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Finding effective therapies against cancers driven by mutant and/or overexpressed hyperactive G-proteins remains an area of active research. Polyisoprenylated cysteinyl amide inhibitors (PCAIs) are agents that mimic the essential posttranslational modifications of G-proteins. It is hypothesized that PCAIs work as anticancer agents by disrupting polyisoprenylation-dependent functional interactions of the G-Proteins. This study tested this hypothesis by determining the effect of the PCAIs on the levels of RAS and related monomeric G-proteins. Following 48 h exposure, we found significant decreases in the levels of KRAS, RHOA, RAC1, and CDC42 ranging within 20-66% after NSL-YHJ-2-27 (5 µM) treatment in all four cell lines tested, A549, NCI-H1299, MDA-MB-231, and MDA-MB-468. However, no significant difference was observed on the G-protein, RAB5A. Interestingly, 38 and 44% decreases in the levels of the farnesylated and acylated NRAS were observed in the two breast cancer cell lines, MDA-MB-231, and MDA-MB-468, respectively, while HRAS levels showed a 36% decrease only in MDA-MB-468 cells. Moreover, after PCAIs treatment, migration, and invasion of A549 cells were inhibited by 72 and 70%, respectively while the levels of vinculin and fascin dropped by 33 and 43%, respectively. These findings implicate the potential role of PCAIs as anticancer agents through their direct interaction with monomeric G-proteins.
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Antineoplásicos , Neoplasias de la Mama , Proteínas de Unión al GTP Monoméricas , Humanos , Femenino , Movimiento Celular , Línea Celular Tumoral , Proteínas de Unión al GTP Monoméricas/farmacología , Amidas/farmacología , Neoplasias de la Mama/tratamiento farmacológico , Antineoplásicos/farmacología , Pulmón , Proliferación CelularRESUMEN
The image quality in clinical PET scan can be severely degraded due to high noise levels in extremely obese patients. Our work aimed to reduce the noise in clinical PET images of extremely obese subjects to the noise level of lean subject images, to ensure consistent imaging quality. The noise level was measured by normalized standard deviation (NSTD) derived from a liver region of interest. A deep learning-based noise reduction method with a fully 3D patch-based U-Net was used. Two U-Nets, U-Nets A and B, were trained on datasets with 40% and 10% count levels derived from 100 lean subjects, respectively. The clinical PET images of 10 extremely obese subjects were denoised using the two U-Nets. The results showed the noise levels of the images with 40% counts of lean subjects were consistent with those of the extremely obese subjects. U-Net A effectively reduced the noise in the images of the extremely obese patients while preserving the fine structures. The liver NSTD improved from 0.13±0.04 to 0.08±0.03 after noise reduction (p = 0.01). After denoising, the image noise level of extremely obese subjects was similar to that of lean subjects, in terms of liver NSTD (0.08±0.03 vs. 0.08±0.02, p = 0.74). In contrast, U-Net B over-smoothed the images of extremely obese patients, resulting in blurred fine structures. In a pilot reader study comparing extremely obese patients without and with U-Net A, the difference was not significant. In conclusion, the U-Net trained by datasets from lean subjects with matched count level can provide promising denoising performance for extremely obese subjects while maintaining image resolution, though further clinical evaluation is needed.
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Hepatocellular carcinoma (HCC), the most common primary liver cancer, is usually diagnosed in its late state. Tyrosine kinase inhibitors such as sorafenib and regorafenib are one of the few treatment options approved for advanced HCC and only prolong the patient's life expectancy by a few months. Therefore, there is a need for novel effective treatments. Cyclophilins are intracellular proteins that catalyze the cis/trans isomerization of peptide bonds at proline residues. Cyclophilins are known to be overexpressed in HCC, affecting therapy resistance and cell proliferation. In the present study, we explored the potential of cyclophilin inhibitors as new therapeutic options for HCC in vitro and in vivo. Our results showed that the novel cyclophilin inhibitor, NV651, was able to significantly decrease proliferation in a diverse set of HCC cell lines. The exposure of HCC cells to NV651 caused an accumulation of cells during mitosis and consequent accumulation in the G2/M phase of the cell cycle. NV651 reduced tumor growth in vivo using an HCC xenograft model without affecting the body weights of the animals. The safety aspects of NV651 were also confirmed in primary human hepatocytes without any cytotoxic effects. Based on the results obtained in this study, we propose NV651 as a potential treatment strategy for HCC.
