Structural proteomics for the development of thrombolytic drug

The structural proteomic approach, using hydrogen-deuterium exchange combined with mass spectrometry (HDX-MS), can provide extensive insights into protein behaviour by serving as a link between structure, conformational dynamics, and function. It can help us identify the sites involved in the protein-protein interactions. Stroke is currently the second leading cause of death, with alteplase as the sole FDA-approved drug for its treatment. Alteplase, however, has several side effects, including intracranial bleeding and neurotoxicity. The development of the new generation of thrombolytics is highly desirable.

Our research focuses on developing an alternative thrombolytic drug, staphylokinase (SAK), which is highly promising in terms of its thrombolytic activity, fibrin specificity, and low production cost. SAK is a 15.5 kDa prokaryotic protein derived from lysogenic strains of Staphylococcus aureus. It is a well-recognized thrombolytic agent that has demonstrated its clinical efficacy at par with alteplase. We aim to improve the thrombolytic efficiency of SAK by understanding its molecular structure, biochemical properties, and interaction sites. The structure of SAK has been studied through X-ray crystallography and nuclear magnetic resonance, revealing that its conformation is crucial for driving thrombolysis by activating plasminogen and interacting with plasmin.

Using the HDX-MS approach, we aim to understand the complete in-solution conformation of wild-type and rationally designed SAK proteins. This analysis identifies the solvent-exposed regions of folded SAK proteins to detect structural changes. Subsequently, its interaction with plasmin and assistance in the activation of plasminogen will be studied in the holo form of SAK proteins. Thus far, we have conducted a comparative HDX study of a few non-immunogenic SAK mutants with 3-point, 8-point, and 12-point mutations. The results indicate significant differences in deuterium uptake in the bulge region comprising residues Leu40-Glu46 and the C-terminus that are known for their role in plasminogen activation. Deuterium uptake is higher in the solvent-exposed residues which is directly related to protein flexibility and affinity. The extent of their involvement in plasminogen activation will be studied through interactive HDX analysis. These results, complemented by biochemical assays, will help explain the impact of SAK conformation on the thrombolytic mechanism.