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1.
J Med Chem ; 62(22): 10321-10341, 2019 11 27.
Artigo em Inglês | MEDLINE | ID: mdl-31670515

RESUMO

The human luteinizing hormone receptor (hLH-R) is a member of the glycoprotein hormone family of G-protein-coupled receptors (GPCRs), activated by luteinizing hormone (hLH) and essentially involved in the regulation of sex hormone production. Thus, hLH-R represents a valid target for the treatment of sex hormone-dependent cancers and diseases (polycystic ovary syndrome, uterine fibroids, endometriosis) as well as contraception. Screening of the Bayer compound library led to the discovery of tetrahydrothienopyridine derivatives as novel, small-molecule (SMOL) hLH-R inhibitors and to the development of BAY-298, the first nanomolar hLH-R antagonist reducing sex hormone levels in vivo. Further optimization of physicochemical, pharmacokinetic, and safety parameters led to the identification of BAY-899 with an improved in vitro profile and proven efficacy in vivo. BAY-298 and BAY-899 serve as valuable tool compounds to study hLH-R signaling in vitro and to interfere with the production of sex hormones in vivo.


Assuntos
Estradiol/sangue , Naftiridinas/química , Receptores do LH/antagonistas & inibidores , Administração Oral , Animais , Disponibilidade Biológica , Relação Dose-Resposta a Droga , Canal de Potássio ERG1/metabolismo , Feminino , Células da Granulosa/efeitos dos fármacos , Ensaios de Triagem em Larga Escala , Humanos , Masculino , Camundongos , Microssomos Hepáticos/efeitos dos fármacos , Ovulação/efeitos dos fármacos , Ovulação/genética , Progesterona/sangue , Ratos Wistar , Receptores do FSH/antagonistas & inibidores , Receptores do LH/metabolismo , Relação Estrutura-Atividade , Testosterona/sangue
2.
Nucleic Acids Res ; 46(W1): W257-W263, 2018 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-29718510

RESUMO

Advancement in the field of computational research has made it possible for the in silico methods to offer significant benefits to both regulatory needs and requirements for risk assessments, and pharmaceutical industry to assess the safety profile of a chemical. Here, we present ProTox-II that incorporates molecular similarity, pharmacophores, fragment propensities and machine-learning models for the prediction of various toxicity endpoints; such as acute toxicity, hepatotoxicity, cytotoxicity, carcinogenicity, mutagenicity, immunotoxicity, adverse outcomes pathways (Tox21) and toxicity targets. The predictive models are built on data from both in vitro assays (e.g. Tox21 assays, Ames bacterial mutation assays, hepG2 cytotoxicity assays, Immunotoxicity assays) and in vivo cases (e.g. carcinogenicity, hepatotoxicity). The models have been validated on independent external sets and have shown strong performance. ProTox-II provides a freely available webserver for in silico toxicity prediction for toxicologists, regulatory agencies, computational and medicinal chemists, and all users without login at http://tox.charite.de/protox_II. The webserver takes a two-dimensional chemical structure as an input and reports the possible toxicity profile of the chemical for 33 models with confidence scores, and an overall toxicity radar chart along with three most similar compounds with known acute toxicity.


Assuntos
Biologia Computacional , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos , Software , Humanos , Internet , Aprendizado de Máquina , Medição de Risco
3.
Food Chem Toxicol ; 107(Pt A): 150-166, 2017 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-28558974

RESUMO

Immunotoxicity, defined as adverse effects of xenobiotics on the immune system, is gaining increasing attention in the approval process of industrial chemicals and drugs. In-vivo and ex-vivo experiments have been the gold standard in immunotoxicity assessment so far, so the development of in-vitro and in-silico alternatives is an important issue. In this paper we describe a widely applicable, easy-to use computational approach which can serve as an initial immunotoxicity screen of new chemical entities. Molecular fingerprints describing chemical structure were used as parameters in a machine-learning approach based on the Naïve-Bayes learning algorithm. The model was trained using blood-cell growth inhibition data from the NCI database and validated externally with several in-house and literature-derived data sets tested in cytotoxicity assays on different types on immune cells. Both cross-validations and external validations resulted in areas under the receiver operator curves (ROC/AUC) of 75% or higher. The classification of the validation data sets occurred with excellent specificities and fair to excellent selectivities, depending on the data set. This means that the probability of actual immunotoxicity is very high for compounds classified as immunotoxic, while the fraction of false negative predictions might vary. Thus, in a multistep immunotoxicity screening scheme, the classification as immunotoxic can be accepted without additional confirmation, while compounds classified as not immunotoxic will have to be subjected to further investigation.


