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1.
Nature ; 550(7674): 114-118, 2017 10 05.
Artículo en Inglés | MEDLINE | ID: mdl-28953874

RESUMEN

The ability to directly uncover the contributions of genes to a given phenotype is fundamental for biology research. However, ostensibly homogeneous cell populations exhibit large clonal variance that can confound analyses and undermine reproducibility. Here we used genome-saturated mutagenesis to create a biobank of over 100,000 individual haploid mouse embryonic stem (mES) cell lines targeting 16,970 genes with genetically barcoded, conditional and reversible mutations. This Haplobank is, to our knowledge, the largest resource of hemi/homozygous mutant mES cells to date and is available to all researchers. Reversible mutagenesis overcomes clonal variance by permitting functional annotation of the genome directly in sister cells. We use the Haplobank in reverse genetic screens to investigate the temporal resolution of essential genes in mES cells, and to identify novel genes that control sprouting angiogenesis and lineage specification of blood vessels. Furthermore, a genome-wide forward screen with Haplobank identified PLA2G16 as a host factor that is required for cytotoxicity by rhinoviruses, which cause the common cold. Therefore, clones from the Haplobank combined with the use of reversible technologies enable high-throughput, reproducible, functional annotation of the genome.


Asunto(s)
Bancos de Muestras Biológicas , Genómica/métodos , Haploidia , Células Madre Embrionarias de Ratones/metabolismo , Mutación , Animales , Vasos Sanguíneos/citología , Linaje de la Célula/genética , Resfriado Común/genética , Resfriado Común/virología , Genes Esenciales/genética , Pruebas Genéticas , Células HEK293 , Homocigoto , Humanos , Ratones , Células Madre Embrionarias de Ratones/citología , Neovascularización Fisiológica/genética , Fosfolipasas A2 Calcio-Independiente/genética , Fosfolipasas A2 Calcio-Independiente/metabolismo , Rhinovirus/patogenicidad
2.
PLoS Genet ; 12(11): e1006374, 2016 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-27855158

RESUMEN

Invasive infections by fungal pathogens cause more deaths than malaria worldwide. We found the ergoline compound NGx04 in an antifungal screen, with selectivity over mammalian cells. High-resolution chemogenomics identified the lipid transfer protein Sec14p as the target of NGx04 and compound-resistant mutations in Sec14p define compound-target interactions in the substrate binding pocket of the protein. Beyond its essential lipid transfer function in a variety of pathogenic fungi, Sec14p is also involved in secretion of virulence determinants essential for the pathogenicity of fungi such as Cryptococcus neoformans, making Sec14p an attractive antifungal target. Consistent with this dual function, we demonstrate that NGx04 inhibits the growth of two clinical isolates of C. neoformans and that NGx04-related compounds have equal and even higher potency against C. neoformans. Furthermore NGx04 analogues showed fungicidal activity against a fluconazole resistant C. neoformans strain. In summary, we present genetic evidence that NGx04 inhibits fungal Sec14p and initial data supporting NGx04 as a novel antifungal starting point.


Asunto(s)
Proteínas Portadoras/química , Criptococosis/tratamiento farmacológico , Cryptococcus neoformans/efectos de los fármacos , Ergolinas/farmacología , Metabolismo de los Lípidos/efectos de los fármacos , Antifúngicos/farmacología , Proteínas Portadoras/genética , Criptococosis/microbiología , Cryptococcus neoformans/genética , Cryptococcus neoformans/patogenicidad , Ergolinas/química , Humanos , Pruebas de Sensibilidad Microbiana , Conformación Proteica , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/genética
3.
J Cell Sci ; 128(6): 1217-29, 2015 Mar 15.
Artículo en Inglés | MEDLINE | ID: mdl-25616894