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Changing wildfire regimes are causing rapid shifts in forests worldwide. In particular, forested landscapes that burn repeatedly in relatively quick succession may be at risk of conversion when pre-fire vegetation cannot recover between fires. Fire refugia (areas that burn less frequently or severely than the surrounding landscape) support post-fire ecosystem recovery and the persistence of vulnerable species in fire-prone landscapes. Observed and projected fire-induced forest losses highlight the need to understand where and why forests persist in refugia through multiple fires. This research need is particularly acute in the Klamath-Siskiyou ecoregion of southwest Oregon and northwest California, USA, where expected increases in fire activity and climate warming may result in the loss of up to one-third of the region's conifer forests, which are the most diverse in western North America. Here, we leverage recent advances in fire progression mapping and weather interpolation, in conjunction with a novel application of satellite smoke imagery, to model the key controls on fire refugia occurrence and persistence through one, two, and three fire events over a 32-year period. Hotter-than-average fire weather was associated with lower refugia probability and higher fire severity. Refugia that persisted through three fire events appeared to be partially entrained by landscape features that offered protection from fire, suggesting that topographic variability may be an important stabilizing factor as forests pass through successive fire filters. In addition, smoke density strongly influenced fire effects, with fire refugia more likely to occur when smoke was moderate or dense in the morning, a relationship attributable to reduced incoming solar radiation resulting from smoke shading. Results from this study could inform management strategies designed to protect fire-resistant portions of biologically and topographically diverse landscapes.
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Refugio de Fauna , Tracheophyta , Ecosistema , Bosques , América del Norte , OregonRESUMEN
Technical advances in genome sequencing, in particular whole-genome sequencing (WGS), provide adequate tools to understanding cancer at the molecular level while specifically focusing on genetic variants that contribute to the causation and progression of pathogenic cancers. Multiple myeloma (MM), a malignant disease of plasma cells that is marked as rare yet incurable, may be diagnosed by WGS tools, as this cancer is associated with chromosomal translocations and mutations in specific protein-coding genes. Among these protein-coding genes, many are known to be responsible for cell cycle regulation in MM. The initial significant protein-coding mutations were found in NRAS, KRAS and TP53 and later reported in FAM46C, DIS3, CCND1, PNRC1, ALOX12B, HLA-A and MAGED1. Here, we report gene network associations of MM using Qiagen's Ingenuity Pathway Analysis (IPA) software and compared biomarker information reported in IPA for these protein-coding genes (NRAS, TP53 and KRAS). Using Qiagen's Ingenuity Variant Analysis (IVA), we characterized cancer driver variants in MT-ND1 as likely pathogenic or variants of uncertain significance.
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Biomarcadores de Tumor/genética , Variación Genética , Mieloma Múltiple/genética , NADH Deshidrogenasa/genética , Progresión de la Enfermedad , Redes Reguladoras de Genes , Genes p53 , Genes ras , Humanos , Mieloma Múltiple/patología , Programas InformáticosRESUMEN
Hepatic fibrosis can result as a pathological response to nonalcoholic steatohepatitis (NASH). Cirrhosis, the late stage of fibrosis, has been linked to poor survival and an increased risk of developing hepatocellular carcinoma, with limited treatment options available. Therefore, there is an unmet need for novel effective antifibrotic compounds. Cyclophilins are peptidyl-prolyl cis-trans isomerases that facilitate protein folding and conformational changes affecting the function of the targeted proteins. Due to their activity, cyclophilins have been presented as key factors in several stages of the fibrotic process. In this study, we investigated the antifibrotic effects of NV556, a novel potent sanglifehrin-based cyclophilin inhibitor, in vitro and in vivo. NV556 potential antifibrotic effect was evaluated in two well-established animal models of NASH, STAM, and methionine-choline-deficient (MCD) mice, as well as in an in vitro 3D human liver ECM culture of LX2 cells, a human hepatic stellate cell line. We demonstrate that NV556 decreased liver fibrosis in both STAM and MCD in vivo models and decreased collagen production in TGFß1-activated hepatic stellate cells in vitro. Taken together, these results present NV556 as a potential candidate for the treatment of liver fibrosis.
