RESUMO
Bivalent proximity-inducing compounds represent a novel class of small molecule therapeutics with exciting potential and new challenges. The most prominent examples of such compounds are utilized in targeted protein degradation where E3 ligases are hijacked to recruit a substrate protein to the proteasome via ubiquitination. In this review we provide an overview of the current state of E3 ligases used in targeted protein degradation, their respective ligands as well as challenges and opportunities that present themselves with these compounds.
Assuntos
Complexo de Endopeptidases do Proteassoma , Ubiquitina , Proteólise , Complexo de Endopeptidases do Proteassoma/metabolismo , Ubiquitina/metabolismo , Ubiquitinação , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Genetic screening technologies to identify and validate macromolecular interactions (MMIs) essential for complex pathways remain an important unmet need for systems biology and therapeutics development. Here, we use a library of peptides from diverse prokaryal genomes to screen MMIs promoting the nuclear relocalization of Forkhead Box O3 (FOXO3a), a tumor suppressor more frequently inactivated by post-translational modification than mutation. A hit peptide engages the 14-3-3 family of signal regulators through a phosphorylation-dependent interaction, modulates FOXO3a-mediated transcription, and suppresses cancer cell growth. In a crystal structure, the hit peptide occupies the phosphopeptide-binding groove of 14-3-3ε in a conformation distinct from its natural peptide substrates. A biophysical screen identifies drug-like small molecules that displace the hit peptide from 14-3-3ε, providing starting points for structure-guided development. Our findings exemplify "protein interference," an approach using evolutionarily diverse, natural peptides to rapidly identify, validate, and develop chemical probes against MMIs essential for complex cellular phenotypes.
Assuntos
Descoberta de Drogas , Proteína Forkhead Box O3/antagonistas & inibidores , Bibliotecas de Moléculas Pequenas/farmacologia , Células Cultivadas , Feminino , Proteína Forkhead Box O3/genética , Proteína Forkhead Box O3/metabolismo , Genes Supressores de Tumor/efeitos dos fármacos , Humanos , Biblioteca de Peptídeos , Fosforilação , Bibliotecas de Moléculas Pequenas/químicaRESUMO
Lung cancer is the leading cause of cancer deaths, and effective treatments are urgently needed. Loss-of-function mutations in the DNA damage response kinase ATM are common in lung adenocarcinoma but directly targeting these with drugs remains challenging. Here we report that ATM loss-of-function is synthetic lethal with drugs inhibiting the central growth factor kinases MEK1/2, including the FDA-approved drug trametinib. Lung cancer cells resistant to MEK inhibition become highly sensitive upon loss of ATM both in vitro and in vivo. Mechanistically, ATM mediates crosstalk between the prosurvival MEK/ERK and AKT/mTOR pathways. ATM loss also enhances the sensitivity of KRAS- or BRAF-mutant lung cancer cells to MEK inhibition. Thus, ATM mutational status in lung cancer is a mechanistic biomarker for MEK inhibitor response, which may improve patient stratification and extend the applicability of these drugs beyond RAS and BRAF mutant tumours.
Assuntos
Proteínas Mutadas de Ataxia Telangiectasia/genética , Proliferação de Células/efeitos dos fármacos , Neoplasias Pulmonares/prevenção & controle , Mutação , Inibidores de Proteínas Quinases/farmacologia , Animais , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Benzamidas/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/genética , Difenilamina/análogos & derivados , Difenilamina/farmacologia , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Camundongos Nus , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas B-raf/metabolismo , Piridonas/farmacologia , Pirimidinonas/farmacologia , Interferência de RNA , Tiofenos/farmacologia , Ureia/análogos & derivados , Ureia/farmacologia , Ensaios Antitumorais Modelo de Xenoenxerto , Proteínas ras/genética , Proteínas ras/metabolismoRESUMO
Patterns of somatic mutations in cancer genes provide information about their functional role in tumourigenesis, and thus indicate their potential for therapeutic exploitation. Yet, the classical distinction between oncogene and tumour suppressor may not always apply. For instance, TP53 has been simultaneously associated with tumour suppressing and promoting activities. Here, we uncover a similar phenomenon for GATA3, a frequently mutated, yet poorly understood, breast cancer gene. We identify two functional classes of frameshift mutations that are associated with distinct expression profiles in tumours, differential disease-free patient survival and gain- and loss-of-function activities in a cell line model. Furthermore, we find an estrogen receptor-independent synthetic lethal interaction between a GATA3 frameshift mutant with an extended C-terminus and the histone methyltransferases G9A and GLP, indicating perturbed epigenetic regulation. Our findings reveal important insights into mutant GATA3 function and breast cancer, provide the first potential therapeutic strategy and suggest that dual tumour suppressive and oncogenic activities are more widespread than previously appreciated.
