Jenny J. Fischer

Probing small molecule–protein interactions: A new perspective for functional proteomics

The isolation of proteome subsets on the basis of the interactions of small molecules with proteins is an emerging paradigm in proteomics. Depending on the nature of the small molecule used as a bait, entire protein families can be monitored in biological samples, or new functions can be attributed to previously uncharacterized proteins. With pharmaceutical compounds as baits, drug targets and toxicity-relevant off-targets can be discovered in an unbiased proteomic screen. At the heart of this strategy are synthetic bi- or trifunctional small molecule probes. These probes carry the small molecules of interest as baits (selectivity function), as well as a sorting function for the isolation of small molecule-protein complexes or conjugates from complex protein mixtures. In some designs, a covalent linkage of the bound protein to the probe is established through a separate reactivity function or a combined selectivity/reactivity function. The covalent linkage allows for isolation or detection of probe-protein conjugates also under harsh or denaturing conditions. Ultimately, specifically isolated proteins are commonly identified by mass spectrometry. This review summarizes probe designs, workflows, and published applications of the three dominant approaches in the field, namely affinity pulldown, activity-based protein profiling, and Capture Compound Mass Spectrometry.

Capture Compound Mass Spectrometry Sheds Light on the Molecular Mechanisms of Liver Toxicity of Two Parkinson Drugs

Capture compound mass spectrometry (CCMS) is a novel technology that helps understand the molecular mechanism of the mode of action of small molecules. The Capture Compounds are trifunctional probes: A selectivity function (the drug) interacts with the proteins in a biological sample, a reactivity function (phenylazide) irreversibly forms a covalent bond, and a sorting function (biotin) allows the captured protein(s) to be isolated for mass spectrometric analysis. Tolcapone and entacapone are potent inhibitors of catechol-O-methyltransferase (COMT) for the treatment of Parkinsons disease. We aimed to understand the molecular basis of the difference of both drugs with respect to side effects. Using Capture Compounds with these drugs as selectivity functions, we were able to unambiguously and reproducibly isolate and identify their known target COMT. Tolcapone Capture Compounds captured five times more proteins than entacapone Capture Compounds. Moreover, tolcapone Capture Compounds isolated mitochondrial and peroxisomal proteins. The major tolcapone-protein interactions occurred with components of the respiratory chain and of the fatty acid beta-oxidation. Previously reported symptoms in tolcapone-treated rats suggested that tolcapone might act as decoupling reagent of the respiratory chain (Haasio et al., 2002b). Our results demonstrate that CCMS is an effective tool for the identification of a drugs potential off targets. It fills a gap in currently used in vitro screens for drug profiling that do not contain all the toxicologically relevant proteins. Thereby, CCMS has the potential to fill a technological need in drug safety assessment and helps reengineer or to reject drugs at an early preclinical stage.

Dasatinib, imatinib and staurosporine capture compounds - Complementary tools for the profiling of kinases by Capture Compound Mass Spectrometry (CCMS).

Capture Compound Mass Spectrometry (CCMS) is a platform technology for the functional isolation of subproteomes. Here we report the synthesis of two new kinase Capture Compounds (CCs) based on the tyrosine-kinase specific inhibitors dasatinib and imatinib and compare their interaction profiles to that of our previously reported staurosporine-CCs. CCs are tri-functional molecules: they comprise a sorting function (e.g. the small molecule or drug of interest) which interacts with target proteins, a photo-activatable reactivity function to covalently trap the interacting proteins, and a sorting function to isolate the CC-protein conjugates from complex biological samples for protein identification by liquid chromatography/mass spectrometry (LC-MS/MS). We present data of CCMS experiments from human HepG2 cells and compare the profiles of the kinases isolated with dasatinib, imatinib and staurosporine CC, respectively. Dasatinib and imatinib have a more selective kinase binding profile than staurosporine. Moreover, the new CCs allow isolation and identification of additional kinases, complementing the staurosporine CC. The family of kinase CCs will be a valuable tool for the proteomic profiling of this important protein class. Besides sets of expected kinases we identified additional specific interactors; these off-targets may be of relevance in the view of the pharmacological profile of dasatinib and imatinib.

SAHA Capture Compound - A novel tool for the profiling of histone deacetylases and the identification of additional vorinostat binders

Suberoylanilide hydroxamic acid (SAHA) is a potent histone deacetylase (HDAC) inhibitor. Inhibitors of HDACs are used in cancer therapy based on the role HDACs play in transcription by regulating chromatin compaction and non‐histone proteins such as transcription factors. Profiling of HDAC expression is of interest in the functional proteomics analysis of cancer. Also, non‐HDAC proteins may interact with HDAC inhibitor drugs and contribute to the drug mode of action. We here present a tool for the unbiased chemical proteomic profiling of proteins that specifically interact with SAHA. We designed and synthesized a trifunctional Capture Compound containing SAHA as selectivity and identified HDACs1, 2, 3 and 6, known and predicted HDAC interactors from human‐derived HepG2 cell lysate, as well as a set of new potential non‐HDAC targets of SAHA. One of these non‐HDAC targets, isochorismatase domain‐containing protein 2 (ISOC2) is putative hydrolase associated with the negative regulation of the tumor‐suppressor p16(INK4a). We demonstrated the direct and dose‐dependent interaction of SAHA to the purified recombinant ISOC2 protein. Using SAHA Capture Compound mass spectrometry, we thus identified potential new SAHA target proteins in an entirely unbiased chemical proteomics approach.