RESUMEN

A new cyclic decadepsipeptide was isolated from Chaetosphaeria tulasneorum with potent bioactivity on mammalian and yeast cells. Chemogenomic profiling in S. cerevisiae indicated that the Sec61 translocon complex, the machinery for protein translocation and membrane insertion at the endoplasmic reticulum, is the target. The profiles were similar to those of cyclic heptadepsipeptides of a distinct chemotype (including HUN-7293 and cotransin) that had previously been shown to inhibit cotranslational translocation at the mammalian Sec61 translocon. Unbiased, genome-wide mutagenesis followed by full-genome sequencing in both fungal and mammalian cells identified dominant mutations in Sec61p (yeast) or Sec61α1 (mammals) that conferred resistance. Most, but not all, of these mutations affected inhibition by both chemotypes, despite an absence of structural similarity. Biochemical analysis confirmed inhibition of protein translocation into the endoplasmic reticulum of both co- and post-translationally translocated substrates by both chemotypes, demonstrating a mechanism independent of a translating ribosome. Most interestingly, both chemotypes were found to also inhibit SecYEG, the bacterial Sec61 translocon homolog. We suggest 'decatransin' as the name for this new decadepsipeptide translocation inhibitor.


Asunto(s)
Productos Biológicos/farmacología , Retículo Endoplásmico/efectos de los fármacos , Proteínas de la Membrana/metabolismo , Transporte de Proteínas/efectos de los fármacos , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Animales , Ascomicetos/metabolismo , Células COS , Células Cultivadas , Chlorocebus aethiops , Células HCT116 , Humanos , Proteínas de la Membrana/antagonistas & inhibidores , Péptidos Cíclicos/farmacología , Polimorfismo de Nucleótido Simple/genética , Canales de Translocación SEC , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crecimiento & desarrollo
4.
Angew Chem Int Ed Engl ; 54(35): 10149-54, 2015 Aug 24.
Artículo en Inglés | MEDLINE | ID: mdl-26179970

RESUMEN

Cultivation of myxobacteria of the Nannocystis genus led to the isolation and structure elucidation of a class of novel cyclic lactone inhibitors of elongation factor 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. Nannocystin 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 didemnin B on EF-1α. This myxobacterial chemotype thus offers an interesting starting point for further investigations of the potential of therapeutics targeting elongation factor 1.


Asunto(s)
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 Cultivadas
5.
Antimicrob Agents Chemother ; 57(5): 2272-80, 2013 May.
Artículo en Inglés | MEDLINE | ID: mdl-23478965

RESUMEN

High-throughput phenotypic screening against the yeast Saccharomyces cerevisiae revealed a series of triazolopyrimidine-sulfonamide compounds with broad-spectrum antifungal activity, no significant cytotoxicity, and low protein binding. To elucidate the target of this series, we have applied a chemogenomic profiling approach using the S. cerevisiae deletion collection. All compounds of the series yielded highly similar profiles that suggested acetolactate synthase (Ilv2p, which catalyzes the first common step in branched-chain amino acid biosynthesis) as a possible target. The high correlation with profiles of known Ilv2p inhibitors like chlorimuron-ethyl provided further evidence for a similar mechanism of action. Genome-wide mutagenesis in S. cerevisiae identified 13 resistant clones with 3 different mutations in the catalytic subunit of acetolactate synthase that also conferred cross-resistance to established Ilv2p inhibitors. Mapping of the mutations into the published Ilv2p crystal structure outlined the chlorimuron-ethyl binding cavity, and it was possible to dock the triazolopyrimidine-sulfonamide compound into this pocket in silico. However, fungal growth inhibition could be bypassed through supplementation with exogenous branched-chain amino acids or by the addition of serum to the medium in all of the fungal organisms tested except for Aspergillus fumigatus. Thus, these data support the identification of the triazolopyrimidine-sulfonamide compounds as inhibitors of acetolactate synthase but suggest that targeting may be compromised due to the possibility of nutrient bypass in vivo.


