RESUMO
Cardiac hypertrophy resulting from sympathetic nervous system activation triggers the development of heart failure. The transcription factor Y-box binding protein 1 (YB-1) can interact with transcription factors involved in cardiac hypertrophy and may thereby interfere with the hypertrophy growth process. Therefore, the question arises as to whether YB-1 influences cardiomyocyte hypertrophy and might thereby influence the development of heart failure. YB-1 expression is downregulated in human heart biopsies of patients with ischemic cardiomyopathy (n = 8), leading to heart failure. To study the impact of reduced YB-1 in cardiac cells, we performed small interfering RNA (siRNA) experiments in H9C2 cells as well as in adult cardiomyocytes (CMs) of rats. The specificity of YB-1 siRNA was analyzed by a miRNA-like off-target prediction assay identifying potential genes. Testing three high-scoring genes by transfecting cardiac cells with YB-1 siRNA did not result in downregulation of these genes in contrast to YB-1, whose downregulation increased hypertrophic growth. Hypertrophic growth was mediated by PI3K under PE stimulation, as well by downregulation with YB-1 siRNA. On the other hand, overexpression of YB-1 in CMs, caused by infection with an adenovirus encoding YB-1 (AdYB-1), prevented hypertrophic growth under α-adrenergic stimulation with phenylephrine (PE), but not under stimulation with growth differentiation factor 15 (GDF15; n = 10-16). An adenovirus encoding the green fluorescent protein (AdGFP) served as the control. YB-1 overexpression enhanced the mRNA expression of the Gq inhibitor regulator of G-protein signaling 2 (RGS2) under PE stimulation (n = 6), potentially explaining its inhibitory effect on PE-induced hypertrophic growth. This study shows that YB-1 protects cardiomyocytes against PE-induced hypertrophic growth. Like in human end-stage heart failure, YB-1 downregulation may cause the heart to lose its protection against hypertrophic stimuli and progress to heart failure. Therefore, the transcription factor YB-1 is a pivotal signaling molecule, providing perspectives for therapeutic approaches.
Assuntos
Adrenérgicos , Insuficiência Cardíaca , Adulto , Humanos , Animais , Ratos , Fenilefrina , Insuficiência Cardíaca/genética , Miócitos Cardíacos , RNA Interferente Pequeno/genética , Adenoviridae , Cardiomegalia/genética , Fatores de TranscriçãoRESUMO
The Hedgehog (Hh) signaling pathway is crucial for vertebrate embryonic development, tissue homeostasis and regeneration. Hh signaling is upregulated in basal cell carcinoma and medulloblastoma and Hh pathway inhibitors targeting the Smoothened (SMO) protein are in clinical use. However, the signaling cascade is incompletely understood and novel druggable proteins in the pathway are in high demand. We describe the discovery of the Hh-pathway modulator Pipinib by means of cell-based screening. Target identification and validation revealed that Pipinib selectively inhibits phosphatidylinositol 4-kinase IIIß (PI4KB) and suppresses GLI-mediated transcription and Hh target gene expression by impairing SMO translocation to the cilium. Therefore, inhibition of PI4KB and, consequently, reduction in phosphatidyl-4-phosphate levels may be considered an alternative approach to inhibit SMO function and thus, Hedgehog signaling.
Assuntos
Antineoplásicos/farmacologia , Proteínas Hedgehog/antagonistas & inibidores , Antígenos de Histocompatibilidade Menor/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Transdução de Sinais/efeitos dos fármacos , Tiofenos/farmacologia , Animais , Antineoplásicos/química , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Cílios/metabolismo , Expressão Gênica/efeitos dos fármacos , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Humanos , Camundongos , Antígenos de Histocompatibilidade Menor/genética , Morfolinas/farmacologia , Osteogênese/efeitos dos fármacos , Fosfotransferases (Aceptor do Grupo Álcool)/antagonistas & inibidores , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Purinas/farmacologia , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Receptor Smoothened/genética , Receptor Smoothened/metabolismo , Relação Estrutura-Atividade , Tiofenos/químicaRESUMO
Cell-based assays enable monitoring of small-molecule bioactivity in a target-agnostic manner and help uncover new biological mechanisms. Subsequent identification and validation of the small-molecule targets, typically employing proteomics techniques, is very challenging and limited, in particular if the targets are membrane proteins. Herein, we demonstrate that the combination of cell-based bioactive-compound discovery with cheminformatic target prediction may provide an efficient approach to accelerate the process and render target identification and validation more efficient. Using a cell-based assay, we identified the pyrazolo-imidazole smoothib as a new inhibitor of hedgehog (Hh) signaling and an antagonist of the protein smoothened (SMO) with a novel chemotype. Smoothib targets the heptahelical bundle of SMO, prevents its ciliary localization, reduces the expression of Hh target genes, and suppresses the growth of Ptch+/- medulloblastoma cells.