Assuntos
Linfócitos B/efeitos dos fármacos , Biologia Computacional/métodos , Linfócitos T/efeitos dos fármacos , Xenobióticos/toxicidade , Linfócitos B/imunologia , Humanos , Sistema Imunitário/citologia , Sistema Imunitário/efeitos dos fármacos , Sistema Imunitário/imunologia , Linfócitos T/imunologia
4.
Chembiochem ; 18(16): 1639-1649, 2017 08 17.
Artigo em Inglês | MEDLINE | ID: mdl-28557180

RESUMO

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.


Assuntos
Antagonistas dos Receptores de Dopamina D2/química , Imidazóis/química , Indóis/química , Receptores de Dopamina D2/análise , Animais , Antagonistas dos Receptores de Dopamina D2/síntese química , Antagonistas dos Receptores de Dopamina D2/efeitos da radiação , Células HEK293 , Humanos , Imidazóis/síntese química , Imidazóis/efeitos da radiação , Indóis/síntese química , Indóis/efeitos da radiação , Ligantes , Simulação de Acoplamento Molecular , Ensaio Radioligante , Ratos , Receptores de Dopamina D2/efeitos da radiação , Espiperona/química , Relação Estrutura-Atividade , Suínos , Espectrometria de Massas em Tandem , Raios Ultravioleta
5.
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
6.
Anal Biochem ; 467: 14-21, 2014 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-25172130

RESUMO

S-Adenosyl-l-methionine (SAM) is recognized as an important cofactor in a variety of biochemical reactions. As more proteins and pathways that require SAM are discovered, it is important to establish a method to quickly identify and characterize SAM binding proteins. The affinity of S-adenosyl-l-homocysteine (SAH) for SAM binding proteins was used to design two SAH-derived capture compounds (CCs). We demonstrate interactions of the proteins COMT and SAHH with SAH-CC with biotin used in conjunction with streptavidin-horseradish peroxidase. After demonstrating SAH-dependent photo-crosslinking of the CC to these proteins, we used a CC labeled with a fluorescein tag to measure binding affinity via fluorescence anisotropy. We then used this approach to show and characterize binding of SAM to the PR domain of PRDM2, a lysine methyltransferase with putative tumor suppressor activity. We calculated the Kd values for COMT, SAHH, and PRDM2 (24.1 ± 2.2 µM, 6.0 ± 2.9 µM, and 10.06 ± 2.87 µM, respectively) and found them to be close to previously established Kd values of other SAM binding proteins. Here, we present new methods to discover and characterize SAM and SAH binding proteins using fluorescent CCs.


Assuntos
Catecol O-Metiltransferase/análise , Proteínas de Ligação a DNA/análise , Polarização de Fluorescência/métodos , Histona-Lisina N-Metiltransferase/análise , Hidrolases/análise , Proteínas Nucleares/análise , S-Adenosil-Homocisteína/metabolismo , S-Adenosilmetionina/metabolismo , Fatores de Transcrição/análise , Catecol O-Metiltransferase/metabolismo , Proteínas de Ligação a DNA/metabolismo , Histona-Lisina N-Metiltransferase/metabolismo , Humanos , Hidrolases/metabolismo , Proteínas Nucleares/metabolismo , Fatores de Transcrição/metabolismo
7.
PLoS One ; 7(11): e50350, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-23209719

