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Computer-Aided Engineering of Staphylokinase Toward Enhanced Affinity and Selectivity for Plasmin

Publication year: 2022
Authors: Nikitin D. 1 2, Mican J. 1 2 3, Toul M. 1 2, Bednar D. 1 2, Peskova M. 4 5, Kittova P. 4 6, Thalerova S. 1 4 5, Vitecek J. 1 4 3, Damborsky J. 1 2, Mikulik R. 1 3, Fleishman S. 7, Prokop Z. 1 2, Marek M. 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.
Published in: Computational and Structural Biotechnology Journal, 2022, Vol. 20 p. 1366-1377
doi: 10.1016/j.csbj.2022.03.004

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.