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Chemical proteomics and structural biology define EPHA2 inhibition by clinical kinase drugs Stephanie Heinzlmeir, Denis Kudlinzki, Sridhar Sreeramulu, Susan Klaeger, Santosh Lakshmi Gande, Verena Linhard, Mathias Wilhelm, Huichao Qiao, Dominic Helm, Benjamin Ruprecht, Krishna Saxena, Guillaume Médard, Harald Schwalbe, and Bernhard Kuster ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.6b00709 • Publication Date (Web): 21 Oct 2016 Downloaded from http://pubs.acs.org on October 24, 2016
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Munchen Fakultat Wissenschaftszentrum Weihenstephan fur Ernahrung Landnutzung und Umwelt, Bavarian Biomolecular Mass Spectrometry Center
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Chemical proteomics and structural biology define EPHA2 inhibition by clinical kinase drugs
Authors
Stephanie Heinzlmeir1,3,4,#, Denis Kudlinzki2,3,4,#, Sridhar Sreeramulu2, Susan Klaeger1,3,4, Santosh Lakshmi Gande2,3,4, Verena Linhard2, Mathias Wilhelm1, Huichao Qiao1, Dominic Helm1, Benjamin Ruprecht1, Krishna Saxena2,3,4, Guillaume Médard1, Harald Schwalbe2,3,4,*, Bernhard Kuster1,3,4,5,6,*
1
Chair of Proteomics and Bioanalytics, Technical University of Munich, 85354 Freising,
Germany 2
Center for Biomolecular Magnetic Resonance, Johann Wolfgang Goethe-University, 60438
Frankfurt, Germany 3
German Cancer Consortium (DKTK), 69120 Heidelberg, Germany
4
German Cancer Research Center (DKFZ), 69120 Heidelberg, Germany
5
Center for Protein Science Munich (CIPSM), 85354 Freising, Germany
6
Bavarian Biomolecular Mass Spectrometry Center, Technical University of Munich, 85354
Freising, Germany #
Co-first authors
* Correspondence:
[email protected],
[email protected] ACS Paragon Plus Environment
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ABSTRACT The receptor tyrosine kinase EPHA2 (Ephrin type-A receptor 2) plays important roles in oncogenesis, metastasis and treatment resistance yet therapeutic targeting, drug discovery or investigation of EPHA2 biology is hampered by the lack of appropriate inhibitors and structural information. Here, we used chemical proteomics to survey 235 clinical kinase inhibitors for their kinase selectivity and identified 24 drugs with sub-micromolar affinities for EPHA2. NMR-based conformational dynamics together with nine new co-crystal structures delineated drug-EPHA2 interactions in full detail. The combination of selectivity profiling, structure determination and kinome wide sequence alignment allowed the development of a classification system in which amino acids in the drug binding site of EPHA2 are categorized into key, scaffold, potency and selectivity residues. This scheme should be generally applicable in kinase drug discovery and we anticipate that the provided information will greatly facilitate the development of selective EPHA2 inhibitors in particular and the repurposing of clinical kinase inhibitors in general.
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The ephrin type-A receptor 2 (EPHA2) belongs to the largest family of receptor tyrosine kinases and is emerging as a potential therapeutic target 1, 2. It was found to be overexpressed in several cancer entities (e.g. breast 3, head and neck 4, non-small cell lung cancer 5) and altered expression correlated with increased malignancy and poor prognosis 5, 6. The molecular signaling of EPHA2 is complex; the protein can act in a ligand-dependent or independent 7 as well as kinase-dependent or -independent fashion 7, 8. For example, the binding of ephrin-A1 ligands to clustered EPHA2 receptors leads to autophosphorylation and subsequent internalization, thereby mediating tumor-suppressive effects owing to the abrogation of RAS and AKT oncogenic signaling. In contrast, ligand-independent signaling induces a reciprocal regulatory feedback loop involving the AKT kinase and leading to an oncogenic phenotype due to activation of the RAS/ERK and PI3K/AKT signaling pathways 7. This interplay can eventually result in epithelial-mesenchymal transition and progression to a more malignant invasive tumor phenotype 9. EPHA2 has also been implicated in the development of resistance against small molecule 9, 10 or antibody-based cancer therapies 11. In a previous study, we showed that EPHA2 overexpression can confer resistance against the EGFR drug Gefitinib in a non-small lung cancer cell line 12, which was recently confirmed for another EGFR inhibitor, Erlotinib 13. The potential clinical benefits of utilizing small molecule EPHA2 inhibitors are controversially discussed in the field. This is not surprising, given the complexity and the contextual dependencies of EPHA2 biology. However, a number of studies and clinical trials suggest that small molecule EPHA2 inhibitors could be used to suppress pro-oncogenic pathways such as RAS/ERK signaling 14, 15. Drug discovery specifically targeting the EPHA2 kinase domain is in its infancy and very few designated EPHA2 inhibitors have been reported in the literature 16. However, EPHA2 inhibition has been observed as an off-target effect for a number of ATPcompetitive kinase inhibitors, notably the leukemia drug Dasatinib 15, 16. On this basis, we reasoned that a systematic survey of clinically used kinase inhibitors for off-target binding to EPHA2 could serve to identify high value molecules with optimized pharmacological profiles for
