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Biosynthesis of glycosylphosphatidylinositol (GPI) is required for anchoring proteins to the plasma membrane, and is essential for the integrity of the fungal cell wall. Here, we use a reporter gene-based screen in Saccharomyces cerevisiae for the discovery of antifungal inhibitors of GPI-anchoring of proteins, and identify the oligocyclopropyl-containing natural product jawsamycin (FR-900848) as a potent hit. The compound targets the catalytic subunit Spt14 (also referred to as Gpi3) of the fungal UDP-glycosyltransferase, the first step in GPI biosynthesis, with good selectivity over the human functional homolog PIG-A. Jawsamycin displays antifungal activity in vitro against several pathogenic fungi including Mucorales, and in vivo in a mouse model of invasive pulmonary mucormycosis due to Rhyzopus delemar infection. Our results provide a starting point for the development of Spt14 inhibitors for treatment of invasive fungal infections.
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Antifúngicos/farmacología , Glicosiltransferasas/antagonistas & inhibidores , Policétidos/farmacología , Proteínas de Saccharomyces cerevisiae/antagonistas & inhibidores , Animales , Proliferación Celular , Modelos Animales de Enfermedad , Fermentación , Genes Reporteros , Glicosilfosfatidilinositoles/biosíntesis , Células HCT116 , Células Hep G2 , Humanos , Concentración de Iones de Hidrógeno , Concentración 50 Inhibidora , Células K562 , Pulmón/microbiología , Masculino , Ratones , Ratones Endogámicos ICR , Mucorales , Familia de Multigenes , Rhizopus , Saccharomyces cerevisiaeRESUMEN
FR171456 is a natural product with cholesterol-lowering properties in animal models, but its molecular target is unknown, which hinders further drug development. Here we show that FR171456 specifically targets the sterol-4-alpha-carboxylate-3-dehydrogenase (Saccharomyces cerevisiae--Erg26p, Homo sapiens--NSDHL (NAD(P) dependent steroid dehydrogenase-like)), an essential enzyme in the ergosterol/cholesterol biosynthesis pathway. FR171456 significantly alters the levels of cholesterol pathway intermediates in human and yeast cells. Genome-wide yeast haploinsufficiency profiling experiments highlight the erg26/ERG26 strain, and multiple mutations in ERG26 confer resistance to FR171456 in growth and enzyme assays. Some of these ERG26 mutations likely alter Erg26 binding to FR171456, based on a model of Erg26. Finally, we show that FR171456 inhibits an artificial Hepatitis C viral replicon, and has broad antifungal activity, suggesting potential additional utility as an anti-infective. The discovery of the target and binding site of FR171456 within the target will aid further development of this compound.
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3-Hidroxiesteroide Deshidrogenasas/antagonistas & inhibidores , Antifúngicos/química , Colesterol/análogos & derivados , Proteínas de Saccharomyces cerevisiae/antagonistas & inhibidores , Saccharomyces cerevisiae/genética , 3-Hidroxiesteroide Deshidrogenasas/genética , Candida albicans , Colesterol/química , Farmacorresistencia Fúngica/genética , Ergosterol/biosíntesis , Mutación , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
Cultivation of myxobacteria of the Nannocystis genus led to the isolation and structure elucidation of a class of novel cyclic lactone inhibitors of elongation factorâ 1. Whole genome sequence analysis and annotation enabled identification of the putative biosynthetic cluster and synthesis process. In biological assays the compounds displayed anti-fungal and cytotoxic activity. Combined genetic and proteomic approaches identified the eukaryotic translation elongation factor 1α (EF-1α) as the primary target for this compound class. Nannocystinâ A (1) displayed differential activity across various cancer cell lines and EEF1A1 expression levels appear to be the main differentiating factor. Biochemical and genetic evidence support an overlapping binding site of 1 with the anti-cancer compound didemninâ B on EF-1α. This myxobacterial chemotype thus offers an interesting starting point for further investigations of the potential of therapeutics targeting elongation factorâ 1.
