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Author Spotlight: Affinity Purification of a Fibrinolytic Enzyme from Sipunculus nudus
Published on: June 2, 2023
Computer-aided engineering of staphylokinase toward enhanced affinity and selectivity for plasmin
Dmitri Nikitin1,2, Jan Mican1,2,3, Martin Toul1,2
1International Clinical Research Center, St. Anne's University Hospital, Pekarska 53, 656 91 Brno, Czech Republic.
Insights
Researchers redesigned staphylokinase (SAK) using computer modeling to improve its effectiveness. The engineered SAK shows significantly higher plasmin affinity and selectivity, offering potential for better thrombolytic drugs.
Area of Science:
- Biochemistry
- Molecular Biology
- Pharmacology
Background:
- Cardio- and cerebrovascular diseases are major causes of death and disability, imposing a significant socio-economic burden.
- Plasminogen activators, including bacterial staphylokinase (SAK), are crucial thrombolytic agents.
- Fibrin-specific agents offer improved safety profiles compared to non-specific thrombolytics.
Purpose of the Study:
- To computationally redesign the molecular surface of staphylokinase (SAK) to enhance its affinity for plasmin.
- To develop improved SAK variants for next-generation thrombolytic therapies.
Main Methods:
- Computer-assisted molecular surface re-design of staphylokinase.
- Recombinant production of computationally designed SAK mutants.
- Biochemical characterization and screening of SAK mutants for plasmin affinity and selectivity.
Main Results:
- A novel SAK mutant demonstrated approximately 7-fold increased affinity for plasmin.
- The engineered SAK mutant exhibited around 10-fold greater plasmin selectivity.
- The mutant showed moderately enhanced plasmin-generating efficiency in vitro.
Conclusions:
- Computational affinity-design is an effective strategy for engineering staphylokinase.
- The developed SAK mutant represents a promising candidate for next-generation thrombolytic drugs.
- Further development could lead to more effective, selective, and safer treatments for cardiovascular and cerebrovascular diseases.
Abstract:
Cardio- and cerebrovascular diseases are leading causes of death and disability, resulting in one of the highest socio-economic burdens of any disease type. The discovery of bacterial and human plasminogen activators and their use as thrombolytic drugs have revolutionized treatment of these pathologies. Fibrin-specific agents have an advantage over non-specific factors because of lower rates of deleterious side effects. Specifically, staphylokinase (SAK) is a pharmacologically attractive indirect plasminogen activator protein of bacterial origin that forms stoichiometric noncovalent complexes with plasmin, promoting the conversion of plasminogen into plasmin. Here we report a computer-assisted re-design of the molecular surface of SAK to increase its affinity for plasmin. A set of computationally designed SAK mutants was produced recombinantly and biochemically characterized. Screening revealed a pharmacologically interesting SAK mutant with ∼7-fold enhanced affinity toward plasmin, ∼10-fold improved plasmin selectivity and moderately higher plasmin-generating efficiency in vitro. Collectively, the results obtained provide a framework for SAK engineering using computational affinity-design that could pave the way to next-generation of effective, highly selective, and less toxic thrombolytics.
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