Assuntos
Neoplasias da Mama/genética , Epigênese Genética , Fator de Transcrição GATA3/genética , Antígenos de Histocompatibilidade/genética , Histona-Lisina N-Metiltransferase/genética , Neoplasias da Mama/patologia , Intervalo Livre de Doença , Resistencia a Medicamentos Antineoplásicos/genética , Estrogênios/genética , Estrogênios/metabolismo , Feminino , Mutação da Fase de Leitura , Regulação Neoplásica da Expressão Gênica , Humanos , Células MCF-7 , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/uso terapêuticoRESUMO
High-throughput live-cell screens are intricate elements of systems biology studies and drug discovery pipelines. Here, we demonstrate an optogenetics-assisted method that avoids the need for chemical activators and reporters, reduces the number of operational steps and increases information content in a cell-based small-molecule screen against human protein kinases, including an orphan receptor tyrosine kinase. This blueprint for all-optical screening can be adapted to many drug targets and cellular processes.
Assuntos
Ensaios de Triagem em Larga Escala , Luz , Inibidores de Proteínas Quinases/farmacologia , Proteínas Quinases/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Células HEK293 , Humanos , Inibidores de Proteínas Quinases/química , Bibliotecas de Moléculas Pequenas/química , Relação Estrutura-AtividadeRESUMO
Some mutations in cancer cells can be exploited for therapeutic intervention. However, for many cancer subtypes, including triple-negative breast cancer (TNBC), no frequently recurring aberrations could be identified to make such an approach clinically feasible. Characterized by a highly heterogeneous mutational landscape with few common features, many TNBCs cluster together based on their 'basal-like' transcriptional profiles. We therefore hypothesized that targeting TNBC cells on a systems level by exploiting the transcriptional cell state might be a viable strategy to find novel therapies for this highly aggressive disease. We performed a large-scale chemical genetic screen and identified a group of compounds related to the drug PKC412 (midostaurin). PKC412 induced apoptosis in a subset of TNBC cells enriched for the basal-like subtype and inhibited tumor growth in vivo. We employed a multi-omics approach and computational modeling to address the mechanism of action and identified spleen tyrosine kinase (SYK) as a novel and unexpected target in TNBC. Quantitative phosphoproteomics revealed that SYK inhibition abrogates signaling to STAT3, explaining the selectivity for basal-like breast cancer cells. This non-oncogene addiction suggests that chemical SYK inhibition may be beneficial for a specific subset of TNBC patients and demonstrates that targeting cell states could be a viable strategy to discover novel treatment strategies.
Assuntos
Antineoplásicos/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Terapia de Alvo Molecular , Proteínas Tirosina Quinases/metabolismo , Fator de Transcrição STAT3/metabolismo , Estaurosporina/análogos & derivados , Neoplasias de Mama Triplo Negativas/metabolismo , Animais , Apoptose/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Sistemas de Liberação de Medicamentos , Feminino , Perfilação da Expressão Gênica , Humanos , Camundongos , Simulação de Acoplamento Molecular , Domínios e Motivos de Interação entre Proteínas , Proteômica/métodos , Análise de Sequência de RNA , Transdução de Sinais , Estaurosporina/farmacologia , Quinase Syk , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND: Measuring the impact of combinations of genetic or chemical perturbations on cellular fitness, sometimes referred to as synthetic lethal screening, is a powerful method for obtaining novel insights into gene function and drug action. Especially when performed at large scales, gene-gene or gene-drug interaction screens can reveal complex genetic interactions or drug mechanism of action or even identify novel therapeutics for the treatment of diseases.The result of such large-scale screen results can be represented as a matrix with a numeric score indicating the cellular fitness (e.g. viability or doubling time) for each double perturbation. In a typical screen, the majority of combinations do not impact the cellular fitness. Thus, it is critical to first discern true "hits" from noise. Subsequent data exploration and visualization methods can assist to extract meaningful biological information from the data. However, despite the increasing interest in combination perturbation screens, no user friendly open-source program exists that combines statistical analysis, data exploration tools and visualization. RESULTS: We developed TOPS (Tool for Combination Perturbation Screen Analysis), a Java and R-based software tool with a simple graphical user interface that allows the user to import, analyze, filter and plot data from double perturbation screens as well as other compatible data. TOPS was designed in a modular fashion to allow the user to add alternative importers for data formats or custom analysis scripts not covered by the original release.We demonstrate the utility of TOPS on two datasets derived from functional genetic screens using different methods. Dataset 1 is a gene-drug interaction screen and is based on Luminex xMAP technology. Dataset 2 is a gene-gene short hairpin (sh)RNAi screen exploring the interactions between deubiquitinating enzymes and a number of prominent oncogenes using massive parallel sequencing (MPS). CONCLUSIONS: TOPS provides the benchtop scientist with a free toolset to analyze, filter and visualize data from functional genomic gene-gene and gene-drug interaction screens with a flexible interface to accommodate different technologies and analysis algorithms in addition to those already provided here. TOPS is freely available for academic and non-academic users and is released as open source.
