BACKGROUND: Snake venoms are rich sources of bioactive molecules, and several venom-derived proteins have entered clinical trials for use in ischemic disorders; however, late-stage failure of a recent drug candidate due to low in vivo efficacy demonstrated the need for new sources of fibrinogenolytic drug candidates. METHODS: A 51.3kDa thrombin-like serine protease (Russelobin) purified from the venom of Russell's Viper (Daboia russelii russelii) was subjected to extensive biochemical characterization, including N-terminal sequencing, substrate specificity, kinetic and inhibitor assays, glycosylation analysis and stability assays. Toxicity and pathology analyses were conducted in NSA mice. RESULTS: Russelobin has extensive N-terminus identity with a beta-fibrinogenase-like serine proteinase precursor from Daboia russelii siamensis venom, a mass of 51.3kDa and contains extensive N-linked oligosaccharides. Serine protease inhibitors and heparin significantly decreased activity, with much lower inhibition by DTT, antithrombin-III and α2-macroglobulin. Russelobin preferentially released FPA and slowly released FPB from human fibrinogen, forming a labile fibrin clot readily hydrolyzed by plasmin. The partially deglycosylated enzyme showed significantly lower activity toward fibrinogen and less resistance against neutralization by plasma α2MG and antithrombin-III. Russelobin was non-cytotoxic, non-lethal and produced no histopathologies in mice, and it demonstrated in vivo dose-dependent defibrinogenating activity. CONCLUSIONS: Russelobin is an A/B fibrinogenase with high specificity toward fibrinogen, both in vitro and in vivo. Extensive glycosylation appears to protect the molecule against endogenous protease inhibitors, prolonging its in vivo efficacy. GENERAL SIGNIFICANCE: Due to its low toxicity, stability and activity as a defibrinogenating agent, Russelobin shows high potential for cardiovascular drug development.
BACKGROUND: Snake venoms are rich sources of bioactive molecules, and several venom-derived proteins have entered clinical trials for use in ischemic disorders; however, late-stage failure of a recent drug candidate due to low in vivo efficacy demonstrated the need for new sources of fibrinogenolytic drug candidates. METHODS: A 51.3kDa thrombin-like serine protease (Russelobin) purified from the venom of Russell's Viper (Daboia russelii russelii) was subjected to extensive biochemical characterization, including N-terminal sequencing, substrate specificity, kinetic and inhibitor assays, glycosylation analysis and stability assays. Toxicity and pathology analyses were conducted in NSA mice. RESULTS:Russelobin has extensive N-terminus identity with a beta-fibrinogenase-like serine proteinase precursor from Daboia russelii siamensis venom, a mass of 51.3kDa and contains extensive N-linked oligosaccharides. Serine protease inhibitors and heparin significantly decreased activity, with much lower inhibition by DTT, antithrombin-III and α2-macroglobulin. Russelobin preferentially released FPA and slowly released FPB from humanfibrinogen, forming a labile fibrin clot readily hydrolyzed by plasmin. The partially deglycosylated enzyme showed significantly lower activity toward fibrinogen and less resistance against neutralization by plasma α2MG and antithrombin-III. Russelobin was non-cytotoxic, non-lethal and produced no histopathologies in mice, and it demonstrated in vivo dose-dependent defibrinogenating activity. CONCLUSIONS:Russelobin is an A/B fibrinogenase with high specificity toward fibrinogen, both in vitro and in vivo. Extensive glycosylation appears to protect the molecule against endogenous protease inhibitors, prolonging its in vivo efficacy. GENERAL SIGNIFICANCE: Due to its low toxicity, stability and activity as a defibrinogenating agent, Russelobin shows high potential for cardiovascular drug development.
Authors: Kayena D Zaqueo; Anderson M Kayano; Rodrigo Simões-Silva; Leandro S Moreira-Dill; Carla F C Fernandes; André L Fuly; Vinícius G Maltarollo; Kathia M Honório; Saulo L da Silva; Gerardo Acosta; Maria Antonia O Caballol; Eliandre de Oliveira; Fernando Albericio; Leonardo A Calderon; Andreimar M Soares; Rodrigo G Stábeli Journal: Biomed Res Int Date: 2014-02-26 Impact factor: 3.411
Authors: Barbara M Zietek; Morwarid Mayar; Julien Slagboom; Ben Bruyneel; Freek J Vonk; Govert W Somsen; Nicholas R Casewell; Jeroen Kool Journal: Anal Bioanal Chem Date: 2018-08-09 Impact factor: 4.142