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Ciclofilinas/antagonistas & inhibidores , Cirrosis Hepática/metabolismo , Animales , Deficiencia de Colina , Colágeno Tipo I/metabolismo , Dieta , Modelos Animales de Enfermedad , Células Estrelladas Hepáticas/efectos de los fármacos , Células Estrelladas Hepáticas/metabolismo , Humanos , Cirrosis Hepática/tratamiento farmacológico , Cirrosis Hepática/etiología , Cirrosis Hepática/patología , Masculino , Metionina/deficiencia , Ratones , Terapia Molecular Dirigida , Enfermedad del Hígado Graso no Alcohólico/tratamiento farmacológico , Enfermedad del Hígado Graso no Alcohólico/metabolismo , Enfermedad del Hígado Graso no Alcohólico/patologíaRESUMEN
BACKGROUND: Satellite-based aboveground forest biomass maps commonly form the basis of forest biomass and carbon stock mapping and monitoring, but biomass maps likely vary in performance by region and as a function of spatial scale of aggregation. Assessing such variability is not possible with spatially-sparse vegetation plot networks. In the current study, our objective was to determine whether high-resolution lidar-based and moderate-resolution Landsat-base aboveground live forest biomass maps converged on similar predictions at stand- to landscape-levels (10 s to 100 s ha) and whether such differences depended on biophysical setting. Specifically, we examined deviations between lidar- and Landsat-based biomass mapping methods across scales and ecoregions using a measure of error (normalized root mean square deviation), a measure of the unsystematic deviations, or noise (Pearson correlation coefficient), and two measures related to systematic deviations, or biases (intercept and slope of a regression between the two sets of predictions). RESULTS: Compared to forest inventory data (0.81-ha aggregate-level), lidar and Landsat-based mean biomass predictions exhibited similar performance, though lidar predictions exhibited less normalized root mean square deviation than Landsat when compared with the reference plot data. Across aggregate-levels, the intercepts and slopes of regression equations describing the relationships between lidar- and Landsat-based biomass predictions stabilized (i.e., little additional change with increasing area of aggregates) at aggregate-levels between 10 and 100 ha, suggesting a consistent relationship between the two maps at landscape-scales. Differences between lidar- and Landsat-based biomass maps varied as a function of forest canopy heterogeneity and composition, with systematic deviations (regression intercepts) increasing with mean canopy cover and hardwood proportion within forests and correlations decreasing with hardwood proportion. CONCLUSIONS: Deviations between lidar- and Landsat-based maps indicated that satellite-based approaches may represent general gradients in forest biomass. Ecoregion impacted deviations between lidar and Landsat biomass maps, highlighting the importance of biophysical setting in determining biomass map performance across aggregate scales. Therefore, regardless of the source of remote sensing (e.g., Landsat vs. lidar), factors affecting the measurement and prediction of forest biomass, such as species composition, need to be taken into account whether one is estimating biomass at the plot, stand, or landscape scale.