Assuntos
Proteínas Hedgehog/metabolismo , Imidazóis/química , Animais , Sítios de Ligação , Linhagem Celular , Descoberta de Drogas , Células HEK293 , Proteínas Hedgehog/antagonistas & inibidores , Humanos , Imidazóis/metabolismo , Imidazóis/farmacologia , Camundongos , Simulação de Acoplamento Molecular , Células NIH 3T3 , Osteoblastos/citologia , Osteoblastos/efeitos dos fármacos , Osteoblastos/metabolismo , Domínios Proteicos , Pirazóis/química , Transdução de Sinais/efeitos dos fármacos , Receptor Smoothened/antagonistas & inibidores , Receptor Smoothened/metabolismo , Alcaloides de Veratrum/química , Alcaloides de Veratrum/metabolismoRESUMO
Biology-oriented synthesis employs the structural information encoded in complex natural products to guide the synthesis of compound collections enriched in bioactivity. The trans-hydrindane dehydro-δ-lactone motif defines the characteristic scaffold of the steroid-like withanolides, a plant-derived natural product class with a diverse pattern of bioactivity. A withanolide-inspired compound collection was synthesized by making use of three key intermediates that contain this characteristic framework derivatized with different reactive functional groups. Biological evaluation of the compound collection in cell-based assays that monitored biological signal-transduction processes revealed a novel class of Hedgehog signaling inhibitors that target the protein Smoothened.
Assuntos
Produtos Biológicos/farmacologia , Ouriços/metabolismo , Transdução de Sinais/efeitos dos fármacos , Vitanolídeos/farmacologia , Animais , Produtos Biológicos/síntese química , Produtos Biológicos/química , Relação Dose-Resposta a Droga , Conformação Molecular , Relação Estrutura-Atividade , Vitanolídeos/síntese química , Vitanolídeos/químicaRESUMO
Cousins you can count on: An iridoid-inspired compound collection was synthesized efficiently by the resolution of cyclic enones in an asymmetric cycloaddition with azomethine ylides. The collection contained novel potent inhibitors of the Wnt and Hedgehog signaling pathways.
Assuntos
Proteínas Hedgehog/metabolismo , Iridoides/química , Iridoides/farmacologia , Transdução de Sinais/efeitos dos fármacos , Proteínas Wnt/metabolismo , Animais , Catálise , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Reação de Cicloadição , Avaliação Pré-Clínica de Medicamentos , Células HEK293 , Proteínas Hedgehog/antagonistas & inibidores , Humanos , Iridoides/síntese química , Camundongos , Estereoisomerismo , Relação Estrutura-Atividade , Proteínas Wnt/antagonistas & inibidoresRESUMO
Aberrant hedgehog (Hh) signaling contributes to the pathogenesis of multiple cancers. Available inhibitors target Smoothened (Smo), which can acquire mutations causing drug resistance. Thus, compounds that inhibit Hh signaling downstream of Smo are urgently needed. We identified dynarrestin, a novel inhibitor of cytoplasmic dyneins 1 and 2. Dynarrestin acts reversibly to inhibit cytoplasmic dynein 1-dependent microtubule binding and motility in vitro without affecting ATP hydrolysis. It rapidly and reversibly inhibits endosome movement in living cells and perturbs mitosis by inducing spindle misorientation and pseudoprometaphase delay. Dynarrestin reversibly inhibits cytoplasmic dynein 2-dependent intraflagellar transport (IFT) of the cargo IFT88 and flux of Smo within cilia without interfering with ciliogenesis and suppresses Hh-dependent proliferation of neuronal precursors and tumor cells. As such, dynarrestin is a valuable tool for probing cytoplasmic dynein-dependent cellular processes and a promising compound for medicinal chemistry programs aimed at development of anti-cancer drugs.
Assuntos
Dineínas do Citoplasma/antagonistas & inibidores , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Animais , Transporte Biológico/efeitos dos fármacos , Linhagem Celular , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cílios/efeitos dos fármacos , Cílios/metabolismo , Dineínas do Citoplasma/metabolismo , Proteínas Hedgehog/antagonistas & inibidores , Proteínas Hedgehog/metabolismo , Humanos , Camundongos , Mitose/efeitos dos fármacos , Células NIH 3T3 , Transporte Proteico/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacosRESUMO
The activity of a new class of antimicrobials-polyoxometalate ionic liquids (POM-ILs)-is systematically investigated. The prototype POM-ILs feature Keggin-type anions (α-SiW11 O39 8- ) and tetraalkylammonium ions as active cationic species. Antimicrobial tests of the POM-ILs against important human pathogens show that variation of the alkyl chain length of the cation leads to significant changes in antimicrobial activity against the medically relevant Gram-negative bacteria Escherichia coli and Pseudomonas aeruginosa, and especially against the Gram-positive Staphylococcus aureus. Owing to the unique materials properties of the POM-ILs, such as high viscosity and water immiscibility, applications of antimicrobial surface coatings against airborne pathogens or for water decontamination can be envisaged. Furthermore, the combination of antimicrobially active cations with POM anions might afford new POM-ILs with two active components.
RESUMO
The limited structural diversity that a compound library represents severely restrains the discovery of bioactive small molecules for medicinal chemistry and chemical biology research, and thus calls for developing new divergent synthetic approaches to structurally diverse and complex scaffolds. Here we present a de novo branching cascades approach wherein simple primary substrates follow different cascade reactions to create various distinct molecular frameworks in a scaffold diversity phase. Later, the scaffold elaboration phase introduces further complexity to the scaffolds by creating a number of chiral centres and incorporating new hetero- or carbocyclic rings. Thus, employing N-phenyl hydroxylamine, dimethyl acetylenedicarboxylate and allene ester as primary substrates, a compound collection of sixty one molecules representing seventeen different scaffolds is built up that delivers a potent tubulin inhibitor, as well as inhibitors of the Hedgehog signalling pathway. This work highlights the immense potential of cascade reactions to deliver compound libraries enriched in structural and functional diversity.