RESUMEN
To identify new potential therapeutic targets for neurodegenerative diseases, we initiated activity-based protein profiling studies with withanolide A (WitA), a known neuritogenic constituent of Withania somnifera root with unknown mechanism of action. Molecular probes were designed and synthesized, and led to the discovery of the glucocorticoid receptor (GR) as potential target. Molecular modeling calculations using the VirtualToxLab predicted a weak binding affinity of WitA for GR. Neurite outgrowth experiments in human neuroblastoma SH-SY5Y cells further supported a glucocorticoid-dependent mechanism, finding that WitA was able to reverse the outgrowth inhibition mediated by dexamethasone (Dex). However, further GR binding and transactivation assays found no direct interference of WitA. Further molecular modeling analysis suggested that WitA, although forming several contacts with residues in the GR binding pocket, is lacking key stabilizing interactions as observed for Dex. Taken together, the data suggest that WitA-dependent induction of neurite outgrowth is not through a direct effect on GR, but might be mediated through a closely related pathway. Further experiments should evaluate a possible role of GR modulators and/or related signaling pathways such as ERK, Akt, NF-κB, TRα, or Hsp90 as potential targets in the WitA-mediated neuromodulatory effects.
Asunto(s)
Receptores de Glucocorticoides/metabolismo , Witanólidos/metabolismo , Sitios de Unión , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Dexametasona/química , Dexametasona/metabolismo , Dexametasona/farmacología , Glucocorticoides/química , Glucocorticoides/metabolismo , Glucocorticoides/farmacología , Proteínas HSP90 de Choque Térmico/metabolismo , Humanos , Simulación del Acoplamiento Molecular , FN-kappa B/metabolismo , Neuritas/efectos de los fármacos , Neuritas/metabolismo , Enfermedades Neurodegenerativas/tratamiento farmacológico , Enfermedades Neurodegenerativas/metabolismo , Enfermedades Neurodegenerativas/patología , Unión Proteica , Estructura Terciaria de Proteína , Receptores de Glucocorticoides/antagonistas & inhibidores , Transducción de Señal/efectos de los fármacos , Witanólidos/farmacología , Witanólidos/uso terapéuticoRESUMEN
Combination chemotherapy has proven to be a favorable strategy to treat acute leukemia. However, the introduction of novel compounds remains challenging and is hindered by a lack of understanding of their mechanistic interactions with established drugs. In the present study, we demonstrate a highly increased response of various acute leukemia cell lines, drug-resistant cells and patient-derived xenograft cells by combining the recently introduced protein disulfide isomerase inhibitor PS89 with cytostatics. In leukemic cells, a proteomics-based target fishing approach revealed that PS89 affects a whole network of endoplasmic reticulum homeostasis proteins. We elucidate that the strong induction of apoptosis in combination with cytostatics is orchestrated by the PS89 target B-cell receptor-associated protein 31, which transduces apoptosis signals at the endoplasmic reticulum -mitochondria interface. Activation of caspase-8 and cleavage of B-cell receptor-associated protein 31 stimulate a pro-apoptotic crosstalk including release of calcium from the endoplasmic reticulum and an increase in the levels of reactive oxygen species resulting in amplification of mitochondrial apoptosis. The findings of this study promote PS89 as a novel chemosensitizing agent for the treatment of acute leukemia and uncovers that targeting the endoplasmic reticulum - mitochondrial network of cell death is a promising approach in combination therapy.
Asunto(s)
Citostáticos/farmacología , Retículo Endoplásmico/metabolismo , Leucemia/metabolismo , Mitocondrias/metabolismo , Transducción de Señal/efectos de los fármacos , Animales , Apoptosis/efectos de los fármacos , Caspasa 8/metabolismo , Línea Celular Tumoral , Modelos Animales de Enfermedad , Resistencia a Antineoplásicos/efectos de los fármacos , Humanos , Leucemia/tratamiento farmacológico , Leucemia/patología , Ratones , Modelos Biológicos , Proteoma , Proteómica/métodos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Staphylococcus aureus is a major bacterial pathogen that invades and damages host tissue by the expression of devastating toxins. We here performed a phenotypic screen of 35 molecules that were structurally inspired by previous hydroxyamide-based S. aureus virulence inhibitors compiled from commercial sources or designed and synthesized de novo. One of the most potent compounds, AV73, not only reduced hemolytic alpha-hemolysin production in S. aureus but also impeded in vitro biofilm formation. The effect of AV73 on bacterial proteomes and extracellular protein levels was analyzed by quantitative proteomics and revealed a significant down-regulation of major virulence and biofilm promoting proteins. To elucidate the mode of action of AV73, target identification was performed using affinity-based protein profiling (AfBPP), where among others YidC was identified as a target.