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Wildfires pose a unique challenge to conservation in fire-prone regions, yet few studies quantify the cumulative effects of wildfires on forest dynamics (i.e., changes in structural conditions) across landscape and regional scales. We assessed the contribution of wildfire to forest dynamics in the eastern Cascade Mountains, USA from 1985 to 2010 using imputed maps of forest structure (i.e., tree size and canopy cover) and remotely sensed burn severity maps. We addressed three questions: (1) How do dynamics differ between the region as a whole and the unburned portion of the region? (2) How do dynamics vary among vegetation zones differing in biophysical setting and historical fire frequency? (3) How have forest structural conditions changed in a network of late successional reserves (LSRs)? Wildfires affected 10% of forests in the region, but the cumulative effects at this scale were primarily slight losses of closed-canopy conditions and slight gains in open-canopy conditions. In the unburned portion of the region (the remaining 90%), closed-canopy conditions primarily increased despite other concurrent disturbances (e.g., harvest, insects). Although the effects of fire were largely dampened at the regional scale, landscape scale dynamics were far more variable. The warm ponderosa pine and cool mixed conifer zones experienced less fire than the region as a whole despite experiencing the most frequent fire historically. Open-canopy conditions increased slightly in the mixed conifer zone, but declined across the ponderosa pine zone even with wildfires. Wildfires burned 30% of the cold subalpine zone, which experienced the greatest increase in open-canopy conditions and losses of closed-canopy conditions. LSRs were more prone to wildfire than the region as a whole, and experienced slight declines in late seral conditions. Despite losses of late seral conditions, wildfires contributed to some conservation objectives by creating open habitats (e.g., sparse early seral and woodland conditions) that otherwise generally decreased in unburned landscapes despite management efforts to increase landscape diversity. This study demonstrates the potential for wildfires to contribute to regional scale conservation objectives, but implications for management and biodiversity at landscape scales vary geographically among biophysical settings, and are contingent upon historical dynamics and individual species habitat preferences.
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Incendios , Bosques , Estados del Pacífico , Pinus ponderosaRESUMEN
Erythromycin, avermectin and rapamycin are clinically useful polyketide natural products produced on modular polyketide synthase multienzymes by an assembly-line process in which each module of enzymes in turn specifies attachment of a particular chemical unit. Although polyketide synthase encoding genes have been successfully engineered to produce novel analogues, the process can be relatively slow, inefficient, and frequently low-yielding. We now describe a method for rapidly recombining polyketide synthase gene clusters to replace, add or remove modules that, with high frequency, generates diverse and highly productive assembly lines. The method is exemplified in the rapamycin biosynthetic gene cluster where, in a single experiment, multiple strains were isolated producing new members of a rapamycin-related family of polyketides. The process mimics, but significantly accelerates, a plausible mechanism of natural evolution for modular polyketide synthases. Detailed sequence analysis of the recombinant genes provides unique insight into the design principles for constructing useful synthetic assembly-line multienzymes.
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Vías Biosintéticas/genética , Evolución Molecular , Variación Genética , Familia de Multigenes , Bioingeniería , Sintasas Poliquetidas/genética , Sirolimus/química , Sirolimus/metabolismoRESUMEN
BACKGROUND: Sequence specific polyamide HxIP 1, targeted to the inverted CCAAT Box 2 (ICB2) on the topoisomerase IIα (topo IIα) promoter can inhibit NF-Y binding, re-induce gene expression and increase sensitivity to etoposide. To enhance biological activity, diamino-containing derivatives (HxI*P 2 and HxIP* 3) were synthesised incorporating an alkyl amino group at the N1-heterocyclic position of the imidazole/pyrrole. METHODS: DNase I footprinting was used to evaluate DNA binding of the diamino Hx-polyamides, and their ability to disrupt the NF-Y:ICB2 interaction assessed using EMSAs. Topo IIα mRNA (RT-PCR) and protein (Immunoblotting) levels were measured following 18h polyamide treatment of confluent A549 cells. γH2AX was used as a marker for etoposide-induced DNA damage after pre-treatment with HxIP* 3 and cell viability was measured using Cell-Titer Glo®. RESULTS: Introduction of the N1-alkyl amino group reduced selectivity for the target sequence 5'-TACGAT-3' on the topo IIα promoter, but increased DNA binding affinity. Confocal microscopy revealed both fluorescent diamino polyamides localised in the nucleus, yet HxI*P 2 was unable to disrupt the NF-Y:ICB2 interaction and showed no effect against the downregulation of topo IIα. In contrast, inhibition of NF-Y binding by HxIP* 3 stimulated dose-dependent (0.1-2µM) re-induction of topo IIα and potentiated cytotoxicity of topo II poisons by enhancing DNA damage. CONCLUSIONS: Polyamide functionalisation at the N1-position offers a design strategy to improve drug-like properties. Dicationic HxIP* 3 increased topo IIα expression and chemosensitivity to topo II-targeting agents. GENERAL SIGNIFICANCE: Pharmacological modulation of topo IIα expression has the potential to enhance cellular sensitivity to clinically-used anticancer therapeutics. This article is part of a Special Issue entitled: Nuclear Factor Y in Development and Disease, edited by Prof. Roberto Mantovani.
