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1.
J Med Chem ; 59(10): 4664-75, 2016 05 26.
Artigo em Inglês | MEDLINE | ID: mdl-27074629

RESUMO

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 Parkinson's 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.


Assuntos
Inibidores de Catecol O-Metiltransferase/farmacologia , Catecol O-Metiltransferase/metabolismo , Inibidores de Catecol O-Metiltransferase/síntese química , Inibidores de Catecol O-Metiltransferase/química , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Células Hep G2 , Humanos , Espectrometria de Massas , Modelos Moleculares , Estrutura Molecular , Relação Estrutura-Atividade
2.
J Med Chem ; 55(8): 3934-44, 2012 Apr 26.
Artigo em Inglês | MEDLINE | ID: mdl-22494098

RESUMO

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.


Assuntos
Benzimidazóis/farmacologia , Inibidores Enzimáticos/farmacologia , Piridinas/farmacologia , Quinona Redutases/antagonistas & inibidores , beta-Alanina/análogos & derivados , Anticoagulantes/farmacologia , Benzimidazóis/química , Dabigatrana , Inibidores Enzimáticos/química , Células Hep G2 , Humanos , Células K562 , Espectrometria de Massas , Modelos Químicos , Ligação Proteica , Proteômica/métodos , Piridinas/química , Trombina/antagonistas & inibidores , beta-Alanina/química , beta-Alanina/farmacologia
3.
Proteomics ; 11(20): 4096-104, 2011 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-21898820

RESUMO

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.


Assuntos
Inibidores de Histona Desacetilases/química , Histona Desacetilases/química , Histona Desacetilases/metabolismo , Ácidos Hidroxâmicos/química , Proteômica/métodos , Células Cultivadas , Relação Dose-Resposta a Droga , Sistemas de Liberação de Medicamentos , Desenho de Fármacos , Humanos , Modelos Moleculares , Estrutura Molecular , Vorinostat
4.
J Biomed Biotechnol ; 2011: 850589, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21941435

RESUMO

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.


Assuntos
Cromatografia Líquida/métodos , Fosfotransferases/isolamento & purificação , Proteômica/métodos , Espectrometria de Massas em Tandem/métodos , Células Hep G2 , Humanos , Modelos Moleculares , Fragmentos de Peptídeos/química , Fosfotransferases/química , Fosfotransferases/classificação , Estaurosporina/química
5.
J Proteomics ; 75(1): 160-8, 2011 Dec 10.
Artigo em Inglês | MEDLINE | ID: mdl-21664307

RESUMO

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.


Assuntos
Perfilação da Expressão Gênica/métodos , Piperazinas/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Quinases/metabolismo , Proteômica/métodos , Pirimidinas/farmacologia , Estaurosporina/farmacologia , Tiazóis/farmacologia , Benzamidas , Cromatografia Líquida/métodos , Dasatinibe , Células Hep G2 , Humanos , Mesilato de Imatinib , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Espectrometria de Massas/métodos , Piperazinas/química , Inibidores de Proteínas Quinases/química , Proteínas Quinases/genética , Pirimidinas/química , Estaurosporina/química , Tiazóis/química
6.
Toxicol Sci ; 113(1): 243-53, 2010 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-19783845

RESUMO

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 Parkinson's 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 drug's 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.


Assuntos
Antiparkinsonianos/toxicidade , Benzofenonas/toxicidade , Inibidores de Catecol O-Metiltransferase , Catecóis/toxicidade , Doença Hepática Induzida por Substâncias e Drogas/etiologia , Inibidores Enzimáticos/toxicidade , Fígado/efeitos dos fármacos , Espectrometria de Massas , Nitrilas/toxicidade , Nitrofenóis/toxicidade , Testes de Toxicidade/métodos , Animais , Antiparkinsonianos/química , Benzofenonas/química , Catecol O-Metiltransferase/metabolismo , Catecóis/química , Doença Hepática Induzida por Substâncias e Drogas/enzimologia , Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Desenho Assistido por Computador , Transporte de Elétrons , Inibidores Enzimáticos/química , Ácidos Graxos/metabolismo , Células Hep G2 , Humanos , Fígado/enzimologia , Fígado/metabolismo , Microssomos Hepáticos/metabolismo , Mitocôndrias Hepáticas/efeitos dos fármacos , Mitocôndrias Hepáticas/metabolismo , Proteínas Mitocondriais/metabolismo , Modelos Moleculares , Estrutura Molecular , Nitrilas/química , Nitrofenóis/química , Oxirredução , Fosforilação Oxidativa , Peroxissomos/efeitos dos fármacos , Peroxissomos/metabolismo , Ratos , Reprodutibilidade dos Testes , Tolcapona
7.
J Proteomics ; 73(4): 815-9, 2010 Feb 10.
Artigo em Inglês | MEDLINE | ID: mdl-20026263

RESUMO

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.


Assuntos
Células Eucarióticas/enzimologia , GTP Fosfo-Hidrolases/análise , GTP Fosfo-Hidrolases/química , Guanosina Difosfato/química , Células Procarióticas/enzimologia , Extratos Celulares , Linhagem Celular , Cromatografia Líquida , Bases de Dados de Proteínas , Eletroforese em Gel de Poliacrilamida , Proteínas de Escherichia coli/análise , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/isolamento & purificação , Proteínas de Escherichia coli/metabolismo , GTP Fosfo-Hidrolases/isolamento & purificação , GTP Fosfo-Hidrolases/metabolismo , Guanosina Difosfato/metabolismo , Humanos , Espectrometria de Massas , Proteômica , Tripsina/farmacologia
8.
J Proteome Res ; 9(2): 806-17, 2010 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-20028079

RESUMO

The central role of kinases in cell signaling has set them in the focus of biomedical research. In functional proteomics analyses, large- scale profiling of kinases has become feasible through the use of affinity pulldown beads that carry immobilized kinase inhibitors. As an alternative approach to solid phase beads, Capture Compound Mass Spectrometry (CCMS) enables the functional isolation of protein-classes on the basis of small molecule-protein interactions in solution. Capture Compounds are trifunctional probes: a selectivity function interacts with the native target proteins in equilibrium, upon irradiation a photoactivatable reactivity function forms an irreversible covalent bond to the target proteins, and a sorting function allows the captured proteins to be isolated from a complex protein mixture. We report the design and application of a novel, fully water-soluble Capture Compound that carries the broadband kinase inhibitor staurosporine as selectivity function. We used this Capture Compound to profile the kinome of the human liver-derived cell line HepG2 and identified one hundred kinases. HepG2 cells are a widely used model system for hepatocarcinoma, hepatitis, and for investigation of drug toxicity effects. CCMS experiments in membrane fractions of human placenta are given as example for the applicability to human tissue.


Assuntos
Hepatócitos/efeitos dos fármacos , Espectrometria de Massas/métodos , Fosfotransferases/metabolismo , Estaurosporina/farmacologia , Linhagem Celular , Eletroforese em Gel de Poliacrilamida , Hepatócitos/citologia , Hepatócitos/metabolismo , Humanos , Modelos Moleculares , Estaurosporina/metabolismo
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