Simon Pitchford, Ryan McGuinness & Joshua Salafsky
Biodesy LLC, 863 Mitten Rd, Suite 101, Burlingame, CA 94010
Direct, Real-time Detection of kinase type II inhibitors using Second Harmonic Generation (SHG) Detection
Demand for inactive conformation inhibitors of kinases (so-called type II compounds) is high. Interest is fueled by the striking clinical success of Gleevec, targeted primarily to Abl kinase, against chronic myeloid leukemia (CML). However, traditional methods for identifying these inhibitors rely on techniques which are slow and labor intensive. To address this shortcoming, we applied SHG detection to develop a rapid and facile assay for detecting type II inhibitors in Abl kinase. Wild-type Abl kinase is labeled with a cysteine-specific probe and bound via a His tag to a proprietary PEG surface. We measured a specific and reproducible response upon exposure to Gleevec, but do not detect signal change if the protein is exposed dasatinib, a type I inhibitor. Titration data confirm that binding occurs in a simple bimolecular fashion. Data is collected in real time, so conformational changes which accompany inhibitor binding are detected immediately. Exposure to other putative type II inhibitors also resulted in a specific changes in the SHG signal. We have adapted this technique in a platform consistent with discovery requirements for identifying specific kinase inhibitors for development and validation.
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