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Antígenos de Neoplasias/biosíntesis , Factor de Unión a CCAAT/metabolismo , Daño del ADN , ADN-Topoisomerasas de Tipo II/biosíntesis , Proteínas de Unión al ADN/biosíntesis , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Nylons/farmacología , Regiones Promotoras Genéticas , Células A549 , Animales , Antígenos de Neoplasias/genética , Factor de Unión a CCAAT/genética , ADN-Topoisomerasas de Tipo II/genética , Proteínas de Unión al ADN/genética , Etopósido/efectos adversos , Etopósido/farmacología , Regulación Enzimológica de la Expresión Génica/genética , Ratones , Células 3T3 NIH , Nylons/químicaRESUMEN
UNLABELLED: Streptomyces iranensis HM 35 is an alternative rapamycin producer to Streptomyces rapamycinicus Targeted genetic modification of rapamycin-producing actinomycetes is a powerful tool for the directed production of rapamycin derivatives, and it has also revealed some key features of the molecular biology of rapamycin formation in S. rapamycinicus. The approach depends upon efficient conjugational plasmid transfer from Escherichia coli to Streptomyces, and the failure of this step has frustrated its application to Streptomyces iranensis HM 35. Here, by systematically optimizing the process of conjugational plasmid transfer, including screening of various media, and by defining optimal temperatures and concentrations of antibiotics and Ca(2+) ions in the conjugation media, we have achieved exconjugant formation for each of a series of gene deletions in S. iranensis HM 35. Among them were rapK, which generates the starter unit for rapamycin biosynthesis, and hutF, encoding a histidine catabolizing enzyme. The protocol that we have developed may allow efficient generation of targeted gene knockout mutants of Streptomyces species that are genetically difficult to manipulate. IMPORTANCE: The developed protocol of conjugational plasmid transfer from Escherichia coli to Streptomyces iranensis may allow efficient generation of targeted gene knockout mutants of other genetically difficult to manipulate, but valuable, Streptomyces species.
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Antibacterianos/metabolismo , Técnicas de Inactivación de Genes/métodos , Sirolimus/metabolismo , Streptomyces/genética , Streptomyces/metabolismo , Conjugación Genética , Escherichia coli/genética , Eliminación de Gen , Técnicas de Transferencia de Gen , Plásmidos/metabolismoRESUMEN
Bacteria in the genus Streptomyces and its close relatives are prolific producers of secondary metabolites with antibiotic activity. Genome sequencing of these bacteria has revealed a rich source of potentially new antibiotic pathways, whose products have never been observed. Moreover, these new pathways can provide novel genes that could be used in combinatorial biosynthesis approaches to generate unnatural analogues of existing antibiotics. We explore here the use of multiple orthologous integrating plasmid systems, based on the int/attP loci from phages TG1, SV1, and ÏBT1, to express the polyketide synthase (PKS) for erythromycin in a heterologous Streptomyces host. Streptomyces strains containing the three polyketide synthase genes eryAI, eryAII, and eryAIII expressed from three different integrated plasmids produced the aglycone intermediate, 6-deoxyerythronolide B (6-dEB). A further pair of integrating plasmids, both derived from the ÏC31 int/attP locus, were constructed carrying a gene cassette for glycosylation of the aglycone intermediates, with or without the tailoring gene, eryF, required for the synthesis of erythronolide B (EB). Liquid chromatography-mass spectrometry of the metabolites indicated the production of angolosaminyl-6-dEB and angolosaminyl-EB. The advantages of using multiplexed integrating plasmids for engineering expression and for combinatorial biosynthesis were demonstrated.
