Overcoming Gatekeeper Mutations in Kinases by Hybrid Compound Design

Most commercially available anti-cancer drugs that target kinases bind within the ATP binding site (type I inhibitors). Although such inhibitors are very successful in treating the early stages of disease control, the emergence of drug-resistance is becoming an ever increasing challenge. One of the most prominent mutations is the replacement of the gatekeeper residue with a bulkier and often aliphatic residue (e.g. in Bcr-Abl_T315I and cSrc_T338M). Therefore, current efforts in kinase research are now focusing on overcoming these mutant variants by developing inhibitors which exclusively bind outside the ATP binding site and lock the kinase in its inactive form (type III inhibitors).^[1,2] We recently designed type II hybrid compounds active against cSrc_T338M by fusing type I and III kinase inhibitors.^[3] Here we report on the enhancement of these type II inhibitors. Chemical modifications to the preliminary small molecules allowed us to improve their inhibitory effect in an activity-based assay (HTRF^®) and reduce IC₅₀ values to ~1 nM. Using protein X-ray crystallography, we demonstrate the binding mode of the newly designed molecules bound to cSrc, which is consistent with that proposed in previously completed in silico modeling experiments. While cSrc_T338M served as an initial model system, we further explored the potency of these compounds in biochemical and cellular assay systems against Abl_wt and Abl_T315I as a clinically-relevant resistant kinase mutatant.