Design and production of new chimeric reteplase with enhanced fibrin affinity: a theoretical and experimental study

Plasminogen activators (PAs) are widely used for treatment of disorders caused by clot formation. Fibrin specific PAs are safe drugs from this group because of reducing the incidence of hemorrhage. The newer generation of PAs like tenecteplase, reteplase and desmoteplase were designed with the aim of achieving desirable properties such as improving specificity and affinity to fibrin and increasing half-life. Protein engineering and using of theoretical methods can help to rational and reliable design of new PAs with a set of favorable properties. In the present study, two new chimeric reteplase named M1-chr and M2-chr were designed with the aim of enhancing fibrin affinity also some potential properties include of increasing resistance to plasminogen activator inhibitor-1 and decreasing neurotoxicity. So, finger domain of desmoteplase was added to reteplase as a high fibrin specific domain. Some other point mutations were considering to achieve other mentioned properties. Three dimensional structure of wild-type reteplase and mutants were created by homology modeling and were evaluated by molecular dynamic simulation. Then, mutants docked to fibrin by HADDOCK web tools. Result of theoretical section verified the stability of mutants' structures. Also showed better interaction between M1-chr with fibrin than M2-chr. Wild-type and mutants were produced in bacterial expression system. Experimental assessment showed both mutants have appropriate enzymatic activity also 1.9-fold fibrin binding ability compared to wild-type. Therefore, this study offers new thrombolytic drugs with desirable properties specially enhanced fibrin affinity so they can represent a promising future in cost-effective production of favorable thrombolytic drugs. Communicated by Ramaswamy H. Sarma

Automated summary

Plasminogen activators (PAs) are commonly used for treating clot-related disorders. Newer generations of PAs like tenecteplase, reteplase and desmoteplase aim to improve specificity and affinity to fibrin, resulting in safer drugs. This study introduces two new chimeric reteplase with enhanced fibrin affinity and other beneficial properties, showing better interaction between M1-chr and fibrin compared to M2-chr. Experimental assessments confirmed the mutants' appropriate enzymatic activity and increased fibrin binding ability, indicating potential cost-effective production of promising thrombolytic drugs.