Towards unlocking the potential of staphylokinase for ischemic stroke treatment via a structure-based protein engineering

L. Kašiarová1,2, A. Strunga1, K. Monková3, J. Nováček3, J. Damborský1, 2, Z. Prokop1,2, M. Marek 1,2  

1 Loschmidt Laboratories, Department of Experimental Biology and RECETOX, Masaryk University, Kamenice 5, 62500 Brno, Czech Republic

2 International Clinical Research Center, St. Anne’s University Hospital Brno, Pekarska 53, 65691 Brno, Czech Republic

3 Central European Institute of Technology (CEITEC), Masaryk University, Kamenice 5, 62500 Brno, Czech Republic

linda.kasiarova@recetox.muni.cz

Stroke, the second leading global cause of death, leaves up to 50% of its survivors disabled, emphasizing the critical need for improved treatment. Alteplase, the primary thrombolytic drug for ischemic stroke faces clinical limitations, like high production costs, short half-life, inadequate recanalization, and the risk of haemorrhage due to low fibrin specificity.

Therefore, staphylokinase (SAK), a cost-effective protein encoded by Staphylococcus aureus, emerges as a potential alternative. Despite its promise, immunogenicity and affinity issues with its binding partner pose a significant drawback. To fill this gap, we apply protein engineering methods to overcome the limitations of SAK. Our kinetic studies reveal that optimizing the binding affinity towards plasmin could enhance the efficiency of staphylokinase up to ten thousand times1,2, and its immunogenicity can be mitigated by site-targeted mutagenesis as demonstrated by multiple non-immunogenic variants with no inferiority to alteplase in clinical trials3.

In this project, we aim to visualize the macromolecular complexes formed between SAK and its protein partners (plasmin and plasminogen) through an integrative structural biology approach. We have successfully produced recombinant proteins, carried out biochemical characterization and found first crystallization hits. Preliminary electron microscopy imaging has also been conducted. These experiments represent pivotal steps towards capturing near-atomic resolution structural snapshots that should provide crucial insights into the SAK action, guiding protein engineering efforts towards its enhanced safety and efficacy. Moreover, the combination of cryo-electron microscopy and x-ray crystallography will unveil the structural nuances of non-immunogenic SAK variants to illuminate the mitigated immunogenicity and will be critical to engineering the next-wave of SAK-based thrombolytics.

 

1.       M. Toul, D. Nikitin, M. Marek, J. Damborsky, Z. Prokop, ACS Catalysis 12, no.7 (2022)

2.       M. Toul, J. Mican, V. Slonkova, D. Nikitin, M. Marek, D. Bednar, J. Damborsky, Z. Prokop, Stroke 53, no.10

  1. S. Vanderschueren, L. Barrios, P. Kerdsinchai, P. Van den Heuvel, L. Hermans, M. Vrolix, F. De Man, E. Benit, L. Muyldermans, D. Collen , Circulation (1995)

This work was supported by the Czech Science Foundation (22-09853S).