Chemical Proteomics and Structural Biology Define EPHA2 Inhibition by Clinical Kinase Drugs

Heinzlmeir, Stephanie; Kudlinzki, D.; Sreeramulu, Sridhar; Klaeger, Susan; Gande, S.L.; Linhard, V.L.; Wilhelm, Mathias; Qiao, Huichao; Helm, Barbara; Ruprecht, Benjamin; Saxena, Krishna; Médard, Guillaume; Schwalbe, Harald; Küster, Bernhard

doi:10.1021/acschembio.6b00709

Cited by 45 publications

(52 citation statements)

References 53 publications

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“…25,26 Recently, Heinzlmeir et al . 27 utilized chemical proteomics and crystal structures along with an NMR experiment to facilitate the development of receptor tyrosine kinase EPHA2 inhibitors. Their approach is widely applicable to a variety of kinases.…”

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confidence: 99%

A Chemical Probe Strategy for Interrogating Inhibitor Selectivity Across the MEK Kinase Family

et al. 2017

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MEK4 is an upstream kinase in MAPK signaling pathways where it phosphorylates p38 MAPK and JNK in response to mitogenic and cellular stress queues. MEK4 is overexpressed and induces metastasis in advanced prostate cancer lesions. However, the value of MEK4 as an oncology target has not been pharmacologically validated because selective chemical probes targeting MEK4 have not been developed. Despite a high level of sequence homology in the ATP-binding site, most reported MEK inhibitors are selective for MEK1/2 and display reduced potency toward other MEKs. Here, we present the first functional and binding selectivity-profiling platform of the MEK family. We applied the platform to profile a set of known kinase inhibitors and used the results to develop an in silico approach for small molecule docking against MEK proteins. The docking studies identified molecular features of the ligands and corresponding amino acids in MEK proteins responsible for high affinity binding versus those driving selectivity. WaterLOGSY and saturation transfer difference (STD) NMR spectroscopy techniques were utilized to understand the binding modes of active compounds. Further minor synthetic manipulations provide a proof of concept by showing how information gained through this platform can be utilized to perturb selectivity across the MEK family. This inhibitor-based approach pinpoints key features governing MEK family selectivity and clarifies empirical selectivity profiles for a set of kinase inhibitors. Going forward, the platform provides a rationale for facilitating the development of MEK-selective inhibitors, particularly MEK4 selective inhibitors, and repurposing of kinase inhibitors for probing the structural selectivity of isoforms.

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confidence: 99%

A Chemical Probe Strategy for Interrogating Inhibitor Selectivity Across the MEK Kinase Family

et al. 2017

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“…The DFG-in conformer is rigid as it is ready for substrate ATP binding, whereas the DFG-out conformers as autoinhibition states are divergent, particularly those of TRK-A markedly increase the flexibility of the DFG motif through Gly667. Docking-simulated models based on the crystal structures of the foretinib/EphA2 and entrectinib/Alk complexes (27,28) support the inhibitory effects of foretinib and entrectinib on the wild-type and G667C mutant of TRK-A. Entrectinib binds to the DFG-in conformation of the wild-type TRK-A, and the difluorobenzene group of the inhibitor closely matches and forms a van der Waals contact with the Ca atom of Gly667 (Fig.…”

Section: Docking Analysis Of Trk-a With or Without G667c Mutation Andmentioning

confidence: 81%

Foretinib Overcomes Entrectinib Resistance Associated with the NTRK1 G667C Mutation in NTRK1 Fusion–Positive Tumor Cells in a Brain Metastasis Model

