In Vitro Characterization of a Multifunctional Staphylokinase Variant with Reduced Reocclusion, Produced from Salt Inducible E. coli GJ1158

The thrombolytic therapy with clinically approved drugs often ensues with recurrent thrombosis caused by thrombin-induced platelet aggregation from the clot debris. In order to minimize these problems, a staphylokinase (SAK)-based bacterial friendly multifunctional recombinant protein SRH (staphylokinase (SAK) linked with tripeptide RGD and dodecapeptide Hirulog (SRH)) was constructed to have Hirulog as an antithrombin agent and RGD (Arg-Gly-Asp) as an antiplatelet agent in the present study. This multifunctional fusion protein SRH was expressed in osmotically inducible E. coli GJ1158 as soluble form and purified with a yield of 0.27 g/L and functionally characterized in vitro. SRH retained the fibrinolytic activity and plasminogen activation rate comparable to the parental counterpart SAK. The antithrombin activity of SRH was significantly higher than SAK. The platelet rich clot lysis assay indicated that SRH had enhanced platelet binding activity and T50% and C50 of SRH were significantly lower than that of SAK. Furthermore, SRH inhibited the ADP-induced platelet aggregation in dose-dependent manner while SAK had no significant effect on platelet aggregation. Thus, the current study suggests that the SAK variant produced from osmotically inducible GJ1158 is more potent thrombolytic agent with antithrombin and antiplatelet aggregation activities for reduction of reocclusion in thrombolytic therapy

Characterization of a Multifunctional Staphylokinase Variant with Reduced Reocclusion, Produced from Salt Inducible E. coli GJ1158

The thrombolytic therapy with clinically approved drugs often ensues with recurrent thrombosis caused by thrombin-induced platelet aggregation from the clot debris. In order to minimize these problems, a staphylokinase (SAK)-based bacterial friendly multifunctional recombinant protein SRH (staphylokinase (SAK) linked with tripeptide RGD and dodecapeptide Hirulog (SRH)) was constructed to have Hirulog as an antithrombin agent and RGD (Arg-Gly-Asp) as an antiplatelet agent in the present study. This multifunctional fusion protein SRH was expressed in osmotically inducible E. coli GJ1158 as soluble form and purified with a yield of 0.27 g/L and functionally characterized in vitro. SRH retained the fibrinolytic activity and plasminogen activation rate comparable to the parental counterpart SAK. The antithrombin activity of SRH was significantly higher than SAK. The platelet rich clot lysis assay indicated that SRH had enhanced platelet binding activity and T 50% and C 50 of SRH were significantly lower than that of SAK. Furthermore, SRH inhibited the ADP-induced platelet aggregation in dose-dependent manner while SAK had no significant effect on platelet aggregation. Thus, the current study suggests that the SAK variant produced from osmotically inducible GJ1158 is more potent thrombolytic agent with antithrombin and antiplatelet aggregation activities for reduction of reocclusion in thrombolytic therapy

Maxwell A: F1000Prime Recommendation of Evaluation [Neres J et al., ACS Chem Biol 2014]. In F1000Prime, 09 Jan 2015; DOI: 10.3410/f.725248139.793502885. F1000Prime.com/725248139#eval793502885

Tuberculosis (TB) is still a serious problem worldwide, particularly with the advent of drug-resistant strains. Using phenotypic screening, the authors have discovered quinoxaline compounds (especially Ty38c) that are active against extracellular and intracellular Mycobacterium tuberculosis, targeting DprE1, an enzyme involved in cell wall synthesis. The structural work reported in this paper will underpin further efforts to find other compounds that inhibit this target. There is no doubt that TB is a scourge of humankind, killing >1 million people each year. Current therapy is problematic owing to resistance issues and the long times over which drugs have to be taken. Current work has tended to move away from target-based screening to phenotypic screening. Using this approach, the authors have discovered the quinoxaline compound Ty38c as a TB drug candidate. Initial mutations were found in rv3405c, a transcriptional regulator that controls expression of rv3406, whose gene product inactivates Ty38c. Using an rv3406-deficient strain, the authors show that the actual target of Ty38c is DprE1, which is a known target for other TB inhibitors. Using a range of Ty38c analogues, the authors perform a series of structure-activity experiments, and using X-ray crystallography they reveal the molecular details of the Ty38c-DprE1 interactions. Taken together, the work represents a tour de force in TB drug discovery research and provides significant hope that new TB agents can be developed from such approaches