Several commercially available small molecule sets are used to dissect signal transduction pathways, though their potential off target effects haven’t been carefully examined. Thus we seek to improve the information base regarding kinase inhibitor selectivity, particularly pertaining to understanding MAPK function potential off target results from the AGC family. For this end we have screened a collection of 80 previously known kinase inhibitors against a section of 27 protein kinases. This panel was composed of the three Aurora kinase isoforms along with 23 AGC kinases and STK32B for their relatively high identity to this group. Of the 80 compounds tested, only 10 of them have already been noted to selectively target members of the AGC group. We employed a recently reported cell-free kinase inhibition assay which relies upon competitive active site interactions to effect luminescence technology. 22 This method allows for the interrogation of many kinases without first having to boost recombinant protein expression or recognize substrates for badly analyzed kinases. The selectivities of each and every element Urogenital pelvic malignancy were assessed by examining how similarly structured little substances influenced extremely similar kinases. In order to determine the partnership between inhibitor promiscuity and kinase identity, kinase identity groups of either the kinase domain or only active site residues were won for inhibition frequency and compared between identity groups. In order to utilize the afore-mentioned competitive binding assay, each kinase was prepared by first fusing the protein kinase domain of 27 kinases to the C terminal half of firefly luciferase through a 13 residue linker. where appropriate, were included for these constructs only the kinase domain and the AGC D terminal domain,23. Because we were interested e3 ubiquitin ligase complex in interactions at the active site of the kinases, and particularly the ATP binding site, peripheral areas were excluded to stop potential interference. A number of the kinases utilized in this study contain two kinase domains, namely the ribosomal protein S6 kinases, and in these circumstances only the N terminal kinase domain was attached with the right luciferase half. A second construct consisting of the complementary N terminal half of luciferase was attached to the coiled coil Fos and translated in reticulocyte lysate along with each Cfluc kinase chimera. The Jun peptide, which binds Fos, was conjugated to an ATP competitive kinase inhibitor, a staurosporine analog, and added to a combination of these two proteins, causing increased luminescence due to a functional ternary complex. Because of its promiscuity, staurosporine has an ideal active site point, enabling us to interrogate any kinase that binds our modified staurosporine conjugated to Jun. 24,25 Following the development of the lightgenerating ternary complex, the addition of free kinase inhibitors targeting the ATP binding site could be used to outcompete staurosporine binding, resulting in a lack of luminescence.