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
- 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).