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.
Protein kinase pathways are traditionally mapped by monitoring downstream phosphorylation. Meanwhile, the non-catalytic functions of protein kinases remain under-appreciated as critical components of kinase signaling. c-Src is a protein kinase known to have non-catalytic signaling function important in healthy and disease cell signaling. Large conformational changes in the regulatory domains regulate c-Src's non-catalytic functions. Herein, we demonstrate that changes in the global conformation of c-Src can be monitored using a selective proteolysis methodology. Further, we use this methodology to investigate changes in the global conformation of several clinical and non-clinical mutations of c-Src. Significantly, we identify a novel activating mutation observed clinically, W121R, that can escape down-regulation mechanisms. Our methodology can be expanded to monitor the global conformation of other tyrosine kinases, including c-Abl, and represents an important tool toward the study of elucidating the non-catalytic functions of protein kinases.
We have developed a modular approach to bisubstrate inhibition of protein kinases. We apply our methodology to c-Src and and identify a highly selective bisubstrate inhibitor for this target. Our approach has yielded the most selective c-Src inhibitor to date, and methodology to render the bisubstrate inhibitor cell-permeable provides a highly valuable tool for the study of c-Src signaling. In addition, we have applied our bisubstrate inhibitor to develop a novel screening methodology to identify non-ATP-competitive inhibitors of c-Src. Using this methodology, we have discovered the most potent non-ATP-competitive inhibitor reported to date. Our methodology is designed to be general and could be applicable to additional kinases inhibited by the promiscuous ATP-competitive fragment used in our studies.
The studies culminating in the total synthesis of the glutarimide-containing eukaryote translation elongation inhibitor lactimidomycin are described. The optimized synthetic route features a Zn(II)-mediated intramolecular Horner-Wadsworth-Emmons (HWE) reaction resulting in a highly stereoselective formation of the strained 12-membered macrolactone of lactimidomycin on a 423 mg scale. The presence of the E,Z-diene functionality was found to be key for effective macrocyclizations as a complete removal of these unsaturation units resulted in exclusive formation of the dimer rather than monocyclic enoate. The synthetic route features a late-stage installation of the glutarimide functionality via an asymmetric catalytic Mukaiyama aldol reaction, which allows for a quick generation of lactimidomycin homolog 55 containing two additional carbons in the glutarimide side chain. Similar to lactimidomycin, this analog was found to possess cytotoxicity against MDA-MB-231 breast cancer cells (GI50 =1-3 μM) using in vitro 2D and 3D assays. Although lactimidomycin was found to be the most potent compound in terms of anticancer activity, 55 as well as truncated analogues 50-52 lacking the glutarimide side-chain were found to be significantly less toxic against human mammary epithelial cells.
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