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 compound's protein-interaction profile can be determined and used to tune the binding affinity.

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.

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.