(25b) High Throughput Screening of Kinases with Ruthenium Inhibitors

Authors: 
Wong, E. Y., Penn Center for Molecular Discovery, University of Pennsylvania
Diamond, S. L., University of Pennsylvania


Microarrays are capable of productive, low-cost high-throughput screening. Kinases are important targets in drug discovery as they have been implicated as a major contributor in the cause and proliferation of numerous cancers. A focused library toward kinase inhibition can be synthesized with Ruthenium metallo-organic chemistry centered-around staurosporine. Nanoliter reactions containing the substrate and 32P-labeled ATP (ATP*) run in micro-spots on glass microscope slides are activated by an aerosol deposition of enzyme. Following initiation of the reaction by aerosolization, there is an incubation period, and phosphor-storage imaging technology is utilized to detect the amount of radio-phosphate transferred to the substrate by reaction. The reactions can be carried out two ways: 1) on regular glass slides with the completed reaction spots transferred over to ion-exchange paper for phosphor-imaging or 2) the reactions can be run on streptavidin-coated microscope slides with biotinylated substrate, with the slides phosphor-imaged after completion of reaction. In both cases a wash would be necessary to remove nonspecifically-bound ATP* before phosphor-imaging. The Ruthenium inhibitors are synthesized by a mix and heat procedure with a Ruthenium precursor that contains the pharmacophore of staurosporine. These Ruthenium compounds have demonstrated the ability to inhibit specific kinases with Km values in the low nanomolar range. For substrate and ATP* concentrations of 500 nM and 1 uM respectively, even at only 1% conversion of the substrate, a signal of 1.7E6 is generated, with a background value less than 0.08E6 (signal to noise ratio greater than 20) for each of the 200-250 micron spots after washing. Phosphor-images of the microarrays show linearity over a 5000-fold change in ATP* concentration, ranging from less than 1 decay per minute to over 2200 decays per minute in each spot. Reactions conducted with the transfer method utilizing the kinase inhibitor staurosporine resulted in slowed kinetics as expected, and shows that the method has promise to become a tool that can determine important kinetic parameters. Both methods allow a chemical library to be screened against any kinase.