Targeted drug delivery to the thrombus by fusing streptokinase with a fibrin-binding peptide (CREKA): an <i>in silico</i> study

<b>Aim:</b> Streptokinase has poor selectivity and provokes the immune response. In this study, we used <i>in silico</i> studies to design a fusion protein to achieve targeted delivery to the thrombus. <b>Materials &amp; methods:</b> Streptokinase was analyzed computationally for mapping. The fusion protein modeling and quality assessment were carried out on several servers. The enzymatic activity and the stability of the fusion protein and its complex with plasminogen were assessed through molecular docking analysis and molecular dynamics simulation respectively. <b>Results:</b> Physicochemical properties analysis, protein quality assessments, protein–protein docking and molecular dynamics simulations predicted that the designed fusion protein is functionally active. <b>Conclusion:</b> Our results showed that this fusion protein might be a prospective candidate as a novel thrombolytic agent with better selectivity. Thrombolytic agents are categorized into two classes based on their mechanisms: eukaryotic serine proteases and prokaryotic proteins like streptokinase (SK), which forms binary complexes with plasmin(ogen). Current SK versions lack selectivity, leading to non-specific activation of fibrin and systemic hemorrhage, highlighting the need for targeted drug delivery to thrombus sites. Peptides offer promise for fibrin targeting due to their small size, ease of manufacturing and decreased immunogenicity, with CREKA (Cys-Arg-Glu-Lys-Ala) specifically identifying fibrin–fibronectin complexes. <i>In silico</i> research presents a valuable strategy for molecular design and optimization, allowing for the evaluation of fusion proteins like CREKA-SK for targeted thrombolysis. This study aims to design and assess the efficacy of a fusion protein comprising CREKA and SK for targeted thrombolysis, leveraging <i>in silico</i> techniques for modeling and quality assessment of SK's 3D structure, docking simulations with its natural target, and molecular dynamics (MD) simulations to assess stability and flexibility. Extensive mapping of SK was conducted, incorporating experimental data on its structural domains and interaction sites. This mapping provided critical insights into identifying regions suitable for fusion with the fibrin-binding peptide (CREKA). Molecular modeling and analysis revealed the successful fusion of CREKA peptide with SK at region 302, with computational assessments confirming the stability and integrity of the fused protein. The development of targeted thrombolytic agents like CREKA-SK holds promise for improving clinical responses in thrombosis management, potentially reducing adverse effects associated with non-specific fibrinolysis.