Combining Active Site and Allosteric Inhibitors to Study Protein Tyrosine Kinases

Abstract

Kinases play a crucial role in a variety of cell signaling pathways that control processes such as: growth, motility, and angiogenesis. Not surprisingly, dysregulation of proper kinase signaling often leads to cancer. Since the introduction of imatinib, a small molecule kinase inhibitor for the treatment of chronic myelogenous leukemia (CML), kinase inhibitors have become a popular drug class with 28 approved as drugs. All of these inhibitors bind in the conserved ATP-binding pocket, and as a result, many are promiscuous and bind off-target kinases. This dissertation describes our work to target kinases through mechanisms other than the ATP-binding pocket. Through targeting pockets other than the ATP-binding pocket, we also demonstrate the importance of understanding kinase global conformation. We created bivalent inhibitors (inhibitors that can bind two sites within their target simultaneously) for two protein tyrosine kinases: c-Src and c-Abl. In both, the bivalent inhibitors were synthesized by linking an ATP-competitive inhibitor to an allosteric inhibitor of the kinase using azide-alkyne click chemistry. In doing so, we created inhibitors that were both more potent and selective than the separate binding pieces alone. Our c-Src bivalent inhibitor was the first c-Src inhibitor that was selective for the active form of the kinase over the inactive form. Additionally, the bivalent inhibitor of c-Abl was cell permeable and had a cellular therapeutic index of 14. We also discovered an allosteric modulator of c-Src via a screen of 1,000 compounds. We determined that this small molecule stabilizes the open conformation of c-Src and, accordingly, is an activator of c-Src. Finally, we investigated the interplay between an allosteric inhibitor of c-Abl (GNF-2) and various ATP-competitive inhibitors. None of the FDA approved, ATP-competitive inhibitors tested showed synergy with GNF-2 because they stabilized different global conformations in c-Abl. Only one ATP-competitive inhibitor stabilized the same conformation as GNF-2, and gratifyingly, this was the only combination that showed synergy. Additionally, this combination was capable of overcoming an imatinib resistant mutant in CML. These findings will play a crucial role in understanding drug combinations. This dissertation provides novel findings about the important role that global conformation plays in kinase inhibition.