Asunto(s)
Acetolactato Sintasa/antagonistas & inhibidores , Antifúngicos/farmacología , Pirimidinas/farmacología , Proteínas de Saccharomyces cerevisiae/antagonistas & inhibidores , Saccharomyces cerevisiae/efectos de los fármacos , Sulfonamidas/farmacología , Compuestos de Sulfonilurea/farmacología , Acetolactato Sintasa/química , Acetolactato Sintasa/genética , Acetolactato Sintasa/metabolismo , Aminoácidos de Cadena Ramificada/metabolismo , Aminoácidos de Cadena Ramificada/farmacología , Antifúngicos/química , Dominio Catalítico/efectos de los fármacos , Ensayos Analíticos de Alto Rendimiento , Humanos , Pruebas de Sensibilidad Microbiana , Simulación del Acoplamiento Molecular , Mutación , Unión Proteica , Pirimidinas/química , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Suero/química , Suero/metabolismo , Sulfonamidas/química , Compuestos de Sulfonilurea/química
7.
Nat Commun ; 11(1): 3387, 2020 07 07.
Artículo en Inglés | MEDLINE | ID: mdl-32636417

RESUMEN

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.


Asunto(s)
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 cerevisiae
8.
Structure ; 14(8): 1293-302, 2006 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-16905103

RESUMEN

Deubiquitinating proteases reverse protein ubiquitination and rescue their target proteins from destruction by the proteasome. USP2, a cysteine protease and a member of the ubiquitin specific protease family, is overexpressed in prostate cancer and stabilizes fatty acid synthase, which has been associated with the malignancy of some aggressive prostate cancers. Here, we report the structure of the human USP2 catalytic domain in complex with ubiquitin. Ubiquitin uses two major sites for the interaction with the protease. Both sites are required simultaneously, as shown by USP2 inhibition assays with peptides and ubiquitin mutants. In addition, a layer of ordered water molecules mediates key interactions between ubiquitin and USP2. As several of those molecules are found at identical positions in the previously solved USP7/ubiquitin-aldehyde complex structure, we suggest a general mechanism of water-mediated ubiquitin recognition by USPs.


Asunto(s)
Endopeptidasas/química , Endopeptidasas/metabolismo , Modelos Moleculares , Ubiquitina/metabolismo , Secuencia de Aminoácidos , Cristalización , Cartilla de ADN , Endopeptidasas/genética , Humanos , Cinética , Metales/metabolismo , Datos de Secuencia Molecular , Estructura Terciaria de Proteína , Ubiquitina/química , Ubiquitina Tiolesterasa , Agua/metabolismo
9.
Cell Chem Biol ; 25(3): 279-290.e7, 2018 03 15.
Artículo en Inglés | MEDLINE | ID: mdl-29307839

RESUMEN

Invasive fungal infections are accompanied by high mortality rates that range up to 90%. At present, only three different compound classes are available for use in the clinic, and these often suffer from low bioavailability, toxicity, and drug resistance. These issues emphasize an urgent need for novel antifungal agents. Herein, we report the identification of chemically versatile benzamide and picolinamide scaffolds with antifungal properties. Chemogenomic profiling and biochemical assays with purified protein identified Sec14p, the major phosphatidylinositol/phosphatidylcholine transfer protein in Saccharomyces cerevisiae, as the sole essential target for these compounds. A functional variomics screen identified resistance-conferring residues that localized to the lipid-binding pocket of Sec14p. Determination of the X-ray co-crystal structure of a Sec14p-compound complex confirmed binding in this cavity and rationalized both the resistance-conferring residues and the observed structure-activity relationships. Taken together, these findings open new avenues for rational compound optimization and development of novel antifungal agents.


