Insights into the modular design of kinase inhibitors and application to Abl and Axl.

Scaffold hopping is a common strategy for generating kinase inhibitors that bind to the DFG-out inactive conformation. Small structural differences in inhibitor scaffolds can have significant effects on potency and selectivity across the kinome, however, these effects are often not studied in detail. Herein, we outline a design strategy to generate an array of DFG-out conformation inhibitors with three different hinge-binders and two DFG-pocket groups. We studied inhibitor selectivity across a large segment of the kinome and elucidated binding preferences that can be used in scaffold hopping campaigns. Using these analyses, we identified two selective inhibitors that display low nanomolar potency against Axl or wild-type and clinically relevant mutants of Abl.

Molecular determinants of drug response in TNBC cell lines.

PURPOSE: There is a need for biomarkers of drug efficacy for targeted therapies in triple-negative breast cancer (TNBC). As a step toward this, we identify multi-omic molecular determinants of anti-TNBC efficacy in cell lines for a panel of oncology drugs. METHODS: Using 23 TNBC cell lines, drug sensitivity scores (DSS3) were determined using a panel of investigational drugs and drugs approved for other indications. Molecular readouts were generated for each cell line using RNA sequencing, RNA targeted panels, DNA sequencing, and functional proteomics. DSS3 values were correlated with molecular readouts using a FDR-corrected significance cutoff of p* < 0.05 and yielded molecular determinant panels that predict anti-TNBC efficacy. RESULTS: Six molecular determinant panels were obtained from 12 drugs we prioritized based on their efficacy. Determinant panels were largely devoid of DNA mutations of the targeted pathway. Molecular determinants were obtained by correlating DSS3 with molecular readouts. We found that co-inhibiting molecular correlate pathways leads to robust synergy across many cell lines. CONCLUSIONS: These findings demonstrate an integrated method to identify biomarkers of drug efficacy in TNBC where DNA predictions correlate poorly with drug response. Our work outlines a framework for the identification of novel molecular determinants and optimal companion drugs for combination therapy based on these correlates.

Selective Proteolysis to Study the Global Conformation and Regulatory Mechanisms of c-Src Kinase.

Protein kinase pathways are traditionally mapped by monitoring downstream phosphorylation. Meanwhile, the noncatalytic functions of protein kinases remain under-appreciated as critical components of kinase signaling. c-Src is a protein kinase known to have noncatalytic signaling function important in healthy and disease cell signaling. Large conformational changes in the regulatory domains regulate c-Src's noncatalytic 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 nonclinical 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 elucidation of the noncatalytic functions of protein kinases.

UM-164: A Potent c-Src/p38 Kinase Inhibitor with In Vivo Activity against Triple-Negative Breast Cancer.

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. Clin Cancer Res; 22(20); 5087-96. ©2016 AACR.

Exquisitely specific bisubstrate inhibitors of c-Src kinase.

We have developed a modular approach to bisubstrate inhibition of protein kinases. We apply our methodology to c-Src and identify a highly selective bisubstrate inhibitor for this target. Our approach has yielded the most selective c-Src inhibitor to date, and the 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.

Substrate activity screening with kinases: discovery of small-molecule substrate-competitive c-Src inhibitors.

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