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Antibacterianos/biosíntesis , Proteínas Bacterianas/genética , Eritromicina/metabolismo , Plásmidos/genética , Streptomyces/metabolismo , Proteínas Bacterianas/biosíntesis , Cromatografía Liquida , Eritromicina/análogos & derivados , Eritromicina/biosíntesis , Ingeniería Genética , Glicosilación , Espectrometría de Masas , Complejos Multienzimáticos/metabolismo , Familia de Multigenes/genética , Streptomyces/genéticaRESUMEN
Inhibition of host-encoded targets, such as the cyclophilins, provides an opportunity to generate potent high barrier to resistance antivirals for the treatment of a broad range of viral diseases. However, many host-targeted agents are natural products, which can be difficult to optimize using synthetic chemistry alone. We describe the orthogonal combination of bioengineering and semisynthetic chemistry to optimize the drug-like properties of sanglifehrin A, a known cyclophilin inhibitor of mixed nonribosomal peptide/polyketide origin, to generate the drug candidate NVP018 (formerly BC556). NVP018 is a potent inhibitor of hepatitis B virus, hepatitis C virus (HCV), and HIV-1 replication, shows minimal inhibition of major drug transporters, and has a high barrier to generation of both HCV and HIV-1 resistance.
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Antivirales/química , Ciclofilinas/antagonistas & inhibidores , Lactonas/química , Oxazinas/química , Animales , Antivirales/farmacología , Antivirales/uso terapéutico , Bioingeniería , Ciclofilinas/metabolismo , Modelos Animales de Enfermedad , Perros , Infecciones por VIH/prevención & control , VIH-1/fisiología , Semivida , Células Hep G2 , Hepacivirus/enzimología , Hepacivirus/fisiología , Virus de la Hepatitis B/fisiología , Humanos , Lactonas/metabolismo , Lactonas/farmacología , Ratones , Ratones SCID , Oxazinas/metabolismo , Oxazinas/farmacología , Ratas , Streptomyces/química , Streptomyces/metabolismo , Replicación Viral/efectos de los fármacosRESUMEN
The genome sequences of eight Streptomyces phages are presented, four of which were isolated for this study. Phages R4, TG1, Hau3, and SV1 were isolated previously and have been exploited as tools for understanding and genetically manipulating Streptomyces spp. We also extracted five apparently intact prophages from recent Streptomyces spp. genome projects and, together with six phage genomes in the database, we analyzed all 19 Streptomyces phage genomes with a view to understanding their relationships to each other and to other actinophages, particularly the mycobacteriophages. Fifteen of the Streptomyces phages group into four clusters of related genomes. Although the R4-like phages do not share nucleotide sequence similarity with other phages, they clearly have common ancestry with cluster A mycobacteriophages, sharing many protein homologues, common gene syntenies, and similar repressor-stoperator regulatory systems. The R4-like phage Hau3 and the prophage StrepC.1 (from Streptomyces sp. strain C) appear to have hijacked a unique adaptation of the streptomycetes, i.e., use of the rare UUA codon, to control translation of the essential phage protein, the terminase. The Streptomyces venezuelae generalized transducing phage SV1 was used to predict the presence of other generalized transducing phages for different Streptomyces species.
