Site-Specific Thiol-mediated PEGylation of Streptokinase Leads to Improved Properties with Clinical Potential

Streptokinase (SK) is an efficient thrombolytic agent that dissolves fibrin blood clots with clinical efficiency comparable to the high priced drug, tissue plasminogen activator (tPA). However, being of bacterial origin, its major drawbacks are its potentially high antigenicity, and relatively short circulating half-life (approximately 10-15 min). In the present investigation, an attempt has been made to address both these shortcomings by site-specific pegylation, and to obtain longer lasting thrombolytics, which are consistent with clinical requirements. Therefore, we employed available three-dimensional structural information on SK to carry out site-specific cysteine incorporation at 'optimal' surface-exposed sites within all the three domains in streptokinase followed by pegylation with 20KDa PEG groups, and screening for biologically active variants. Interestingly, some of these SK PEG-conjugates exhibited considerably subdued immune-reactivity along with enhanced in-vitro proteolytic stability profiles and extended circulating in-vivo half-lives (2 to 20-fold compared to that of native unconjugated SK) depending upon location and number of PEG-groups per molecule obtained in homogeneous form. The obtained results are a promising approach for favorably modulating immune-reactivity and half-life by cysteine- specific PEGylation of SK to achieve therapeutic attributes desirable for the treatment of different circulatory disorders, such as ischemic stroke, myocardial infarction and pulmonary embolism.