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Chemical Proteomics Reveals the Target Landscape of Clinical Kinase Inhibitors

Susan Kläger - March 2017

Protein kinases are key signaling molecules in the cell and catalyze the phosphate group transfer of ATP to their respective substrates. They have emerged as major drug target class, as they are often aberrant in diseases like cancer or inflammation. Small molecule kinase inhibitors provide one treatment option. Over 250 of these molecules are currently evaluated in clinical trials; over 30 have already been approved for human therapy. Most of them mimic ATP, thus targeting the ATP-binding pocket of kinases and preventing signal transduction via phosphate-transfer. As the ATP-binding pocket is quite conserved across the 518 protein kinases, many inhibitors can bind to more than one target protein. This polypharmacology can be advantageous and lead to the use of one drug in more than one indication. It might also lead to side effects and has influence on the mode of action of a drug. Therefore, thorough evaluation of the target space of a kinase inhibitor and its selectivity is necessary.

In this study, 242 small molecule inhibitors currently tested in clinical trials have been subjected to competitive Kinobeads profiling in a dose dependent manner followed by LC-MS/MS readout. The Kinobeads technology allows enrichment of over 300 kinases from cell or tissue lysate by binding of the ATP pocket. Competition with a free inhibitor for the ATP pocket leads to a dose dependent decrease of potential targets on the beads. Thus, drug-protein interaction profiles for each drug and all proteins bound by Kinobeads can be obtained. These allow determination of effective concentrations for half-maximal inhibition EC50 values and apparent binding constants. The selectivity of the investigated panel ranges from very selective drugs to unselective, multi-kinase inhibitors. Selective inhibition is desirable for unambiguous drug-protein interaction studies in basic research and thus, these inhibitors might be directly used as chemical probes, whereas more unselective inhibitors might be beneficial for the therapeutic success of a drug. Kinase inhibitor selectivity can be influenced by a number of factors. No major difference between inhibitors targeting the ‘DFG-in’ confirmation of kinases (type 1) and those binding the ‘DFG-out’ confirmation (type 2) inhibitors could be observed, whereas the allosteric (type 3) inhibitors could be confirmed as selective MAP2K1 and MAP2K2 drugs. Irreversible EGFR inhibitors were not necessarily more selective than their reversible counterparts. Reversible inhibitors can have no further additional targets; irreversible inhibitors are often more affine for EGFR than their off-targets. The observed additional off-targets of some drugs can explain adverse effects or generate rational hypotheses for drug repositioning. This work raises many opportunities for both cases. A set of six inhibitors was examined further for their additional NTRK1 inhibition in colon cancer and the approved MET-inhibitor Cabozantinib was evaluated in FLT3-ITD driven acute myeloid leukemia. Another important finding was the non-kinase off-target Ferrochelatase for 12% of all inhibitors. The protoporphyrin pocket of this enzyme could be determined as the binding site for some of these inhibitors and inhibition is likely linked to the side effect of photosensitivity in some patients receiving these inhibitors, as observed in Vemurafenib therapy.

To conclude, this work revealed the target landscape of small molecule inhibitors with the use of chemical proteomics. This thesis offers new insights into inhibitor selectivity and the druggable kinome. It can help to understand the molecular mode of actions of inhibitors and molecular reasons for side effects. Furthermore, new possibilities for drug repurposing as well as inhibitor design can be generated. It can be anticipated that this study will have impact on multiple disciplines like basic research, medicinal chemistry, cell biology, and medicine. 

Application of mass spectrometry-based proteomics to study cancer drug resistance mechanisms

Heiner Koch - October 2016

Resistance to small molecule kinase inhibitors appears within weeks to months and represents a major drawback in clinical cancer therapies. However, quantitative mass spectrometry became a powerful tool to characterize molecular changes on the proteome and protein modification level on a system-wide scale. In this thesis high resolution mass spectrometry was applied to characterize adaptive and growth factor mediated mechanisms that result in cancer drug resistance. 

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Development of phospho- and chemoproteomic methods to study cellular signaling

Benjamin Ruprecht - 20 May 2016

Kinase mediated protein phosphorylation on serine, threonine and tyrosine side chains is an important post-translational modification which affects and governs a large body of cellular signaling in health and disease. In recent years, mass spectrometry-based proteomics has emerged as the prime technology for the large scale, explorative and proteome-wide analysis of the kinome and associated phosphorylation events.

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Application of multivariate methods to the integrative analysis of high-throughput omics data

Chen Meng - 26 January 2016

More computational efforts are required to interpret biological knowledge from high-throughput omics data, such as genomics, transcriptomics and proteomics. Multivariate methods has shown great potential for this purpose, but the application of these methods to omics data analysis is still in its infancy. This thesis explored several applications of multivariate methods for the integrative analysis of multiple omics datasets, including exploratory analysis, clustering analysis and gene set analysis in an integrative manner. The results underscore the importance of analyzing multiple omics data sets in an integrative scheme. 