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Bacteriófagos/genética , Bacteriófagos/fisiología , Evolución Biológica , Streptomyces/virología , Adaptación Fisiológica , Secuencia de Aminoácidos , Secuencia de Bases , Regulación Viral de la Expresión Génica/fisiología , Genoma Viral , Datos de Secuencia Molecular , Profagos/genética , Profagos/metabolismo , Especificidad de la Especie , Streptomyces/clasificación , Proteínas Virales/genética , Proteínas Virales/metabolismoRESUMEN
Hx-amides are fluorescent hybrids of imidazole (I)- and pyrrole (P)-containing polyamides and Hoechst 33258, and they bind in the minor groove of specific DNA sequences. Synthesis and DNA binding studies of HxII (5) complete our studies on the first set of Hx-amides: Hx-I/P-I/P. HxPP (2), HxIP (3) and HxPI (4) were reported earlier. Results from DNase I footprinting, biosensor-SPR, CD and ΔTM studies showed that Hx-amides interacted with DNA via the anti-parallel and stacked, side-by-side motif. Hx was found to mimic the DNA recognition properties of two consecutive pyrrole units (PP) in polyamides. Accordingly, the stacked Hx/PP pairing binds preferentially to two consecutive AT base pairs, A/T-A/T; Hx/IP prefers C-A/T; Hx/PI prefers A/T-C; and Hx/II prefers C-C. The results also showed that Hx-amides bound their cognate sequence at a higher affinity than their formamido-triamide counterparts.
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Amidas/química , Anisoles/química , Bencimidazoles/química , ADN/química , Imidazoles/química , Pirroles/química , Emparejamiento Base , Dicroismo Circular , ADN/metabolismo , Colorantes Fluorescentes/química , Conformación de Ácido NucleicoRESUMEN
The rapK gene required for biosynthesis of the DHCHC starter acid that initiates rapamycin biosynthesis was deleted from strain BIOT-3410, a derivative of Streptomyces rapamycinicus which had been subjected to classical strain and process development and capable of robust rapamycin production at titres up to 250mg/L. The resulting strain BIOT-4010 could no longer produce rapamycin, but when supplied exogenously with DHCHC produced rapamycin at titres equivalent to its parent strain. This strain enabled mutasynthetic access to new rapalogs that could not readily be isolated from lower titre strains when fed DHCHC analogs. Mutasynthesis of some rapalogs resulted predominantly in compounds lacking late post polyketide synthase biosynthetic modifications. To enhance the relative production of fully elaborated rapalogs, genes encoding late-acting biosynthetic pathway enzymes which failed to act efficiently on the novel compounds were expressed ectopically to give strain BIOT-4110. Strains BIOT-4010 and BIOT-4110 represent valuable tools for natural product lead optimization using biosynthetic medicinal chemistry and for the production of rapalogs for pre-clinical and early stage clinical trials.
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Mejoramiento Genético/métodos , Mutagénesis Sitio-Dirigida/métodos , Recombinación Genética/genética , Sirolimus/metabolismo , Streptomyces/fisiología , Sirolimus/aislamiento & purificación , Especificidad de la Especie , Streptomyces/clasificaciónRESUMEN
In this study, we established a flow cytometry live cell-based assay that permits the screening of hepatitis C virus (HCV) inhibitors. Specifically, we created a stable cell line, which harbors a subgenomic replicon encoding an NS5A-YFP fusion protein. This system allows direct measurement of YFP fluorescence in live hepatoma cells in which the HCV replicon replicates. We demonstrated that this stable fluorescent system permits the rapid and sensitive quantification of HCV replication inhibition by direct-acting antiviral agents (DAA) including protease and NS5A inhibitors and host-targeting antiviral agents (HTA) including cyclophilin inhibitors. This flow cytometry-based live cell assay is well suited for multiple applications such as the evaluation of HCV replication as well as antiviral drug screening.