Assuntos
Avaliação Pré-Clínica de Medicamentos , Genes , Software , Algoritmos , Neoplasias da Mama/genética , Linhagem Celular Tumoral , Gráficos por Computador , Interpretação Estatística de Dados , Feminino , Genômica/métodos , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Modelos Lineares , Interferência de RNARESUMO
Linking the molecular aberrations of cancer to drug responses could guide treatment choice and identify new therapeutic applications. However, there has been no systematic approach for analyzing gene-drug interactions in human cells. Here we establish a multiplexed assay to study the cellular fitness of a panel of engineered isogenic cancer cells in response to a collection of drugs, enabling the systematic analysis of thousands of gene-drug interactions. Applying this approach to breast cancer revealed various synthetic-lethal interactions and drug-resistance mechanisms, some of which were known, thereby validating the method. NOTCH pathway activation, which occurs frequently in breast cancer, unexpectedly conferred resistance to phosphoinositide 3-kinase (PI3K) inhibitors, which are currently undergoing clinical trials in breast cancer patients. NOTCH1 and downstream induction of c-MYC over-rode the dependency of cells on the PI3K-mTOR pathway for proliferation. These data reveal a new mechanism of resistance to PI3K inhibitors with direct clinical implications.
Assuntos
Antineoplásicos/farmacologia , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Resistencia a Medicamentos Antineoplásicos/genética , Inibidores de Fosfoinositídeo-3 Quinase , Linhagem Celular Tumoral , Proliferação de Células , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Mutação , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Transdução de Sinais , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismoRESUMO
Insertional mutagenesis in a haploid background can disrupt gene function. We extend our earlier work by using a retroviral gene-trap vector to generate insertions in >98% of the genes expressed in a human cancer cell line that is haploid for all but one of its chromosomes. We apply phenotypic interrogation via tag sequencing (PhITSeq) to examine millions of mutant alleles through selection and parallel sequencing. Analysis of pools of cells, rather than individual clones enables rapid assessment of the spectrum of genes involved in the phenotypes under study. This facilitates comparative screens as illustrated here for the family of cytolethal distending toxins (CDTs). CDTs are virulence factors secreted by a variety of pathogenic Gram-negative bacteria responsible for tissue damage at distinct anatomical sites. We identify 743 mutations distributed over 12 human genes important for intoxication by four different CDTs. Although related CDTs may share host factors, they also exploit unique host factors to yield a profile characteristic for each CDT.