Asunto(s)
Adhesinas Bacterianas/biosíntesis , Antibacterianos/farmacología , Toxinas Bacterianas/biosíntesis , Biopelículas/efectos de los fármacos , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Staphylococcus aureus , Biopelículas/crecimiento & desarrollo , Staphylococcus aureus/patogenicidad , Staphylococcus aureus/fisiologíaRESUMEN
ß-Lactones have recently been introduced as the first selective ClpP inhibitors that attenuate virulence of both sensitive Staphylococcus aureus and multiresistant strains (MRSA). Although previous knockout studies showed that ClpP is essential for S. aureus alpha-toxin production, a link between ß-lactone inhibition and molecular virulence mechanisms has been lacking so far. We here perform a chemical-proteomic approach to elucidate antivirulence pathways. First, we demonstrate by gel-free activity-based protein profiling that ClpP is the predominant target of ß-lactones. Only a few off-targets were discovered, which, unlike ClpP, were not involved in the reduction of alpha-toxin expression. Second, in-depth mechanistic insight was provided by a full proteomic comparison between lactone treated and untreated S. aureus cells. Quantitative mass-spectrometric analysis revealed increased repressor of toxin (Rot) levels and a corresponding down-regulation of α-toxin, providing the first direct connection between the lactone-dependent phenotype and a corresponding cellular mechanism. By building up a quantitative virulence regulation network, we visualize the impact of ClpP inhibition in a systems biology context. Interestingly, a lack of in vitro Rot degradation by either ClpXP or ClpCP calls either for a proteolysis mechanism with yet unknown adaptor proteins or for an indirect mode of action that may involve ClpX-mediated RNA signaling and feedback circuits.
Asunto(s)
Lactonas/farmacología , Staphylococcus aureus/patogenicidad , Virulencia/efectos de los fármacos , Proteínas Bacterianas/análisis , Proteínas Bacterianas/genética , Toxinas Bacterianas/genética , Endopeptidasa Clp/efectos de los fármacos , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Proteómica , Proteínas Represoras/análisis , Biología de SistemasRESUMEN
Gram-negative bacteria represent a challenging task for antibacterial drug discovery owing to their impermeable cell membrane and restricted uptake of small molecules. We herein describe the synthesis of natural-product-derived epoxycyclohexenones and explore their antibiotic activity against several pathogenic bacteria. A compound with activity against Salmonella Typhimurium was identified, and the target enzymes were unraveled by quantitative chemical proteomics. Importantly, two protein hits were linked to bacterial stress response, and corresponding assays revealed an elevated susceptibility to reactive oxygen species upon compound treatment. The consolidated inhibition of these targets provides a rationale for antibacterial activity and highlights epoxycyclohexenones as natural product scaffolds with suitable properties for killing Gram-negative Salmonella.
Asunto(s)
Antibacterianos/farmacología , Benzoquinonas/farmacología , Productos Biológicos/farmacología , Salmonella typhimurium/efectos de los fármacos , Antibacterianos/síntesis química , Antibacterianos/química , Benzoquinonas/síntesis química , Benzoquinonas/química , Productos Biológicos/síntesis química , Productos Biológicos/química , Pruebas de Sensibilidad Microbiana , Simulación de Dinámica Molecular , Estructura MolecularRESUMEN
Methicillin-resistant Staphylococcus aureus (MRSA) causes severe infections with only few effective antibiotic therapies currently available. To approach this challenge, chemical entities with a novel and resistance-free mode of action are desperately needed. Here, we introduce a new hydroxyamide compound that effectively reduces the expression of devastating toxins in various S. aureus and MRSA strains. The molecular mechanism was investigated by transcriptome analysis as well as by affinity-based protein profiling. Down-regulation of several pathogenesis associated genes suggested the inhibition of a central virulence-related pathway. Mass spectrometry-based chemical proteomics revealed putative molecular targets. Systemic treatment with the hydroxyamide showed significant reduction of abscess sizes in a MRSA mouse skin infection model. The absence of resistance development in vitro further underlines the finding that targeting virulence could lead to prolonged therapeutic options in comparison to antibiotics that directly address bacterial survival.
Asunto(s)
Antibacterianos/química , Antibacterianos/farmacología , Proteínas Bacterianas/química , Ácidos Hidroxámicos/química , Ácidos Hidroxámicos/farmacología , Staphylococcus aureus Resistente a Meticilina/química , Staphylococcus aureus Resistente a Meticilina/efectos de los fármacos , Infecciones Estafilocócicas/microbiología , Staphylococcus aureus/efectos de los fármacos , Animales , Proteínas Bacterianas/metabolismo , Staphylococcus aureus Resistente a Meticilina/patogenicidad , Ratones , Pruebas de Sensibilidad Microbiana , Staphylococcus aureus/patogenicidadRESUMEN
Caseinolytic proteaseâ P (ClpP) is an important regulator of Staphylococcus aureus pathogenesis. A high-throughput screening for inhibitors of ClpP peptidase activity led to the identification of the first non-covalent binder for this enzyme class. Co-crystallization of the small molecule with S.â aureus ClpP revealed a novel binding mode: Because of the rotation of the conserved residue prolineâ 125, ClpP is locked in a defined conformational state, which results in distortion of the catalytic triad and inhibition of the peptidase activity. Based on these structural insights, the molecule was optimized by rational design and virtual screening, resulting in derivatives exceeding the potency of previous ClpP inhibitors. Strikingly, the conformational lock is overturned by binding of ClpX, an associated chaperone that enables proteolysis by substrate unfolding in the ClpXP complex. Thus, regulation of inhibitor binding by associated chaperones is an unexpected mechanism important for ClpP drug development.