Assessment of compound interaction with kinases

The inhibitor-kinase binding affinity was assessed in KINOMEscan™ assay (Discoverex) according to service provider’s protocol. In brief, in this binding assay the studied compound in solution is incubated with DNA-tagged kinase and kinase active-site directed ligand bound to the beads. The compound competes for binding to kinase with the immobilized ligand. After incubation beads are removed from the solution and kinase bound to ligand is quantified by qPCR. The strength of compound binding is determined based on its ability to block kinase-ligand binding [18]. 11-point 3-fold serial dilution of CPL302-253 was used in order to determine its binding constants (Kd) against different isotypes of class I PI3K. The single concentration of CPL302-253 was used in order to determine its specificity in abroad panel of 468 human kinases. The measure of compounds affinity to each kinase in the panel was the percentage of kinase activity inhibition by tested ligand in comparison to the control.