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Mass spectrometry based chemical proteomics for drug selectivity profiling

Dominic Helm - 13 July 2015

Mass spectrometry is the standard technology for chemical proteomics. Kinobead technology is an established tool in the field of chemical proteomics. The development of new mass spectrometry technologies is essential to advance the current performance of the study of kinase inhibitors. This thesis aimed to characterise a DIA approach for its use in a large scale chemical proteomics study. Further, the project focused on the development of a new ion mobility assisted DDA approach on a Q-TOF instrument and evaluated it for its application in bottom-up proteomics.
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Towards Comprehensive Identification of Proteins from MALDI imaging

Stefan Maier - 16 July 2014

MALDI imaging mass spectrometry is a powerful tool for the visualization of protein distributions in tissues. But as the molecular identity of masses detected by MALDI IMS often stays elusive the full potential of the technology cannot be unlocked. Based on the development of a method for the targeted isolation of the detectable proteins this thesis addresses the key issue of the field by combining bottom-up and top-down strategies for protein identification.
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Development and application of small molecule probes for kinase affinity purification and quantitative chemical proteomics

Xin Ku - 25 June 2014

Kinobeads technology using immobilized inhibitors has proven an efficient tool for kinase inhibitor profiling. This project aimed at developing new kinobead probes and hence extending the advantages of this technology to profile current and future kinase inhibitors. Known promiscuous kinase inhibitors were chemically modified to generate new kinobeads probes, which were then used to profile the clinical angiogenesis inhibitors.
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Computational Proteomics

Harald Marx - 11 June 2014

The key high-throughput technology to interrogate the proteome on a large scale is mass spectrometry based proteomics. To interpret the resulting experimental data, computational proteomics plays a critical role. The objective of this thesis was to develop novel approaches for database searching, in particular improve aspects of the theoretical search space and means to validate the results. The thesis concentrates on the construction process and composition of sequence databases and the subsequent statistical validation of peptide identifications and phosphorylation site localization.
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Quantitative chemical and phosphoproteomics for studying signaling in cancer

Fiona Pachl - 11 December 2013

Protein kinases are key regulators of major biological signaling processes in cell and a variety of diseases like cancer have been associated with deregulation of kinase activity. As a consequence, protein kinases are among the most intensively studied signaling molecules in pathophysiological biology and received considerable attention as therapeutic targets. This thesis addresses key issues of quantitative mass spectrometry to advance current chemoproteomic approaches to study kinase activity in cancer.
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Studies towards the proteome-wide detection, identification and quantification of protein glycosylation

Hannes Hahne - 5 November 2012

Glycosylation is one of the most abundant post-translational modifications of proteins and involved in virtually any cellular process. Mass spectrometry has evolved as key technology for the proteome-wide analysis of glycosylation. This thesis addressed key issues of tandem mass spectrometry to advance current proteomic and glycomic technologies for the system-wide analysis of O-GlcNAc proteins and N-linked glycosylation and demonstrate their utility using relevant biological models.
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Quantitative chemical proteomics for cancer characterization

Zhixiang Wu - 11 May 2012

Protein kinases are among the most intensively studied cellular signaling molecules in normal biology as well as many pathophysiological events, such as cancer. So far, numerous aberrant kinases have been revealed to be involved in most if not all the stages of tumorigenesis. Therefore, characterization of the cancer in a kinome-centric way may offer new insight of therapeutic invention. In this study, a kinase centric chemical proteomics assay (Kinobead) in conjunction with quantitative mass spectrometry is established and optimized, which enable quantitatively profiling around 150 kinases in parallel within 4% and 8% overall variance between technical and biological replicates respectively. First, it is employed to investigate 34 head and neck squamous cell carcinoma (HNSCC) cell lines resulting in 146 quantified kinases, of which 42 kinases showed statistically significant (p<0.05) expression inter-cell lines. Additional loss of function experiment using siRNA in high- and low- expressing cell lines further identified kinases including EGFR, EPHA2, LYN, JAK1, WEE1 and NEK9 involved in cell survival and proliferation. Among these, EGFR is confirmed as a drug target and EPHA2 is revealed to be novel drug target. Both contribute to around 20% and 15% of the HNSCC cell lines respectively. This notion is underscored by immunohistochemical analyses showing that high EGFR and EPHA2 expression is detected in a subset of HNSCC tissues. Downstream signaling pathway analysis suggests that EPHA2 promotes the cell proliferation via activating AKT and ERK signaling pathway. In addition the several significant candidates are the potential targets of the approved potent pan-SRC family kinase inhibitor dasatinib, which significantly reduces some but not all of HNSCC cell lines. These findings may lead to new therapeutic options for HNSCC patients. This may ultimately lead to a more rational approach to individualized cancer diagnosis and therapy. Second, together with a global approach, kinobead based profiling is applied to study the HSP90 dependent proteome, which enriches in signal transducer including many kinases, by investigating the protein response upon the HSP90 inhibition. Employing stable isotope labeling with amino acids in cell culture (SILAC) and quantitative mass spectrometry, >6,200 proteins are identified from four different human cell lines and ~1,600 proteins showed significant regulation upon drug treatment. Gene ontology and pathway/network analysis revealed common and cell type specific regulatory effects with strong connections to unfolded protein binding and protein kinase activity. Of the 288 identified protein kinases, 98 were downregulated (e.g. EGFR, BTK) and 17 up-regulated (e.g. AURA, AXL), in response to GA treatment, almost half of which are formerly unknown HSP90 client kinases. Furthermore pulsed-SILAC results suggested that protein down-regulation by HSP90 inhibition correlates with protein half life in many cases. Protein kinases show significantly shorter half lives than other proteins highlighting both challenges and opportunities for HSP90 inhibition in cancer therapy. The highly similar proteomic responses to the HSP90 drugs GA and PU-H71 suggest that both drugs work by similar molecular mechanisms. Several kinases (AXL, DDR1, TRIO) and other signaling proteins (BIRC6, ISG15, FLII) are validated as novel clients of HSP90 using HSP90 immunoprecipitation and affinity-based purification. Taken together, the strategy employed in this study is generic and therefore also of more general utility for the identification of novel drug targets and molecular pathway markers in tumors and broadly definition of the cellular proteome response to HSP90 inhibition provides a rich resource for further investigation relevant for the treatment of cancer.
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