Examining the mechanism of action of a kinesin inhibitor using Stable Isotope Labeled Inhibitors for Crosslinking (SILIC)

It is difficult to determine a chemical inhibitor’s binding site in multi-protein mixtures, particularly when high-resolution structural studies are not straightforward. Building upon previous research involving photo-crosslinking and the use of mixtures of stable isotopes, we report a method, S table I sotope L abeled I nhibitors for C rosslinking (SILIC), for mapping a small molecule inhibitor’s binding site in its target protein. In SILIC, structure-activity relationship data is used to design inhibitor analogs that incorporate a photo-crosslinking group along with either natural or ‘heavy’ stable isotopes. An equimolar mixture of these inhibitor analogs is crosslinked to the target protein to yield a robust signature for identifying inhibitor-modified peptide fragments in complex mass spectrometry data. As a proof of concept, we applied this approach to an ATP-competitive inhibitor of kinesin-5, a widely conserved motor protein required for cell division and an anti-cancer drug target. This crosslinking analysis, along with mutagenesis studies, suggests that the inhibitor binds at an allosteric site in the motor protein.