Assuntos
Estudos de Associação Genética/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Fenótipo , Alelos , Proteínas de Bactérias/metabolismo , Toxinas Bacterianas/metabolismo , Toxinas Bacterianas/toxicidade , Linhagem Celular Tumoral , Cromossomos Humanos , Clonagem Molecular , Genoma Humano , Bactérias Gram-Negativas/patogenicidade , Haploidia , Humanos , Mutagênese Insercional , Mutação , Interferência de RNA , Sitios de Sequências Rotuladas , Fatores de Virulência/análise , Fatores de Virulência/metabolismoRESUMO
UNLABELLED: Signal transducer and activator of transcription 3 (Stat3) is activated in a variety of malignancies, including hepatocellular carcinoma (HCC). Activation of Ras occurs frequently at advanced stages of HCC by aberrant signaling through growth factor receptors or inactivation of effectors negatively regulating Ras signaling. Here, we addressed the role of Stat3 in Ras-dependent HCC progression in the presence and absence of p19(ARF) /p14(ARF) . We show that constitutive active (ca) Stat3 is tumor suppressive in Ras-transformed p19(ARF-/-) hepatocytes, whereas the expression of Stat3 lacking Tyr(705) phosphorylation (U-Stat3) enhances tumor formation. Accordingly, Ras-transformed Stat3(Δhc) /p19(ARF-/-) hepatocytes (lacking Stat3 and p19(ARF) ) showed increased tumor growth, compared to those expressing Stat3, demonstrating a tumor-suppressor activity of Stat3 in cells lacking p19(ARF) . Notably, endogenous expression of p19(ARF) in Ras-transformed hepatocytes conveyed oncogenic Stat3 functions, resulting in augmented or reduced HCC progression after the expression of caStat3 or U-Stat3, respectively. In accord with these data, the knockdown of p14(ARF) (the human homolog of p19(ARF) ) in Hep3B cells was associated with reduced pY-Stat3 levels during tumor growth to circumvent the tumor-suppressive effect of Stat3. Inhibition of Janus kinases (Jaks) revealed that Jak causes pY-Stat3 activation independently of p14(ARF) levels, indicating that p14(ARF) controls the oncogenic function of pY-Stat3 downstream of Jak. CONCLUSION: These data show evidence that p19(ARF) /p14(ARF) determines the pro- or anti-oncogenic activity of U-Stat3 and pY-Stat3 in Ras-dependent HCC progression.
Assuntos
Carcinoma Hepatocelular/fisiopatologia , Inibidor p16 de Quinase Dependente de Ciclina/fisiologia , Neoplasias Hepáticas/fisiopatologia , Fator de Transcrição STAT3/fisiologia , Animais , Carcinoma Hepatocelular/patologia , Proliferação de Células , Células Cultivadas , Inibidor p16 de Quinase Dependente de Ciclina/deficiência , Inibidor p16 de Quinase Dependente de Ciclina/genética , Modelos Animais de Doenças , Progressão da Doença , Hepatócitos/patologia , Janus Quinases/fisiologia , Neoplasias Hepáticas/patologia , Camundongos , Camundongos Knockout , Transdução de Sinais/fisiologiaAssuntos
Sistema do Grupo Sanguíneo Rh-Hr/genética , Animais , Técnicas de Cultura de Células/métodos , Divisão Celular , Drosophila melanogaster/citologia , Drosophila melanogaster/genética , Avaliação Pré-Clínica de Medicamentos/métodos , Perfilação da Expressão Gênica , Humanos , RNA de Cadeia Dupla/genética , Sistema do Grupo Sanguíneo Rh-Hr/fisiologiaRESUMO
HIF-1alpha represents the oxygen-regulated sub-unit of the transcription factor HIF-1, which regulates the transcription of numerous genes involved in cellular response to hypoxia and oxidative stress. It is shown here that nitric oxide (NO) induces HIF-1alpha stabilization in human endothelial cells from umbilical cords (HUVECs) under normoxic conditions. HIF-1alpha protein was increased approximately 36-fold after incubation with 500 microM DETA-NO, which releases a steady state NO concentration of roughly one thousandth of the initial concentration of the donor. Loading of the cells with vitamin C counteracted NO-induced HIF-1alpha accumulation. Based on the observations that oxidative and nitrosative stress can influence the activity of the proteasomal system, which is responsible for the non-lysosomal degradation of proteins, among them HIF-1alpha, it was investigated whether NO-induced stabilization of HIF-1alpha might be due to reduced 20S proteasomal activity. This hypothesis could not be proved, because NO concentrations to inhibit 20S proteasomal activity were about one order of magnitude higher than that to inhibit HIF-1alpha degradation.