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Antineoplásicos/química , Proliferación Celular/efectos de los fármacos , Compuestos Macrocíclicos/farmacología , Myxococcales/fisiología , Neoplasias/patología , Factor 1 de Elongación Peptídica/antagonistas & inhibidores , Antifúngicos/química , Antifúngicos/farmacología , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Candida albicans/efectos de los fármacos , Genómica/métodos , Humanos , Compuestos Macrocíclicos/química , Estructura Molecular , Neoplasias/tratamiento farmacológico , Factor 1 de Elongación Peptídica/genética , Factor 1 de Elongación Peptídica/metabolismo , Proteómica/métodos , Relación Estructura-Actividad , Células Tumorales CultivadasRESUMEN
4-Aminothiazolyl analogues of the antibacterial natural product GE2270 A (1) were designed, synthesized, and evaluated for gram positive bacteria growth inhibition. The aminothiazole-based chemical template was evaluated for chemical stability, and its decomposition revealed a novel, structurally simplified, des-thiazole analogue of 1. Subsequent stabilization of the 4-aminothiazolyl functional motif was achieved and initial structure activity relationships defined.
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Antibacterianos/química , Antibacterianos/farmacología , Descubrimiento de Drogas , Péptidos Cíclicos/química , Péptidos Cíclicos/farmacología , Tiazoles/química , Antibacterianos/síntesis química , Bacterias/efectos de los fármacos , Estabilidad de Medicamentos , Pruebas de Sensibilidad Microbiana , Péptidos Cíclicos/síntesis química , Relación Estructura-Actividad , Tiazoles/síntesis química , Tiazoles/farmacologíaRESUMEN
We identified the thiomuracins, a novel family of thiopeptides produced by a rare-actinomycete bacterium typed as a Nonomuraea species, via a screen for inhibition of growth of the bacterial pathogen Staphylococcus aureus. Thiopeptides are a class of macrocyclic, highly modified peptides that are decorated by thiazoles and defined by a central six-membered heterocyclic ring system. Mining the genomes of thiopeptide-producing strains revealed the elusive biosynthetic route for this class of antibiotics. The thiopeptides are chromosomally encoded, ribosomally synthesized proteins, and isolation of gene clusters for production of thiomuracin and the related thiopeptide GE2270A revealed the post-translational machinery required for maturation. The target of the thiomuracins was identified as bacterial Elongation Factor Tu (EF-Tu). In addition to potently inhibiting a target that is unexploited by marketed human therapeutics, the thiomuracins have a low propensity for selecting for antibiotic resistance and confer no measurable cross-resistance to antibiotics in clinical use.
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Antibacterianos/farmacología , Factor Tu de Elongación Peptídica/metabolismo , Péptidos/genética , Péptidos/farmacología , Staphylococcus aureus/efectos de los fármacos , Tiazoles/farmacología , Actinomycetales/química , Secuencia de Aminoácidos , Antibacterianos/química , Antibacterianos/aislamiento & purificación , Proteínas Bacterianas/aislamiento & purificación , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/farmacología , Regulación Bacteriana de la Expresión Génica , Genes Bacterianos , Datos de Secuencia Molecular , Péptidos/química , Péptidos/aislamiento & purificación , Biosíntesis de Proteínas , Staphylococcus aureus/crecimiento & desarrollo , Tiazoles/química , Tiazoles/aislamiento & purificaciónRESUMEN
Epothilones (Epo's) A and B are naturally occurring microtubule-stabilizers, which inhibit the growth of human cancer cells in vitro at low nM or sub-nM concentrations. In contrast to taxol (paclitaxel, Taxol) epothilones are also active against different types of multidrug-resistant cancer cell lines in vitro and against multidrug-resistant tumors in vivo. Their attractive preclinical profile has made epothilones important lead structures in the search for improved cytotoxic anticancer drugs and Epo B (EPO906, patupilone) is currently undergoing Phase III clinical trials. Numerous synthetic and semisynthetic analogs have been prepared since the absolute stereochemistry of epothilones was first disclosed in mid-1996 and their in vitro biological activity has been determined. Apart from generating a wealth of SAR information, these efforts have led to the identification of at least six compounds (in addition to Epo B), which are currently at various stages of clinical evaluation in humans. The most advanced of these compounds, Epo B lactam BMS-247550 (ixabepilone), has recently obtained FDA approval for the treatment of metastatic and advanced breast cancer. This chapter will first provide a summary of the basic features of the biological profile of Epo B in vitro and in vivo. This will be followed by a review of the processes that have been developed for the fermentative production of Epo B. The main part of the chapter will focus on the most relevant aspects of the epothilone SAR with regard to effects on tubulin polymerization, in vitro antiproliferative activity, and in vivo antitumor activity. Particular emphasis will be placed on work conducted in the authors' own laboratories, but data from other groups will also be included. In a final section, the current status of those epothilone analogs undergoing clinical development will be briefly discussed.
