Computer-aided engineering of staphylokinase toward enhanced affinity and selectivity for plasmin

Dmitri Nikitin; Jan Mican; Martin Toul; David Bednar; Michaela Peskova; Patricia Kittova; Sandra Thalerova; Jan Vitecek; Jiri Damborsky; Robert Mikulik; Sarel J Fleishman; Zbynek Prokop; Martin Marek

doi:10.1016/j.csbj.2022.03.004

Computer-aided engineering of staphylokinase toward enhanced affinity and selectivity for plasmin

Comput Struct Biotechnol J. 2022 Mar 12:20:1366-1377. doi: 10.1016/j.csbj.2022.03.004. eCollection 2022.

Authors

Dmitri Nikitin^{1

2}, Jan Mican^{1

2

3}, Martin Toul^{1

2}, David Bednar^{1

2}, Michaela Peskova^{4

5}, Patricia Kittova^{4

6}, Sandra Thalerova^{1

4

5}, Jan Vitecek^{1

4

3}, Jiri Damborsky^{1

2}, Robert Mikulik^{1

3}, Sarel J Fleishman⁷, Zbynek Prokop^{1

2}, Martin Marek^{1

2}

Affiliations

¹ International Clinical Research Center, St. Anne's University Hospital, Pekarska 53, 656 91 Brno, Czech Republic.
² Loschmidt Laboratories, Department of Experimental Biology and RECETOX, Faculty of Science, Masaryk University, Kamenice 5, 625 00 Brno, Czech Republic.
³ Neurology Department, St. Anne's University Hospital and Faculty of Medicine, Masaryk University, Kamenice 5, 625 00 Brno, Czech Republic.
⁴ Institute of Biophysics of the Czech Academy of Sciences, Kralovopolska 135, 612 65 Brno, Czech Republic.
⁵ Department of Biochemistry, Faculty of Science, Masaryk University, Kamenice 5, 625 00 Brno, Czech Republic.
⁶ Department of Biochemistry, Faculty of Medicine, Masaryk University, Kamenice 5, 625 00 Brno, Czech Republic.
⁷ Weizmann Institute of Science, Rehovot 76100, Israel.

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.

Keywords: Acute myocardial infarction; AffiLib; Affinity engineering; Enzyme kinetics; Plasminogen activators; Rational design; SAK, Staphylokinase; Staphylokinase; Stroke treatments; Thrombolytics.