Asunto(s)
Antifúngicos/metabolismo , Benzamidas/química , Ácidos Picolínicos/química , Amidas/química , Amidas/metabolismo , Amidas/farmacología , Secuencia de Aminoácidos , Antifúngicos/química , Antifúngicos/farmacología , Aspergillus/efectos de los fármacos , Benzamidas/metabolismo , Benzamidas/farmacología , Sitios de Unión , Candida albicans/efectos de los fármacos , Cristalografía por Rayos X , Farmacorresistencia Fúngica/efectos de los fármacos , Pruebas de Sensibilidad Microbiana , Simulación de Dinámica Molecular , Mutagénesis Sitio-Dirigida , Proteínas de Transferencia de Fosfolípidos/química , Proteínas de Transferencia de Fosfolípidos/genética , Proteínas de Transferencia de Fosfolípidos/metabolismo , Ácidos Picolínicos/metabolismo , Ácidos Picolínicos/farmacología , Estructura Terciaria de Proteína , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Alineación de Secuencia , Relación Estructura-Actividad
10.
Cell Rep ; 19(3): 451-460, 2017 04 18.
Artículo en Inglés | MEDLINE | ID: mdl-28423309

RESUMEN

Flavivirus infections by Zika and dengue virus impose a significant global healthcare threat with no US Food and Drug Administration (FDA)-approved vaccination or specific antiviral treatment available. Here, we present the discovery of an anti-flaviviral natural product named cavinafungin. Cavinafungin is a potent and selectively active compound against Zika and all four dengue virus serotypes. Unbiased, genome-wide genomic profiling in human cells using a novel CRISPR/Cas9 protocol identified the endoplasmic-reticulum-localized signal peptidase as the efficacy target of cavinafungin. Orthogonal profiling in S. cerevisiae followed by the selection of resistant mutants pinpointed the catalytic subunit of the signal peptidase SEC11 as the evolutionary conserved target. Biochemical analysis confirmed a rapid block of signal sequence cleavage of both host and viral proteins by cavinafungin. This study provides an effective compound against the eukaryotic signal peptidase and independent confirmation of the recently identified critical role of the signal peptidase in the replicative cycle of flaviviruses.


Asunto(s)
Productos Biológicos/farmacología , Virus del Dengue/fisiología , Lipopéptidos/farmacología , Replicación Viral/efectos de los fármacos , Virus Zika/fisiología , Productos Biológicos/química , Sistemas CRISPR-Cas/genética , Virus del Dengue/efectos de los fármacos , Técnicas de Silenciamiento del Gen , Genoma Humano , Genómica , Células HCT116 , Humanos , Lipopéptidos/química , Proteínas de la Membrana , Subunidades de Proteína/metabolismo , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/genética , Serina Endopeptidasas , Proteínas Virales/metabolismo , Virus Zika/efectos de los fármacos
11.
J Biomol Screen ; 21(3): 306-15, 2016 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-26459507

RESUMEN

Phenotypic screens are effective starting points to identify compounds with desirable activities. To find novel antifungals, we conducted a phenotypic screen in Saccharomyces cerevisiae and identified two discrete scaffolds with good growth inhibitory characteristics. Lack of broad-spectrum activity against pathogenic fungi called for directed chemical compound optimization requiring knowledge of the molecular target. Chemogenomic profiling identified effects on geranylgeranyltransferase I (GGTase I), an essential enzyme that prenylates proteins involved in cell signaling, such as Cdc42p and Rho1p. Selection of resistant mutants against both compounds confirmed the target hypothesis and enabled mapping of the compound binding site to the substrate binding pocket. Differential resistance-conferring mutations and selective substrate competition demonstrate distinct binding modes for the two chemotypes. Exchange of the S. cerevisiae GGTase I subunits with those of Candida albicans resulted in an absence of growth inhibition for both compounds, thus confirming the identified target as well as the narrow antifungal spectrum of activity. This prenylation pathway is reported to be nonessential in pathogenic species and challenges the therapeutic value of these leads while demonstrating the importance of an integrated target identification platform following a phenotypic screen.


