrombosis and in patients suffering from hereditary angioedema. The anti-FXII antibody garadacimab discovered by phage-display library technology is actually under phase II clinical evaluation for the prophylactic treatment of hereditary angioedema. The implication of FXII in neuro-inflammatory and neurodegenerative disorders is also an emerging research field. The FXII or FXIIa inhibitors currently under development include peptides, proteins, antibodies, RNA-based technologies, and, to a lesser extent, small-molecular weight inhibitors. Most of them are proteins, mainly isolated from hematophagous arthropods and plants. The discovery and development of these FXII inhibitors and their potential indications are discussed in the review.