Improving the solubility, activity, and stability of reteplase using in silico design of new variants

Abstract

Reteplase (recombinant plasminogen activator, r-PA) is a thrombolytic agent recombined from tissue-type plasminogen activator (t-PA), which has several prominent features such as strong thrombolytic ability and E. coli expressibility. Despite these outstanding features, it demonstrates reduced fibrin binding affinity, reduced stimulation of protease activity, and lower solubility, hence higher aggregation propensity, compared to t-PA. The present study was devoted to design r-PA variants with comparable structural stability, enhanced biological activity, and high solubility. For this purpose, computational molecular modeling techniques were utilized. The supercharging technique was applied for r-PA to designing new species of the protein. Based on the results from in silico evaluation of selected mutations in comparison to the wild-type r-PA, the designed supercharged mutant (S7 variant) exhibited augmented stability, decreased solvation energy, as well as enhanced binding affinity to fibrin. The data also implied increased plasminogen cleavage activity of the new variant. These findings have implications to therapies which involve removal of intravascular blood clots, including the treatment of acute myocardial infarction.