Friedrich Kroll

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.

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.

Identification of Potential Off-target Toxicity Liabilities of Catechol-O-methyltransferase Inhibitors by Differential Competition Capture Compound Mass Spectrometry

Structurally related inhibitors of a shared therapeutic target may differ regarding potential toxicity issues that are caused by different off-target bindings. We devised a differential competition capture compound mass spectrometry (dCCMS) strategy to effectively differentiate off-target profiles. Tolcapone and entacapone are potent inhibitors of catechol-O-methyl transferase (COMT) for the treatment of Parkinsons disease. Tolcapone is also known for its hepatotoxic side effects even though it is therapeutically more potent than entacapone. Here, we identified 3-hydroxyisobutyryl-CoA hydrolase (HIBCH) as a possible toxicity-causing off-target of tolcapone, and this protein is not bound by the less toxic COMT inhibitor entacapone. Moreover, two novel compounds from a focused library synthesized in-house, N(2),N(2),N(3),N(3)-tetraethyl-6,7-dihydroxy-5-nitronaphthalene-2,3-dicarboxamide and 5-(3,4-dihydroxy-5-nitrobenzylidene)-3-ethylthiazolidine-2,4-dione, were utilized to gain insight into the structure-activity relationships in binding to COMT and the novel off-target HIBCH. These compounds, especially N(2),N(2),N(3),N(3)-tetraethyl-6,7-dihydroxy-5-nitronaphthalene-2,3-dicarboxamide, could serve as starting point for the development of improved and more specific COMT inhibitors.

Targeting G Protein‐Coupled Receptors by Capture Compound Mass Spectrometry: A Case Study with Sertindole

Unbiased chemoproteomic profiling of small‐molecule interactions with endogenous proteins is important for drug discovery. For meaningful results, all protein classes have to be tractable, including G protein‐coupled receptors (GPCRs). These receptors are hardly tractable by affinity pulldown from lysates. We report a capture compound (CC)‐based strategy to target and identify GPCRs directly from living cells. We synthesized CCs with sertindole attached to the CC scaffold in different orientations to target the dopamine D2 receptor (DRD2) heterologously expressed in HEK 293 cells. The structure–activity relationship of sertindole for DRD2 binding was reflected in the activities of the sertindole CCs in radioligand displacement, cell‐based assays, and capture compound mass spectrometry (CCMS). The activity pattern was rationalized by molecular modelling. The most‐active CC showed activities very similar to that of unmodified sertindole. A concentration of DRD2 in living cells well below 100 fmol used as an experimental input was sufficient for unambiguous identification of captured DRD2 by mass spectrometry. Our new CCMS workflow broadens the arsenal of chemoproteomic technologies to close a critical gap for the comprehensive characterization of drug–protein interactions.