Asunto(s)
Transferasas Alquil y Aril/antagonistas & inhibidores , Antifúngicos/farmacología , Descubrimiento de Drogas/métodos , Inhibidores Enzimáticos/farmacología , Pruebas de Sensibilidad Microbiana , Transferasas Alquil y Aril/química , Transferasas Alquil y Aril/genética , Transferasas Alquil y Aril/metabolismo , Antifúngicos/química , Farmacorresistencia Fúngica , Inhibidores Enzimáticos/química , Perfilación de la Expresión Génica , Metabolómica/métodos , Modelos Moleculares , Conformación Molecular , Mutación , Unión Proteica , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/genética
12.
Chem Biol ; 22(1): 87-97, 2015 Jan 22.
Artículo en Inglés | MEDLINE | ID: mdl-25544045

RESUMEN

The highly conserved 70 kDa heat shock proteins (Hsp70) play an integral role in proteostasis such that dysregulation has been implicated in numerous diseases. Elucidating the precise role of Hsp70 family members in the cellular context, however, has been hampered by the redundancy and intricate regulation of the chaperone network, and relatively few selective and potent tools. We have characterized a natural product, novolactone, that targets cytosolic and ER-localized isoforms of Hsp70 through a highly conserved covalent interaction at the interface between the substrate-binding and ATPase domains. Biochemical and structural analyses indicate that novolactone disrupts interdomain communication by allosterically inducing a conformational change in the Hsp70 protein to block ATP-induced substrate release and inhibit refolding activities. Thus, novolactone is a valuable tool for exploring the requirements of Hsp70 chaperones in diverse cellular contexts.


Asunto(s)
Abietanos/metabolismo , Productos Biológicos/metabolismo , Proteínas HSP70 de Choque Térmico/metabolismo , Abietanos/química , Adenosina Trifosfatasas/metabolismo , Regulación Alostérica , Sitios de Unión , Productos Biológicos/química , Línea Celular , Cristalografía por Rayos X , Retículo Endoplásmico/metabolismo , Genoma Fúngico , Proteínas del Choque Térmico HSP40/metabolismo , Proteínas HSP70 de Choque Térmico/química , Humanos , Simulación de Dinámica Molecular , Unión Proteica , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Estructura Terciaria de Proteína , Saccharomyces cerevisiae/genética , Especificidad por Sustrato
13.
Nat Commun ; 6: 8613, 2015 Oct 12.
Artículo en Inglés | MEDLINE | ID: mdl-26456460

RESUMEN

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.


Asunto(s)
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/genética
14.
Microbiol Res ; 169(2-3): 107-20, 2014.
Artículo en Inglés | MEDLINE | ID: mdl-24360837

RESUMEN

Due to evolutionary conservation of biology, experimental knowledge captured from genetic studies in eukaryotic model organisms provides insight into human cellular pathways and ultimately physiology. Yeast chemogenomic profiling is a powerful approach for annotating cellular responses to small molecules. Using an optimized platform, we provide the relative sensitivities of the heterozygous and homozygous deletion collections for nearly 1800 biologically active compounds. The data quality enables unique insights into pathways that are sensitive and resistant to a given perturbation, as demonstrated with both known and novel compounds. We present examples of novel compounds that inhibit the therapeutically relevant fatty acid synthase and desaturase (Fas1p and Ole1p), and demonstrate how the individual profiles facilitate hypothesis-driven experiments to delineate compound mechanism of action. Importantly, the scale and diversity of tested compounds yields a dataset where the number of modulated pathways approaches saturation. This resource can be used to map novel biological connections, and also identify functions for unannotated genes. We validated hypotheses generated by global two-way hierarchical clustering of profiles for (i) novel compounds with a similar mechanism of action acting upon microtubules or vacuolar ATPases, and (ii) an un-annotated ORF, YIL060w, that plays a role in respiration in the mitochondria. Finally, we identify and characterize background mutations in the widely used yeast deletion collection which should improve the interpretation of past and future screens throughout the community. This comprehensive resource of cellular responses enables the expansion of our understanding of eukaryotic pathway biology.


