Charlotte Bouckaert

Determination of inhibitory potency of argatroban toward thrombin by electrophoretically mediated microanalysis

Developing an EMMA method for enzymatic assay remains a challenge, particularly using UV detection. Indeed, it is necessary to optimize the separation conditions while allowing the enzymatic reaction to occur within the capillary respecting kinetic constraints and achieving enough sensitivity. In this work, such EMMA methodology was set up to evaluate the inhibitory potency of drugs toward thrombin, a serine protease implicated in the coagulation cascade. To achieve our goal, the separation buffer, the injection sequence, the internal standard and the chromogenic substrate were investigated. The newly developed system was then assessed determining the kinetic Km constant for the selected substrate and compared with the results obtained with a continuous spectrophotometer cell assay. Secondly, the Ki inhibitory constant of the thrombin inhibitor argatroban was determined and found in agreement with the published value.

Synthesis, evaluation and structure-activity relationship of new 3-carboxamide coumarins as FXIIa inhibitors

Inhibitors of the coagulation factor XIIa (FXIIa) are attractive to detail the roles of this protease in hemostasis and thrombosis, to suppress artifact due to contact pathway activation in blood coagulation assays, and they are promising as antithrombotic therapy. The 3-carboxamide coumarins have been previously described as small-molecular-weight FXIIa inhibitors. In this study, we report a structure-activity relationship (SAR) study around this scaffold with the aim to discover new selective FXIIa inhibitors with an improved physico-chemical profile. To better understand these SAR, docking experiments were undertaken. For this purpose, we built an original hybrid model of FXIIa. This model has the advantage to gather the best features from the recently published crystal structure of FXIIa in its zymogen form and a more classical homology model. Results with the hybrid model are encouraging as they help understanding the activity and selectivity of our best compounds.

Partial filling affinity capillary electrophoresis as a useful tool for fragment-based drug discovery: A proof of concept on thrombin.

With the emergence of more challenging targets, a relatively new approach, fragment-based drug discovery (FBDD), proved its efficacy and gained increasing importance in the pharmaceutical industry. FBDD identifies low molecular-weight (MW) ligands (fragments) that bind to biologically important macromolecules, then a structure-guided fragment growing or merging approach is performed, contributing to the quality of the lead. However, to select the appropriate fragment to be evolved, sensitive analytical screening methods must be used to measure the affinity in the μM or even mM range. In this particular context, we developed a robust and selective partial filling affinity CE (ACE) method for the direct binding screening of a small fragment library in order to identify new thrombin inhibitors. To demonstrate the accuracy of our assay, the complex dissociation constants of three known thrombin inhibitors, namely benzamidine, p-aminobenzamidine and nafamostat were determined and found to be in good concordance with the previously reported values. Finally, the screening of a small library was performed and demonstrated the high discriminatory power of our method towards weak binders compared to classical spectrophotometric activity assay, proving the interest of our method in the context of FBDD.

Discovery and assessment of water soluble coumarins as inhibitors of the coagulation contact pathway

Over the last decade, the coagulation factor XIIa (FXIIa) has seen renewed interest as a therapeutic target. Indeed, its inhibition could offer a protection against thrombosis without increasing the risk of bleeding. Moreover, it could answer the need for a safe prevention of blood-contacting medical devices-related thrombosis. Among the FXII and FXIIa inhibitors already described in literature, organic small-molecular-weight inhibitors are rather left behind. In this study, we were focused on the discovery and assessment of water soluble small molecules. First, a search within our library of compounds flagged two promising hits. Indeed, enzymes and plasma assays suggested they have a greater activity on the contact factors (FXIa, plasma kallikrein and FXIIa) than on the TF pathway. Then, simple pharmacomodulations were undertaken with the aim to design more selective FXIIa inhibitors. This afforded compounds having different degrees of selectivity. All compounds were finally screened in whole blood using an 8-channel microfluidic model and thromboelastometry measurements. Interestingly, all molecules interfered with the thrombus formation and one of them could be considered as a small organic contact inhibitor.