Interactomics of Blebbistatin

Abstract

Photoreactive molecules like aryl azides play important role in life sciences, as practical tools to achieve precisely timed covalent cross-links between ligands and targets. Since the azido group is small, stable and biologically inert, azido-modified aromatic drug molecules can keep their original pharmacological activity. By photoactivating of such modified molecules in vivo, both their strong and weak partners can be captured. Moreover, applying the azidated drug in a concentration range and utilizing proteomic tools the apparent binding constants of protein-ligand interactions can be determined.We have developed a new and simple technique to azidate aromatic drug molecules in an easy two step reaction. Using our method we have synthesized azidoblebbistatin which is a new derivative of blebbistatin, the most widely used myosin inhibitor. In the absence of UV irradiation azidoblebbistatin and blebbistatin exhibits identical inhibitory properties. Using UV light azidoblebbistatin can be covalently crosslinked to myosin whereas the unbound inhibitor molecules become inactive. Using Dictyostelium discoideum cell lysate we performed interactomic investigation to identify the previously unknown targets of blebbistatin utilizing the self- fluorescence of azidoblebbistatin. Since the crosslinking was performed applying increasing concentration of azidoblebbistatin, densitometry of the fluorescent spots resulting from gelelectrophoresis revealed the apparent binding constant of azidoblebbistatin. In case of myosin II it was the exact value as measured in in vitro tests. With this technique the strongest interactant was found to be myosin II (EC50=5 μM) while eight weak partners (EC50>30 μM) were also detected including vacuolar H+-ATPase (EC50=50 ± 31 μM), malate dehydrogenase (EC50=55 ± 17 μM) and elongation factor 1α (EC50>100 μM).