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Antineoplásicos/farmacología , Productos Biológicos/farmacología , Diseño de Fármacos , Epotilonas/farmacología , Fermentación , Microbiología Industrial , Moduladores de Tubulina/farmacología , Animales , Antineoplásicos/química , Antineoplásicos/metabolismo , Productos Biológicos/biosíntesis , Productos Biológicos/química , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Epotilonas/biosíntesis , Epotilonas/química , Humanos , Concentración 50 Inhibidora , Microtúbulos/efectos de los fármacos , Estructura Molecular , Relación Estructura-Actividad , Moduladores de Tubulina/química , Moduladores de Tubulina/metabolismoRESUMEN
Inducible transgene expression technologies are of unmatched potential for biopharmaceutical manufacturing of unstable, growth-impairing and cytotoxic proteins as well as conditional metabolic engineering to improve desired cell phenotypes. Currently available transgene dosing modalities which rely on physical parameters or small-molecule drugs for transgene fine-tuning compromise downstream processing and/or are difficult to implement technologically. The recently designed gas-inducible acetaldehyde-inducible regulation (AIR) technology takes advantage of gaseous acetaldehyde to modulate product gene expression levels. At regulation effective concentrations gaseous acetaldehyde is physiologically inert and approved as food additive by the Federal Drug Administration (FDA). During standard bioreactor operation, gaseous acetaldehyde could simply be administered using standard/existing gas supply tubing and eventually eliminated by stripping with inducer-free air. We have determined key parameters controlling acetaldehyde transfer in three types of bioreactors and designed a mass balance-based model for optimal product gene expression fine-tuning using gaseous acetaldehyde. Operating a standard stirred-tank bioreactor set-up at 10 L scale we have validated AIR technology using CHO-K1-derived serum-free suspension cultures transgenic for gas-inducible production of human interferon-beta (IFN-beta). Gaseous acetaldehyde-inducible IFN-beta production management was fully reversible while maintaining cell viability at over 95% during the entire process. Compatible with standard bioreactor design and downstream processing procedures AIR-based technology will foster novel opportunities for pilot and large-scale manufacturing of difficult-to-produce protein pharmaceuticals.