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use as starting points to develop chemical probes to study EPHA2 biology, as lead compounds for medicinal chemistry directed against EPHA2 or for re-purposing existing drugs to treat EPHA2 dependent pathologies. To further substantiate this concept, we used a chemical proteomic approach, notably the Kinobeads technology 17, 18, to determine the selectivity of 235 approved or clinically evaluated small molecule kinase inhibitors and identified 24 sub-micromolar EPHA2 inhibitors in this way and characterized their kinase target space in the same experiment. We also obtained, for the first time, high resolution co-crystal structures of nine clinical and tool compounds in complex with EPHA2 to determine the structural basis for kinase inhibition. The combination of target selectivity profiling, structural biology and kinome wide sequence alignments enabled us to develop a classification system in which amino acids in the drug binding site are categorized into key, scaffold, potency and selectivity residues. This scheme should be generally applicable in kinase drug discovery and we anticipate that the provided information will facilitate the development of selective EPHA2 inhibitors in particular and the repurposing of clinical kinase inhibitors in general.
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RESULTS AND DISCUSSION
Chemical proteomics reveals EPHA2 as a common off-target of clinical kinase inhibitors. In order to identify EPHA2 inhibitors, we used the Kinobeads technology to determine the target space and binding affinities of 235 clinical small molecule kinase inhibitors (Table S1a) and three tool compounds known to target EPH family proteins (Cpd66, LDN-211904, PD-173955) 17-19. The clinical kinase inhibitors that were tested are approved drugs or are administered to humans as part of clinical trials (phase 1-3) and therefore provide a highly optimized pharmacological profile. In this chemical proteomic approach, unselective kinase inhibitors are immobilized on beads enabling the enrichment of protein kinases and other proteins from complex native lysates. For our experiments, mixed lysates of MV-4-11, SK-N-BE(2), COLO 205, and K-562 cells were treated with increasing concentrations of a test compound followed by Kinobeads affinity enrichment. Mass spectrometry was then used to identify and quantify bead bound proteins as a function of the applied test compound dose, including more than 300 out of the 518 human kinases (for more detailed information see Figure S1 and supplementary methods). Although none of the clinical drugs tested was originally intended to target EPHA2, we found that 32 molecules showed inhibition of EPHA2 binding to Kinobeads with apparent affinities ranging in KDapp values from 2.8 nM to 6.1 µM (Figure 1a affinities given as pKDapp values: –log10(KDapp); Table 1). Twenty four clinical inhibitors and the three tool compounds showed sub-micromolar KDapp values and, surprisingly, twelve of these compounds had not been previously known to target EPHA2. Dasatinib (2.8 nM), Ponatinib (6.9 nM) and Danusertib (9.0 nM) showed the highest affinity in our screen. Fifteen inhibitors were validated in a HotSpot kinase activity assay, confirming that molecules binding to Kinobeads also showed inhibition of EPHA2 enzymatic activity (Table 1). As far as described in the literature, the identified set of EPHA2 inhibitors comprise ATP competitive type 1 and type 2 inhibitors providing a rich set of molecular features for the analysis of the chemical landscape of EPHA2 inhibition (see Table S1a–b for more
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detailed information about the inhibitor set; and Table S1c–d and Supplementary Data S1 for binding data and respective binding curves).
Target space elucidation of EPHA2 inhibitors allows for rationalized drug repurposing. A Kinobeads experiment determines the range of kinases that can be bound by a test compound and thus provides information about its selectivity. Collectively, the 27 EPHA2 inhibitors targeted 133 protein kinases (KDapp
