Unconventional approaches to kinase inhibition : covalent inhibitors and docking site inhibitors of mitogen-activated protein kinases

The body of work that follows is a description of biochemical screening methods to identify non-ATP competitive and covalent ERK inhibitors, as well as assay design for characterizing covalent inhibitors of JNK in an isoform-specific manner. A covalent ERK inhibitor is presented, as well as a novel class of non-covalent ERK docking site inhibitors. An assay to elucidate the kinetic parameters of covalent JNK inhibitors is described, and the kinetic parameters for the inhibitor JNK-IN-8 are illustrated as a case study. Additionally, a series of JNK-IN-8 analogues were tested in a PyVMT breast cancer cell model for their ability to preferentially inhibit either JNK1 or JNK2. The rationale behind targeting MAPKs at docking sites and focusing on covalent inhibitors is centered on several key elements. First, the proximity-based mechanism of MAPK catalysis depends on docking site interactions to direct substrate consensus sequences to the vicinity of the MAPK active site. MAPK activators, phosphatases, scaffolding proteins, and other molecules also engage docking sites. Therefore, targeting docking sites with inhibitors can not only directly or indirectly block substrate phosphorylation or enzyme activation, but the mechanisms of catalysis themselves can be altered. Secondly, the signal dynamics of MAPKs, such as their switch-like behavior, intra-pathway feedback, and inter-pathway crosstalk makes complete inhibition of MAPKs necessary to stop signaling in a therapeutic context. Covalent, irreversible MAPK inhibitors can induce complete inhibition by being effectively immune to substrate competition, and by forcing recovery of signaling to be fully dependent on either MAPK mutations, protein synthesis, or pathway bypass. Targeting a MAPK at multiple interaction sites can further promote a full blockade of signaling. The collective goal of the work presented in this dissertation is to contribute to the breadth of targeted kinase inhibitors that can be used against cancers, such as BRAF-V600E melanoma. These inhibitors and methods of inhibitor development can be used as tools to probe the functionality of individual protein-protein interaction sites on ERK and JNK, and the principles described here can be adapted to apply to other kinases and binding sites as needed in future studies.