Asunto(s)
Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/genética , Antifúngicos/farmacología , Vías Biosintéticas , Farmacorresistencia Fúngica , Regulación Fúngica de la Expresión Génica , Ensayos Analíticos de Alto Rendimiento , Datos de Secuencia Molecular , Filogenia , Saccharomyces cerevisiae/clasificación , Saccharomyces cerevisiae/efectos de los fármacos , Proteínas de Saccharomyces cerevisiae/metabolismo
15.
ACS Chem Biol ; 8(7): 1519-27, 2013 Jul 19.
Artículo en Inglés | MEDLINE | ID: mdl-23614532

RESUMEN

Translation initiation is an emerging target in oncology and neurobiology indications. Naturally derived and synthetic rocaglamide scaffolds have been used to interrogate this pathway; however, there is uncertainty regarding their precise mechanism(s) of action. We exploited the genetic tractability of yeast to define the primary effect of both a natural and a synthetic rocaglamide in a cellular context and characterized the molecular target using biochemical studies and in silico modeling. Chemogenomic profiling and mutagenesis in yeast identified the eIF (eukaryotic Initiation Factor) 4A helicase homologue as the primary molecular target of rocaglamides and defined a discrete set of residues near the RNA binding motif that confer resistance to both compounds. Three of the eIF4A mutations were characterized regarding their functional consequences on activity and response to rocaglamide inhibition. These data support a model whereby rocaglamides stabilize an eIF4A-RNA interaction to either alter the level and/or impair the activity of the eIF4F complex. Furthermore, in silico modeling supports the annotation of a binding pocket delineated by the RNA substrate and the residues identified from our mutagenesis screen. As expected from the high degree of conservation of the eukaryotic translation pathway, these observations are consistent with previous observations in mammalian model systems. Importantly, we demonstrate that the chemically distinct silvestrol and synthetic rocaglamides share a common mechanism of action, which will be critical for optimization of physiologically stable derivatives. Finally, these data confirm the value of the rocaglamide scaffold for exploring the impact of translational modulation on disease.


Asunto(s)
Benzofuranos/metabolismo , Factor 4F Eucariótico de Iniciación/química , Factor 4F Eucariótico de Iniciación/metabolismo , Saccharomyces cerevisiae/metabolismo , Benzofuranos/química , Sitios de Unión , Modelos Biológicos , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/genética , Triterpenos/química , Triterpenos/metabolismo
16.
Cell Host Microbe ; 11(6): 654-63, 2012 Jun 14.
Artículo en Inglés | MEDLINE | ID: mdl-22704625

RESUMEN

With renewed calls for malaria eradication, next-generation antimalarials need be active against drug-resistant parasites and efficacious against both liver- and blood-stage infections. We screened a natural product library to identify inhibitors of Plasmodium falciparum blood- and liver-stage proliferation. Cladosporin, a fungal secondary metabolite whose target and mechanism of action are not known for any species, was identified as having potent, nanomolar, antiparasitic activity against both blood and liver stages. Using postgenomic methods, including a yeast deletion strains collection, we show that cladosporin specifically inhibits protein synthesis by directly targeting P. falciparum cytosolic lysyl-tRNA synthetase. Further, cladosporin is >100-fold more potent against parasite lysyl-tRNA synthetase relative to the human enzyme, which is conferred by the identity of two amino acids within the enzyme active site. Our data indicate that lysyl-tRNA synthetase is an attractive, druggable, antimalarial target that can be selectively inhibited.