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Acetaldehído/farmacología , Reactores Biológicos , Gases/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Interferón beta/biosíntesis , Ingeniería de Proteínas/métodos , Proteínas Recombinantes/biosíntesis , Animales , Células CHO , Técnicas de Cultivo de Célula/métodos , Simulación por Computador , Cricetinae , Cricetulus , Regulación de la Expresión Génica/fisiología , Humanos , Interferón beta/genética , Modelos Biológicos , Proyectos PilotoRESUMEN
Homeostasis under hypoxic conditions is maintained through a coordinated transcriptional response mediated by the hypoxia-inducible factor (HIF) pathway and requires coactivation by the CBP and p300 transcriptional coactivators. Through a target-based high-throughput screen, we identified chetomin as a disrupter of HIF binding to p300. At a molecular level, chetomin disrupts the structure of the CH1 domain of p300 and precludes its interaction with HIF, thereby attenuating hypoxia-inducible transcription. Systemic administration of chetomin inhibited hypoxia-inducible transcription within tumors and inhibited tumor growth. These results demonstrate a therapeutic window for pharmacological attenuation of HIF activity and further establish the feasibility of disrupting a signal transduction pathway by targeting the function of a transcriptional coactivator with a small molecule.
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Antibacterianos/farmacología , Proteínas de Unión al ADN , Proteínas Nucleares/metabolismo , Receptores de Hidrocarburo de Aril/metabolismo , Transactivadores/metabolismo , Factores de Transcripción/metabolismo , Transcripción Genética/efectos de los fármacos , Animales , Translocador Nuclear del Receptor de Aril Hidrocarburo , Carcinoma Hepatocelular/patología , Hipoxia de la Célula/genética , Neoplasias del Colon/metabolismo , Neoplasias del Colon/patología , Neoplasias del Colon/terapia , Disulfuros , Proteína p300 Asociada a E1A , Eritropoyetina/metabolismo , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia , Alcaloides Indólicos , Neoplasias Hepáticas/patología , Luciferasas/metabolismo , Masculino , Ratones , Ratones Desnudos , Proteínas Nucleares/genética , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patología , Neoplasias de la Próstata/terapia , Unión Proteica/efectos de los fármacos , Receptores de Hidrocarburo de Aril/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal , Transactivadores/genética , Factores de Transcripción/genética , Trasplante Heterólogo , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
A group of cyclic peptides consisting of 8 amino acid residues, named argyrins A to H, were isolated from the culture broth of strains of the myxobacterium Archangium gephyra. Argyrin B was found to be a potent inhibitor of T cell independent antibody formation by murine B cells and strongly inhibited the two way murine mixed lymphocyte reaction. All argyrins had slight antibiotic activity, especially against Pseudomonas sp., and inhibited growth of mammalian cell cultures. The growth inhibition was often incomplete and varied highly with different cell lines.
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Inmunosupresores/aislamiento & purificación , Myxococcales/química , Péptidos Cíclicos/aislamiento & purificación , Animales , Linfocitos B/metabolismo , Fermentación , Fibroblastos , Inmunosupresores/química , Inmunosupresores/farmacología , Concentración 50 Inhibidora , Ratones , Pruebas de Sensibilidad Microbiana , Myxococcales/metabolismo , Péptidos Cíclicos/química , Péptidos Cíclicos/farmacología , Relación Estructura-Actividad , Linfocitos T/metabolismoRESUMEN
As the interest of research is beginning to shift from genomicsto proteomics the number of proteins to be expressed is rapidlyincreasing. To do so, well-established, high-level expressionsystems and rapid, cost-effective production means are needed. For addressing the latter, a novel cultivation system for recombinant cells, the Wave Bioreactortrade mark has recently becomeavailable. We describe the set-up and the optimisation of parameters essential for successful operation and growth of insect cells to high cell densities in the Wave Bioreactor. According to our experience, the Cellbagtrade mark system comparesvery favorably to conventional cultivation vessels such as bioreactors and roller cultures with respect to simplicity ofoperation and cost. Additionally, we developed a rapid and simple protocol for assessing expression and production conditions for the Baculovirus/insect cell system applicable to many different genes/proteins. Important parameters like MOI,TOI, peak cell density (PCD) and expression levels are determinedin pre-experiments on small scale to achieve optimal expressionof a given protein. These conditions are subsequently transformedand applied to large scale cultures grown in nutrient-supplemented medium in the Wave Bioreactor.