Assuntos
Antioxidantes/farmacologia , Ácido Ascórbico/farmacologia , Células Endoteliais/efeitos dos fármacos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Óxido Nítrico/fisiologia , Hipóxia Celular , Células Cultivadas/efeitos dos fármacos , Células Cultivadas/metabolismo , Ácido Desidroascórbico/metabolismo , Células Endoteliais/metabolismo , Humanos , Recém-Nascido , Óxido Nítrico/antagonistas & inibidores , Estresse Oxidativo , Oxigênio/metabolismo , Complexo de Endopeptidases do Proteassoma/efeitos dos fármacos , Complexo de Endopeptidases do Proteassoma/metabolismo , Estabilidade Proteica , Triazenos/farmacologia , Veias UmbilicaisRESUMO
Highly reactive alpha,beta-unsaturated aldehydes like 4-hydroxy-2-nonenal (4-HNE), generated from oxidation of polyunsaturated fatty acids, can bind to proteins, polynucleotides and exert cytotoxicity. 4-HNE is known to react readily with thiol and amino groups on free or bound amino acids. Recently, hydrogen sulfide (H(2)S) has been identified as an endogenous vascular gasotransmitter and neuromodulator which can reach up to 160 micromol/l in the brain. Markedly higher 4-HNE concentrations were reported in the brain of patients suffering from Alzheimer's disease. Assuming that the low molecular thiol H(2)S may react with 4-HNE, we have tested the ability of H(2)S to counteract the cytotoxic and protein-modifying activity of 4-HNE. The results show that H(2)S at physiologically relevant concentrations could effectively protect neuronal cells (SH-SY5Y) from the cytotoxic action of 4-HNE. The HNE-modification of cellular proteins was also inhibited in presence of H(2)S. These data suggest that H(2)S may be an important protective factor against carbonyl stress by inactivating/modulating the action of highly reactive alpha,beta-unsaturated aldehydes like 4-HNE in the brain.
Assuntos
Poluentes Atmosféricos/farmacologia , Aldeídos/metabolismo , Aldeídos/farmacologia , Sulfeto de Hidrogênio/farmacologia , Peroxidação de Lipídeos/efeitos dos fármacos , Análise de Variância , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Interações Medicamentosas , Ensaio de Desvio de Mobilidade Eletroforética/métodos , Humanos , Neuroblastoma/patologiaRESUMO
BACKGROUND: Cardiac glycosides are Na(+)/K(+)-pump inhibitors widely used to treat heart failure. They are also highly cytotoxic, and studies have suggested specific anti-tumor activity leading to current clinical trials in cancer patients. However, a definitive demonstration of this putative anti-cancer activity and the underlying molecular mechanism has remained elusive. METHODOLOGY/PRINCIPAL FINDINGS: Using an unbiased transcriptomics approach, we found that cardiac glycosides inhibit general protein synthesis. Protein synthesis inhibition and cytotoxicity were not specific for cancer cells as they were observed in both primary and cancer cell lines. These effects were dependent on the Na(+)/K(+)-pump as they were rescued by expression of a cardiac glycoside-resistant Na(+)/K(+)-pump. Unlike human cells, rodent cells are largely resistant to cardiac glycosides in vitro and mice were found to tolerate extremely high levels. CONCLUSIONS/SIGNIFICANCE: The physiological difference between human and mouse explains the previously observed sensitivity of human cancer cells in mouse xenograft experiments. Thus, published mouse xenograft models used to support anti-tumor activity for these drugs require reevaluation. Our finding that cardiac glycosides inhibit protein synthesis provides a mechanism for the cytotoxicity of CGs and raises concerns about ongoing clinical trials to test CGs as anti-cancer agents in humans.
Assuntos
Glicosídeos Cardíacos/farmacologia , Biossíntese de Proteínas/efeitos dos fármacos , Inibidores da Síntese de Proteínas/farmacologia , Animais , Morte Celular/efeitos dos fármacos , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Digitoxina/farmacologia , Relação Dose-Resposta a Droga , Humanos , Janus Quinase 2/antagonistas & inibidores , Camundongos , Proteínas Mutantes/metabolismo , ATPase Trocadora de Sódio-PotássioRESUMO
LOOHs (lipid hydroperoxides) in oxLDL [oxidized LDL (low-density lipoprotein)] are potentially atherogenic compounds. Recently, H2S was identified as the third endogenous gasotransmitter in the vasculature. H2O2 is known to be destroyed by H2S. Assuming that H2S may also react with LOOHs, the results show that H2S can destroy LOOHs in oxLDL. The ability of LOOH-enriched LDL to induce HO-1 (haem oxygenase 1) in endothelial cells was abolished by H2S pretreatment. HPLC analysis showed that 9-HPODE [(9S)-hydroperoxy-(10E,12Z)-octadecadienoic acid], a compound found in oxLDL, was reduced to 9-HODE [(9S)-hydroxy-(10E,12Z)-octadecadienoic acid] in the presence of H2S. Thus H2S may act as an antiatherogenic agent by reducing LOOHs to the less reactive LOHs and could abrogate the pathobiological activity of oxLDL.
