Multiobjective Engineering of Fibrin-Selective Thrombolytic Proteases with Enhanced Biocatalytic Efficiency and Inhibition Resistance

Abstract Thrombolytic enzymes represent an important class of proteolytic biocatalysts for medical applications, yet currently used FDA-approved variants, including alteplase and tenecteplase, remain limited by suboptimal catalytic efficiency, off-target activity, and susceptibility to inhibition. These limitations reflect the complexity of enzyme function in physiological environments, where therapeutic performance depends on the simultaneous optimization of multiple catalytic and biophysical properties. Here, we introduce a multiobjective enzyme engineering strategy for the design of next-generation thrombolytic proteases, explicitly targeting multiple properties required for therapeutic performance. Our approach combines computer-aided design, evolutionary reconstruction, database search, and literature-guided mutation selection to improve catalytic activity, fibrin selectivity, inhibition resistance, and functional lifetime within a single workflow. This framework is coupled with systematic biochemical characterization, in vitro evaluation of clot penetration and fibrinolytic activity, and in vivo validation of efficacy and safety. By addressing multiple performance parameters simultaneously, this strategy enables efficient navigation of trade-offs that typically limit enzyme optimization. Using this approach, we identify Brnoteplase as a lead variant with enhanced fibrin selectivity, improved resistance to inhibition, and elevated clot penetration, resulting in increased effective catalytic lifetime and bolus administration compatibility. In vivo studies demonstrate enhanced fibrinolysis and recanalization with lower proportion of severe hemorrhagic transformation under the tested dosing. These findings provide a broadly applicable framework for designing proteolytic biocatalysts suitable for complex biological environments.