Irina Vladimirovna Gribkova

New Synthetic Thrombin Inhibitors: Molecular Design and Experimental Verification

Background The development of new anticoagulants is an important goal for the improvement of thromboses treatments. Objectives The design, synthesis and experimental testing of new safe and effective small molecule direct thrombin inhibitors for intravenous administration. Methods Computer-aided molecular design of new thrombin inhibitors was performed using our original docking program SOL, which is based on the genetic algorithm of global energy minimization in the framework of a Merck Molecular Force Field. This program takes into account the effects of solvent. The designed molecules with the best scoring functions (calculated binding energies) were synthesized and their thrombin inhibitory activity evaluated experimentally in vitro using a chromogenic substrate in a buffer system and using a thrombin generation test in isolated plasma and in vivo using the newly developed model of hemodilution-induced hypercoagulation in rats. The acute toxicities of the most promising new thrombin inhibitors were evaluated in mice, and their stabilities in aqueous solutions were measured. Results New compounds that are both effective direct thrombin inhibitors (the best KI was <1 nM) and strong anticoagulants in plasma (an IC50 in the thrombin generation assay of approximately 100 nM) were discovered. These compounds contain one of the following new residues as the basic fragment: isothiuronium, 4-aminopyridinium, or 2-aminothiazolinium. LD50 values for the best new inhibitors ranged from 166.7 to >1111.1 mg/kg. A plasma-substituting solution supplemented with one of the new inhibitors prevented hypercoagulation in the rat model of hemodilution-induced hypercoagulation. Activities of the best new inhibitors in physiological saline (1 µM solutions) were stable after sterilization by autoclaving, and the inhibitors remained stable at long-term storage over more than 1.5 years at room temperature and at 4°C. Conclusions The high efficacy, stability and low acute toxicity reveal that the inhibitors that were developed may be promising for potential medical applications.

Eculizumab effect on the hemostatic state in patients with paroxysmal nocturnal hemoglobinuria.

Paroxysmal nocturnal hemoglobinuria (PNH) is characterized by a hypercoagulable state associated with acute hemolysis. Eculizumab is used to reduce the intensity of intravascular hemolysis in PNH patients. The hemostatic status of three patients with PNH was assessed during eculizumab treatment by D-dimer assay and the global assays: thromboelastography (TEG), thrombin generation test (TGТ), and thrombodynamics (TD). In the state of hemolytic crisis before the therapy D-dimer concentration was increased in two patients accompanied by hypercoagulation changes in TEG parameter angle (α). TD parameter the clot growth velocity (V) revealed hypercoagulability while TGT parameter ETP was within the normal range in all patients. The lactate dehydrogenase (LDH) activity decreased during the 8months of eculizumab therapy. The physical health was improved, the frequency of hemolytic crisis decreased. Patients periodically exhibited hypercoagulable state: the mean values α=38±11° (with normal range 20-40°), ETP=1311±442nM·min (with normal range 800-1560nM·min), V=31±4μm/min (with normal range 20-29μm/min). During the eculizumab therapy two patients had the repeated clinical manifestation of acute hemolytic crisis, the parameters of the global tests were increased compared to the previous measurement. The global hemostasis tests TEG, TGT and TD revealed hypercoagulability in patients with PNH during eculizumab therapy.

The modification of the thrombin generation test for the clinical assessment of dabigatran etexilate efficiency.

A new oral anticoagulant, dabigatran etexilate (DE, a prodrug of direct thrombin inhibitor (DTI) dabigatran), has been used clinically to prevent thrombosis. The assessment of dabigatran efficiency is necessary in some clinical cases, such as renal insufficiency, risk of bleeding, and drug interactions. However, a specific thrombin generation test (TGT) that is one of the most informative and sensitive to anticoagulant therapy (calibrated automated thrombinography (САТ)) shows a paradoxical increase of test parameters, such as endogenous thrombin potential (ETP) and peak thrombin, in patients receiving DE. The paradoxical behaviour of ETP and peak thrombin in these patients in the presence of DTIs is mostly caused by a decrease in the activity of thrombin in the α2-macroglobulin-thrombin complex that is used as a calibrator in CAT. For a correct estimation of the TGT parameters in patient’s plasma containing DTIs we proposed to use our previously described alternative calibration method that is based on the measurement of the fluorescence signal of a well-known concentration of the reaction product (7-amino-4-methylcoumarin). In this study, the validity of such approach was demonstrated in an ex vivo study in patients with knee replacement and two special patients with multiple myeloma, who received DE for thrombosis prophylaxis.

Application of Molecular Modeling to Development of New Factor Xa Inhibitors

In consequence of the key role of factor Xa in the clotting cascade and absence of its activity in the processes that do not affect coagulation, this protein is an attractive target for development of new blood coagulation inhibitors. Factor Xa is more effective and convenient target for creation of anticoagulants than thrombin, inhibition of which may cause some side effects. This study is aimed at finding new inhibitors of factor Xa by molecular computer modeling including docking SOL and postdocking optimization DISCORE programs. After validation of molecular modeling methods on well-known factor Xa inhibitors the virtual screening of NCI Diversity and Voronezh State University databases of ready-made low molecular weight species has been carried out. Seventeen compounds selected on the basis of modeling results have been tested experimentally in vitro. It has been found that 12 of them showed activity against factor Xa (IC50 = 1.8–40 μM). Based on analysis of the results, the new original compound was synthesized and experimentally verified. It shows activity against factor Xa with IC50 value of 0.7 μM.