Assuntos
Sulfeto de Hidrogênio/farmacologia , Peróxidos Lipídicos/metabolismo , Lipoproteínas LDL/metabolismo , Análise de Variância , Células Cultivadas , Cromatografia Líquida de Alta Pressão , Relação Dose-Resposta a Droga , Células Endoteliais/citologia , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Heme Oxigenase-1/genética , Humanos , Ácidos Linoleicos/metabolismo , Ácidos Linoleicos Conjugados/metabolismo , Malondialdeído/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Substâncias Reativas com Ácido Tiobarbitúrico/metabolismo , Fatores de TempoRESUMO
OBJECTIVE: Beside NO (nitric monoxide) and CO (carbon monoxide), H2S (hydrogen sulfide) has been identified recently as the third gasotransmitter. By acting directly on KATP-channels on smooth muscle cells (SMC) H2S possesses vasorelaxing properties. It has the potential to react with metal ions (i.e. Cu, Fe, Zn) in metalloproteins. Angiotensin-converting enzyme (ACE), responsible for vasoconstriction, is a zinc (Zn) containing enzyme. We therefore hypothesized that H2S may interact with the Zn in the active center of ACE, modulating (inhibiting) enzyme activity. METHODS: ACE activity was measured on the surface of human endothelial cells (HUVECs) monolayers in culture, ex-vivo in umbilical veins and in HUVEC protein extracts. Quantitative real-time polymerase chain reaction (PCR) was used to study the effect of H2S on ACE mRNA expression in HUVECs. RESULTS: H2S inhibited the activity of ACE in HUVEC protein extracts in a dose-dependent manner, and only Zn but not Cd, Ca or Mg could counteract the inhibitory effect. Cell-surface ACE activity was inhibited by H2S on HUVEC monolayers and in ex-vivo umbilical veins. No influence of H2S on ACE mRNA expression was observed. CONCLUSION: H2S exhibits direct inhibitory action on ACE activity in HUVECs, obviously by interfering with the Zn in the active center of the enzyme. Thus, beside the known influence of H2S on SMC KATP-channels, the observed direct ACE inhibitory effect may add to the vasorelaxant effect of H2S in the vasculature by reducing angiotensin II production and inhibiting bradykinin degradation.
Assuntos
Inibidores da Enzima Conversora de Angiotensina/farmacologia , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/enzimologia , Sulfeto de Hidrogênio/farmacologia , Peptidil Dipeptidase A/metabolismo , Aminoácidos Sulfúricos/farmacologia , Sequência de Bases , Células Cultivadas , Primers do DNA/genética , Feminino , Humanos , Técnicas In Vitro , Peptidil Dipeptidase A/genética , Gravidez , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Veias Umbilicais/efeitos dos fármacos , Veias Umbilicais/enzimologia , Vasodilatadores/farmacologia , Zinco/farmacologiaRESUMO
Hypochlorite (HOCl), the product of the activated myeloperoxidase/H(2)O(2)/chloride (MPO/H(2)O(2)/Cl(- )) system is favored as a trigger of LDL modifications, which may play a pivotal role in early atherogenesis. As HOCl has been shown to react with thiol-containing compounds like glutathione and N-acetylcysteine protecting LDL from HOCl modification, we have tested the ability of hydrogen sulfide (H(2)S) - which has recently been identified as an endogenous vasorelaxant - to counteract the action of HOCl on LDL. The results show that H(2)S could inhibit the atherogenic modification of LDL induced by HOCl, as measured by apolipoprotein alterations. Beside its HOCl scavenging potential, H(2)S was found to inhibit MPO (one may speculate that this occurs via H(2)S/heme interaction) and destroy H(2)O(2). Thus, H(2)S may interfere with the reactants and reaction products of the activated MPO/H(2)O(2)/Cl(- ) system. Our data add to the evidence of an anti-atherosclerotic action of this gasotransmitter taking the role of HOCl in the atherogenic modification of LDL into account.