Asunto(s)
Antimaláricos/farmacología , Inhibidores Enzimáticos/farmacología , Hongos/química , Isocumarinas/farmacología , Lisina-ARNt Ligasa/antagonistas & inhibidores , Plasmodium falciparum/enzimología , Antimaláricos/aislamiento & purificación , Línea Celular , Evaluación Preclínica de Medicamentos/métodos , Inhibidores Enzimáticos/aislamiento & purificación , Humanos , Concentración 50 Inhibidora , Isocumarinas/aislamiento & purificación , Pruebas de Sensibilidad Parasitaria , Plasmodium falciparum/efectos de los fármacos , Biosíntesis de Proteínas/efectos de los fármacos , Proteínas Protozoarias/antagonistas & inhibidores
17.
PLoS One ; 7(9): e42657, 2012.
Artículo en Inglés | MEDLINE | ID: mdl-22970117

RESUMEN

Argyrins, produced by myxobacteria and actinomycetes, are cyclic octapeptides with antibacterial and antitumor activity. Here, we identify elongation factor G (EF-G) as the cellular target of argyrin B in bacteria, via resistant mutant selection and whole genome sequencing, biophysical binding studies and crystallography. Argyrin B binds a novel allosteric pocket in EF-G, distinct from the known EF-G inhibitor antibiotic fusidic acid, revealing a new mode of protein synthesis inhibition. In eukaryotic cells, argyrin B was found to target mitochondrial elongation factor G1 (EF-G1), the closest homologue of bacterial EF-G. By blocking mitochondrial translation, argyrin B depletes electron transport components and inhibits the growth of yeast and tumor cells. Further supporting direct inhibition of EF-G1, expression of an argyrin B-binding deficient EF-G1 L693Q variant partially rescued argyrin B-sensitivity in tumor cells. In summary, we show that argyrin B is an antibacterial and cytotoxic agent that inhibits the evolutionarily conserved target EF-G, blocking protein synthesis in bacteria and mitochondrial translation in yeast and mammalian cells.


Asunto(s)
Oligopéptidos/metabolismo , Factor G de Elongación Peptídica/metabolismo , Sitio Alostérico , Secuencia de Aminoácidos , Animales , Burkholderia/efectos de los fármacos , Línea Celular Tumoral , Secuencia Conservada , Cristalografía por Rayos X , Humanos , Mamíferos , Pruebas de Sensibilidad Microbiana , Proteínas Mitocondriales/metabolismo , Datos de Secuencia Molecular , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Oligopéptidos/química , Oligopéptidos/farmacología , Factor G de Elongación Peptídica/antagonistas & inhibidores , Factor G de Elongación Peptídica/química , Unión Proteica/efectos de los fármacos , Pseudomonas aeruginosa/efectos de los fármacos , Saccharomyces cerevisiae/metabolismo , Homología de Secuencia de Aminoácido
18.
New Phytol ; 172(4): 739-52, 2006.
Artículo en Inglés | MEDLINE | ID: mdl-17096799

RESUMEN

Previous studies have shown that arbuscular mycorrhizal fungi (AMF) can influence plant diversity and ecosystem productivity. However, little is known about the effects of AMF and different AMF taxa on other important community properties such as nutrient acquisition, plant survival and soil structure. We established experimental grassland microcosms and tested the impact of AMF and of different AMF taxa on a number of grassland characteristics. We also tested whether plant species benefited from the same or different AMF taxa in subsequent growing seasons. AMF enhanced phosphorus acquisition, soil aggregation and survival of several plant species, but AMF did not increase total plant productivity. Moreover, AMF increased nitrogen acquisition by some plant species, but AMF had no effect on total N uptake by the plant community. Plant growth responses to AMF were temporally variable and some plant species obtained the highest biomass with different AMF in different years. Hence the results indicate that it may be beneficial for a plant to be colonized by different AMF taxa in different seasons. This study shows that AMF play a key role in grassland by improving plant nutrition and soil structure, and by regulating the make-up of the plant community.


Asunto(s)
Ecosistema , Micorrizas/fisiología , Raíces de Plantas/fisiología , Poaceae/fisiología , Suelo , Biomasa , Nitrógeno/metabolismo , Fósforo/metabolismo , Raíces de Plantas/metabolismo , Raíces de Plantas/microbiología , Poaceae/metabolismo , Poaceae/microbiología , Estaciones del Año , Microbiología del Suelo , Simbiosis/fisiología , Factores de Tiempo
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