GDP-capture compound--a novel tool for the profiling of GTPases in pro- and eukaryotes by capture compound mass spectrometry (CCMS).

The functional isolation of proteome subsets based on small molecule-protein interactions is an increasingly popular and promising field in functional proteomics. Entire protein families may be profiled on the basis of their common interaction with a metabolite or small molecule inhibitor. This is enabled by novel multifunctional small molecule probes. One platform approach in this field are Capture Compounds that contain a small molecule of interest to bind target proteins, a photo-activatable reactivity function to covalently trap bound proteins, and a sorting function to isolate Capture Compound-protein conjugates from complex biological samples for direct trypsinisation and protein identification by liquid chromatography/mass spectrometry (CCMS). We here present the synthesis and application of a novel GDP-Capture Compound for the functional enrichment of GTPases, a pivotal protein family that exerts key functions in signal transduction. We present data from CCMS experiments on two biological lysates from Escherichia coli and from human-derived Hek293 cells. The GDP-Capture Compound robustly captures a wide range of different GTPases from both systems and will be a valuable tool for the proteomic profiling of this important protein family.

Dabigatran and dabigatran ethyl ester: potent inhibitors of ribosyldihydronicotinamide dehydrogenase (NQO2).

Recent studies have revealed that compounds believed to be highly selective frequently address multiple target proteins. We investigated the protein interaction profile of the widely prescribed thrombin inhibitor dabigatran (1), resulting in the identification and subsequent characterization of an additional target enzyme. Our findings are based on an unbiased functional proteomics approach called capture compound mass spectrometry (CCMS) and were confirmed by independent biological assays. 1 was shown to specifically bind ribosyldihydronicotinamide dehydrogenase (NQO2), a detoxification oxidoreductase. Molecular dockings predicted and biological experiments confirmed that dabigatran ethyl ester (2) inhibits NQO2 even more effectively than the parent 1 itself. Our data show that 1 and 2 are inhibitors of NQO2, thereby revealing a possible new aspect in the mode of action of 1. We present a workflow employing chemical proteomics, molecular modeling, and functional assays by which a compounds protein-interaction profile can be determined and used to tune the binding affinity.

Improvement of Capture Compound Mass Spectrometry Technology (CCMS) for the Profiling of Human Kinases by Combination with 2D LC-MS/MS

An increasingly popular and promising field in functional proteomics is the isolation of proteome subsets based on small molecule-protein interactions. One platform approach in this field are Capture Compounds that contain a small molecule of interest to bind target proteins, a photo-activatable reactivity function to covalently trap bound proteins, and a sorting function to isolate captured protein conjugates from complex biological samples for direct protein identification by liquid chromatography/mass spectrometry (nLC-MS/MS). In this study we used staurosporine as a selectivity group for analysis in HepG2 cells derived from human liver. In the present study, we combined the functional isolation of kinases with different separation workflows of automated split-free nanoflow liquid chromatography prior to mass spectrometric analysis. Two different CCMS setups, CCMS technology combined with 1D LC-MS and 2D LC-MS, were compared regarding the total number of kinase identifications. By extending the chromatographic separation of the tryptic digested captured proteins from 1D LC linear gradients to 2D LC we were able to identify 97 kinases. This result is similar to the 1D LC setup we previously reported but this time 4 times less input material was needed. This makes CCMS of kinases an even more powerful tool for the proteomic profiling of this important protein family.

Proteome-Wide Identification of Staurosporine-Binding Kinases Using Capture Compound Mass Spectrometry

The enormous diversity of kinases and their pivotal role in cell signaling have set kinases in the focus of biomedical research. Profiling the kinome of tissues of different origin is essential for biomarker discovery. In drug research, it is necessary to comprehend the specificity profile of a given kinase inhibitor. Capture Compound Mass Spectrometry (CCMS) (Koster et al., Assay Drug. Dev. Technol. 5:381-390, 2007) addresses the need for a tool to physically isolate and reliably profile the binders of kinase inhibitors directly in biological samples. Capture Compounds™ are trifunctional probes: a selectivity function consisting of the kinase inhibitor interacts reversibly with the native target proteins in equilibrium, a photoactivatable reactivity function forms an irreversible covalent bond to the target protein upon irradiation, and a sorting function allows the captured protein(s) to be isolated and identified by mass spectrometric analysis in an affinity-driven manner. Capture Compounds™ with any kinase inhibitor as selectivity function can be synthesized. We here used staurosporine as the selectivity function because it targets and, therefore, allows profiling a broad range of kinases (Romano and Giordano, Cell Cycle 7:3364-3668, 2008). Furthermore, we give an example of the application of the staurosporine Capture Compound to isolate kinases from human liver-derived HepG2 cells.