title: Bufadienolides from Kalanchoe daigremontiana as thrombin inhibitors-In vitro and in silico study.
abstract: Thrombin is an active plasma coagulation factor II, critical for the formation of fibrin clot during blood coagulation. For that reason, this protein is also a crucial target for different anti-thrombotic therapies. The work is based on in vitro evaluation of the inhibitory effect of bufadienolide-rich fraction, isolated from roots of Kalanchoe daigremontiana (1-50μg/ml) on enzymatic properties of a serine proteinase - thrombin. The efficacy of the inhibition of amidolytic activity of thrombin (measured as a hydrolysis of the chromogenic substrate S-2238, Chromogenix) attained about 10 and 66%, respectively. The IC50, established for the examined bufadienolide fraction was 2.79μg/ml, while the IC50calculated for argatroban (reference compound) was 0.78μg/ml. Linearization conducted using Lineweaver-Burk plot indicated that the K. daigremontiana fraction contains compounds that are uncompetitive inhibitors of thrombin. K. daigremontiana fraction was also able to reduce the proteolytic activity of thrombin towards its physiological substrate, i.e. fibrinogen. Additionally, this study is supported by in silico analysis of interactions of the most common compounds, identified in the examined in Kalanchoe extract to crystal structure of this enzyme.
authors.affiliation: Department of General Biochemistry, Faculty of Biology and Environmental Protection, University of Lodz, Pomorska 141/143, 90-236 Lodz, Poland. Electronic address: joannak@biol.uni.lodz.pl.
authors.forename: Joanna
authors.initials: J
authors.lastname: Kolodziejczyk-Czepas
authors.affiliation: Department of General Biochemistry, Faculty of Biology and Environmental Protection, University of Lodz, Pomorska 141/143, 90-236 Lodz, Poland.
authors.forename: Malgorzata
authors.initials: M
authors.lastname: Sieradzka
authors.affiliation: Department of Biochemistry and Crop Quality, Institute of Soil Science and Plant Cultivation, State Research Institute, Czartoryskich 8, 24-100 Pulawy, Poland.
authors.forename: Barbara
authors.initials: B
authors.lastname: Moniuszko-Szajwaj
authors.affiliation: Department of Biochemistry and Crop Quality, Institute of Soil Science and Plant Cultivation, State Research Institute, Czartoryskich 8, 24-100 Pulawy, Poland.
authors.forename: Łukasz
authors.initials: Ł
authors.lastname: Pecio
authors.affiliation: Department of General Biochemistry, Faculty of Biology and Environmental Protection, University of Lodz, Pomorska 141/143, 90-236 Lodz, Poland.
authors.forename: Michal B
authors.initials: MB
authors.lastname: Ponczek
authors.affiliation: Department of General Biochemistry, Faculty of Biology and Environmental Protection, University of Lodz, Pomorska 141/143, 90-236 Lodz, Poland.
authors.forename: Pawel
authors.initials: P
authors.lastname: Nowak
authors.affiliation: Department of Biochemistry and Crop Quality, Institute of Soil Science and Plant Cultivation, State Research Institute, Czartoryskich 8, 24-100 Pulawy, Poland.
authors.forename: Anna
authors.initials: A
authors.lastname: Stochmal
mesh_terms: D000577:Amides / Q000737:chemistry / Q000378:metabolism; D002018:Bufanolides / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D003198:Computer Simulation; D031962:Kalanchoe / Q000737:chemistry*; D062105:Molecular Docking Simulation; D011480:Protease Inhibitors / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D011487:Protein Conformation; D059748:Proteolysis / Q000187:drug effects; D013917:Thrombin / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism
chemical_list: D000577:Amides; D002018:Bufanolides; D011480:Protease Inhibitors; C087925:bufadienolide; D013917:Thrombin
keywords: Bufadienolide; Inhibitor; Thrombin