Nishiyama

Yamada

Kita

et al. 2018

Clinical Cancer Research

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Purpose: Rearrangement of the neurotrophic tropomyosin receptor kinase 1 (NTRK1) gene, which encodes tyrosine receptor kinase A (TRK-A), occurs in various cancers, including colon cancer. Although entrectinib is effective in the treatment of central nervous system (CNS) metastases that express NTRK1 fusion proteins, acquired resistance inevitably results in recurrence. The CNS is a sanctuary for targeted drugs; however, the mechanism by which CNS metastases become entrectinib-resistant remains elusive and must be clarified to develop better therapeutics.Experimental Design: The entrectinib-resistant cell line KM12SM-ER was developed by continuous treatment with entrectinib in the brain metastasis-mimicking model inoculated with the entrectinib-sensitive human colon cancer cell line KM12SM, which harbors the TPM3-NTRK1 gene fusion. The mechanism of entrectinib resistance in KM12SM-ER cells was examined by nextgeneration sequencing. Compounds that overcame entrectinib resistance were screened from a library of 122 kinase inhibitors.Results: KM12SM-ER cells, which showed moderate resistance to entrectinib in vitro, had acquired the G667C mutation in NTRK1. The kinase inhibitor foretinib inhibited TRK-A phosphorylation and the viability of KM12SM-ER cells bearing the NTRK1-G667C mutation in vitro. Moreover, foretinib markedly inhibited the progression of entrectinib-refractory KM12SM-ER-derived liver metastases and brain tumors in animal models, predominantly through inhibition of TRK-A phosphorylation.Conclusions: These results suggest that foretinib may be effective in overcoming entrectinib resistance associated with the NTRK1-G667C mutation in NTRK1 fusion-positive tumors in various organs, including the brain, and provide a rationale for clinical trials of foretinib in cancer patients with entrectinibresistant tumors harboring the NTRK1-G667C mutation, including patients with brain metastases. Clin Cancer Res; 1-13. Ó2018AACR.

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“…Our previously reported crystal structure that determined the binding mode of dasatinib to EPHA2 (PDB ID: 5I9Y) established that the pyrimidine moiety of dasatinib is not engaged in EPHA2 binding. By docking experiments, we discovered that two other known EPHA2 inhibitors, CHEMBL249097 and PD‐173955 (Figure b), position a phenyl moiety in lieu of the pyrimidine (Supporting Information Figure 1). We therefore excluded hetero substitutions from this aromatic ring in our design.…”

Section: Resultsmentioning

confidence: 99%

“…In fact, dasatinib is the only drug which is already administered to patients in a number of clinical trials in order to assess the clinical value of EPHA2 inhibition in treating cancer (http://clinicaltrials.gov). Recently, we reported on 24 clinical kinase inhibitors that bind EPHA2 as an unintended off‐target which substantially expanded the landscape of known EPHA2 inhibitors . This collection of compounds should enable research to select more appropriate EPHA2 targeting compounds and foster drug repurposing of clinically evaluated molecules for application in EPHA2‐dependent diseases.…”

Section: Introductionmentioning

confidence: 99%

Chemoproteomics‐Aided Medicinal Chemistry for the Discovery of EPHA2 Inhibitors

et al. 2017

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The receptor tyrosine kinase EPHA2 has gained attention as a therapeutic drug target for cancer and infectious diseases. However, EPHA2 research and EPHA2-based therapies have been hampered by the lack of selective small-molecule inhibitors. Herein we report the synthesis and evaluation of dedicated EPHA2 inhibitors based on the clinical BCR-ABL/SRC inhibitor dasatinib as a lead structure. We designed hybrid structures of dasatinib and the previously known EPHA2 binders CHEMBL249097, PD-173955, and a known EPHB4 inhibitor in order to exploit both the ATP pocket entrance as well as the ribose pocket as binding epitopes in the kinase EPHA2. Medicinal chemistry and inhibitor design were guided by a chemical proteomics approach, allowing early selectivity profiling of the newly synthesized inhibitor candidates. Concomitant protein crystallography of 17 inhibitor co-crystals delivered detailed insight into the atomic interactions that underlie the structure-affinity relationship. Finally, the anti-proliferative effect of the inhibitor candidates was confirmed in the glioblastoma cell line SF-268. In this work, we thus discovered a novel EPHA2 inhibitor candidate that features an improved selectivity profile while maintaining potency against EPHA2 and anticancer activity in SF-268 cells.

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Chemical Proteomics and Structural Biology Define EPHA2 Inhibition by Clinical Kinase Drugs

Cited by 45 publications

References 53 publications

A Chemical Probe Strategy for Interrogating Inhibitor Selectivity Across the MEK Kinase Family

A Chemical Probe Strategy for Interrogating Inhibitor Selectivity Across the MEK Kinase Family

Foretinib Overcomes Entrectinib Resistance Associated with the NTRK1 G667C Mutation in NTRK1 Fusion–Positive Tumor Cells in a Brain Metastasis Model

Chemoproteomics‐Aided Medicinal Chemistry for the Discovery of EPHA2 Inhibitors

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