RESUMO

The primary gestagen of elephants is 5α-dihydroprogesterone (DHP), which is unlike all other mammals studied until now. The level of DHP in elephants equals that of progesterone in other mammals, and elephants are able to bind DHP with similar affinity to progesterone indicating a unique ligand-binding specificity of the elephant progestin receptor (PR). Using site-directed mutagenesis in combination with in vitro binding studies we here report that this change in specificity is due to a single glycine to alanine exchange at position 722 (G722A) of PR, which specifically increases DHP affinity while not affecting binding of progesterone. By conducting molecular dynamics simulations comparing human and elephant PR ligand-binding domains (LBD), we observed that the alanine methyl group at position 722 is able to push the DHP A-ring into a position similar to progesterone. In the human PR, the DHP A-ring position is twisted towards helix 3 of PR thereby disturbing the hydrogen bond pattern around the C3-keto group, resulting in a lower binding affinity. Furthermore, we observed that the elephant PR ligand-binding pocket is more rigid than the human analogue, which probably explains the higher affinity towards both progesterone and DHP. Interestingly, the G722A substitution is not elephant-specific, rather it is also present in five independent lineages of mammalian evolution, suggesting a special role of the substitution for the development of distinct mammalian gestagen systems.


Assuntos
Receptores de Progesterona/química , Receptores de Progesterona/metabolismo , 5-alfa-Di-Hidroprogesterona/metabolismo , Alanina/genética , Animais , Clonagem Molecular , Elefantes , Glicina/genética , Cavalos , Humanos , Ligação de Hidrogênio , Procaviídeos , Ligantes , Modelos Químicos , Modelos Moleculares , Simulação de Dinâmica Molecular , Mutagênese Sítio-Dirigida , Filogenia , Ligação Proteica , Estrutura Terciária de Proteína , Trichechus
8.
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
9.
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
10.
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
11.
Bioorg Med Chem ; 18(21): 7365-79, 2010 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-20932767

RESUMO

Melanin-concentrating hormone (MCH) regulates feeding and energy homeostasis through interaction with its receptor, the melanin-concentrating receptor 1 (MCHR1), making it a target in the treatment of obesity. Molecular modeling and docking studies were performed in order to find a binding model for the docking of two new series of MCHR1 antagonists to the receptor. Results suggested interactions between the ligands and two glutamines (Gln5.42 and Gln6.55) not conserved in many of the GPCRs family members. Histamine 3 receptor (HRH3) presents two apolar residues in the aforementioned positions and the available biological data against this receptor supported the role of the two glutamines in the binding of antagonists to the MCHR1. This knowledge could be useful in the development of new, more active and more selective MCHR1 antagonists.


Assuntos
Fármacos Antiobesidade/química , Receptores de Somatostatina/antagonistas & inibidores , Fármacos Antiobesidade/síntese química , Fármacos Antiobesidade/farmacologia , Sítios de Ligação , Humanos , Simulação de Dinâmica Molecular , Ligação Proteica , Receptores Histamínicos H3/química , Receptores Histamínicos H3/metabolismo , Receptores de Somatostatina/metabolismo , Relação Estrutura-Atividade
12.
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
13.
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
14.
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
15.
Bioorg Med Chem Lett ; 17(8): 2334-7, 2007 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-17293110

RESUMO

New antimicrobial compounds are of major importance because of the growing problem of bacterial resistance and antimicrobial peptides have been gaining a lot of interest. Their mechanism of action, however, is often obscure. Here a set of non-peptidic compounds with antimicrobial activity are presented that have been designed based on criteria derived from three-dimensional structures of antimicrobial peptides. Even though only a small set of compounds has been designed, the activity immediately matches that of the original peptides, supporting the proposed criteria for activity, i.e. not the peptidic nature of antimicrobial peptides is responsible for their activity but rather the proper arrangement of the relevant functional groups.


Assuntos
Anti-Infecciosos/química , Desenho de Fármacos , Oligopeptídeos/química , Anti-Infecciosos/farmacologia , Bactérias/efeitos dos fármacos , Eritrócitos/efeitos dos fármacos , Humanos , Testes de Sensibilidade Microbiana , Conformação Molecular , Oligopeptídeos/farmacologia , Peptídeos Cíclicos/química , Peptídeos Cíclicos/farmacologia
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