New Triazole-Based Potent Inhibitors of Human Factor XIIa as Anticoagulants

Abstract

Factor XIIa (FXIIa) functions as a plasma serine protease within the contact activation pathway. Various animal models have indicated a substantial role for FXIIa in thromboembolic diseases. Interestingly, individuals and animals with FXII deficiency seem to maintain normal hemostasis. Consequently, inhibiting FXIIa could potentially offer a viable therapeutic approach for achieving effective and safer anticoagulation without the bleeding risks associated with the existing anticoagulants. Despite the potential, only a limited number of small molecule inhibitors targeting human FXIIa have been documented. Thus, we combined a small library of 32 triazole and triazole-like molecules to be evaluated for FXIIa inhibition by using a chromogenic substrate hydrolysis assay under physiological conditions. Initial screening at 200 μM involved 18 small molecules, revealing that 4 molecules inhibited FXIIa more than 20%. In addition to being the most potent inhibitor identified in the first round, inhibitor 8 also exhibited a substantial margin of selectivity against related serine proteases, including factors XIa, Xa, and IXa. However, the molecule also inhibited thrombin with a similar potency. It also prolonged the clotting time of human plasma, as was determined in the activated partial thromboplastin time and prothrombin time assays. Subsequent structure−activity relationship studies led to the identification of several inhibitors with submicromolar activity, among which inhibitor 22 appears to demonstrate significant selectivity not only over factors IXa, Xa, and XIa, but also over thrombin. In summary, this study introduces novel triazole-based small molecules, specifically compounds 8 and 22, identified as potent and selective inhibitors of human FXIIa. The aim is to advance these inhibitors for further development as anticoagulants to provide a more effective and safer approach to preventing and/or treating thromboembolic diseases.

Discovery of Heparin Mimetic, Potent, and Selective Inhibitors of Human Clotting Factor XIIIa

“…To further evaluate the selectivity as well as the potential toxicity of inhibitors, we studied their effects on the clotting times of normal human plasma as well as their effects on the proliferation of three cell lines. On the one hand, the inhibitors’ effects on activated partial thromboplastin time (APTT) and prothrombin time (PT) were measured, as described earlier under in vitro conditions, − using variable concentrations (0–2000 μM) (Table ). Results indicated that the inhibitors’ concentrations estimated to double APTT and PT are in the high micromolar range.…”

“…Clotting times, including activated partial thromboplastin time (APTT) and prothrombin time (PT), were assessed in normal human plasma utilizing the BBL Fibrosystem fibrometer from Becton–Dickinson, as detailed in previous publications. − For the PT assay, thromboplastin-D was dissolved in 4 mL of highly purified water and then warmed to 37 °C. Subsequently, 10 μL of the inhibitor was added to 90 μL of normal human plasma and incubated for 30 s. Following this, 200 μL of prewarmed PT reagent was added, and the clotting time was recorded.…”