No abstract

In the kinase field, there are many widely held tenets about conformation-selective inhibitors that have yet to be validated using controlled experiments. We have designed, synthesized, and characterized a series of kinase inhibitor analogues of dasatinib, an FDA-approved kinase inhibitor that binds the active conformation. This inhibitor series includes two Type II inhibitors that bind the DFG-out inactive conformation and two inhibitors that bind the αC-helix-out inactive conformation. Using this series of compounds, we analyze the impact that conformation-selective inhibitors have on target binding and kinome-wide selectivity.

We have developed the first irreversible inhibitors of wild-type c-Src kinase. We demonstrate that our irreversible inhibitors display improved potency and selectivity relative to their reversible counterparts. Our strategy involves modifying a promiscuous kinase inhibitor with an electrophile to generate covalent inhibitors of c-Src. We applied this methodology to two inhibitor scaffolds that exhibit increased cellular efficacy when rendered irreversible. In addition, we have demonstrated the utility of irreversible inhibitors in studying the conformation of an important loop in kinases that can control inhibitor selectivity and cause drug resistance. Together, we have developed a general and robust framework for generating selective irreversible inhibitors from reversible, promiscuous inhibitor scaffolds.

Purpose: c-Src has been shown to play a pivotal role in breast cancer progression, metastasis, and angiogenesis. In the clinic, however, the limited efficacy and high toxicity of existing c-Src inhibitors have tempered the enthusiasm for targeting c-Src. We developed a novel c-Src inhibitor (UM-164) that specifically binds the DFG-out inactive conformation of its target kinases. We hypothesized that binding the inactive kinase conformation would lead to improved pharmacologic outcomes by altering the noncatalytic functions of the targeted kinases.Experimental Design: We have analyzed the anti-triple-negative breast cancer (TNBC) activity of UM-164 in a comprehensive manner that includes in vitro cell proliferation, migration, and invasion assays (including a novel patient-derived xenograft cell line, VARI-068), along with in vivo TNBC xenografts. Results:We demonstrate that UM-164 binds the inactive kinase conformation of c-Src. Kinome-wide profiling of UM-164 identified that Src and p38 kinase families were potently inhibited by UM-164. We further demonstrate that dual c-Src/p38 inhibition is superior to mono-inhibition of c-Src or p38 alone. We demonstrate that UM-164 alters the cell localization of c-Src in TNBC cells. In xenograft models of TNBC, UM-164 resulted in a significant decrease of tumor growth compared with controls, with limited in vivo toxicity.Conclusions: In contrast with c-Src kinase inhibitors used in the clinic (1, 2), we demonstrate in vivo efficacy in xenograft models of TNBC. Our results suggest that the dual activity drug UM-164 is a promising lead compound for developing the first targeted therapeutic strategy against TNBC.

Substrate-competitive kinase inhibitors represent a promising class of kinase inhibitors, however, there is no methodology to selectively identify this type of inhibitor. Herein, we report the application of substrate activity screening to tyrosine kinases. Using this methodology, we have discovered the first small molecule substrates for any protein kinase and the first substrate-competitive inhibitors of c-Src with activity in both biochemical and cellular assays. Characterization of our lead inhibitor demonstrates that substrate-competitive kinase inhibitors possess unique properties, including cellular efficacy that matches biochemical potency and synergy with ATP-competitive inhibitors.