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Alisporivir is the most advanced host-targeting antiviral cyclophilin (Cyp) inhibitor in phase III studies and has demonstrated a great deal of promise in decreasing hepatitis C virus (HCV) viremia in infected patients. In an attempt to further elucidate the mechanism of action of alisporivir, HCV replicons resistant to the drug were selected. Interestingly, mutations constantly arose in domain II of NS5A. To demonstrate that these mutations are responsible for drug resistance, they were reintroduced into the parental HCV genome, and the resulting mutant viruses were tested for replication in the presence of alisporivir or in the absence of the alisporivir target, CypA. We also examined the effect of the mutations on NS5A binding to itself (oligomerization), CypA, RNA, and NS5B. Importantly, the mutations did not affect any of these interactions. Moreover, the mutations did not preserve NS5A-CypA interactions from alisporivir rupture. NS5A mutations alone render HCV only slightly resistant to alisporivir. In sharp contrast, when multiple NS5A mutations are combined, significant resistance was observed. The introduction of multiple mutations in NS5A significantly restored viral replication in CypA knockdown cells. Interestingly, the combination of NS5A mutations renders HCV resistant to all classes of Cyp inhibitors. This study suggests that a combination of multiple mutations in domain II of NS5A rather than a single mutation is required to render HCV significantly and universally resistant to Cyp inhibitors. This in accordance with in vivo data that suggest that alisporivir is associated with a low potential for development of viral resistance.
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Antivirales/farmacología , Ciclosporina/farmacología , Hepacivirus/efectos de los fármacos , Hepacivirus/genética , Proteínas no Estructurales Virales/genética , Secuencia de Aminoácidos , Línea Celular Tumoral , Farmacorresistencia Viral/genética , Genotipo , Humanos , Datos de Secuencia Molecular , Mutación , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Homología de Secuencia de AminoácidoRESUMEN
Cyclophilin inhibitors currently in clinical trials for hepatitis C virus (HCV) are all analogues of cyclosporine (CsA). Sanglifehrins are a group of naturally occurring cyclophilin binding polyketides that are structurally distinct from the cyclosporines and are produced by a microorganism amenable to biosynthetic engineering for lead optimization and large-scale production by fermentation. Preclinical characterization of the potential utility of this class of compounds for the treatment of HCV revealed that the natural sanglifehrins A to D are all more potent than CsA at disrupting formation of the NS5A-CypA, -CypB, and -CypD complexes and at inhibition of CypA, CypB, and CypD isomerase activity. In particular, sanglifehrin B (SfB) was 30- to 50-fold more potent at inhibiting the isomerase activity of all Cyps tested than CsA and was also shown to be a more potent inhibitor of the 1b subgenomic replicon (50% effective concentrations [EC50s] of 0.070 µM and 0.16 µM in Huh 5-2 and Huh 9-13 cells, respectively). Physicochemical and mouse pharmacokinetic analyses revealed low oral bioavailability (F<4%) and low solubility (<25 µM), although the half-lives (t1/2) of SfA and SfB in mouse blood after intravenous (i.v.) dosing were long (t1/2>5 h). These data demonstrate that naturally occurring sanglifehrins are suitable lead compounds for the development of novel analogues that are less immunosuppressive and that have improved metabolism and pharmacokinetic properties.
Asunto(s)
Antivirales/farmacología , Ciclofilinas/antagonistas & inhibidores , Lactonas/farmacología , Animales , Antivirales/química , Western Blotting , Línea Celular , Ensayo de Inmunoadsorción Enzimática , Células Hep G2 , Hepacivirus/efectos de los fármacos , Humanos , Lactonas/química , Masculino , Ratones , Estructura Molecular , Replicación Viral/efectos de los fármacosRESUMEN
The macrocyclic polyketides FK506, FK520, and rapamycin are potent immunosuppressants that prevent T-cell proliferation through initial binding to the immunophilin FKBP12. Analogs of these molecules are of considerable interest as therapeutics in both metastatic and inflammatory disease. For these polyketides the starter unit for chain assembly is (4R,5R)-4,5-dihydroxycyclohex-1-enecarboxylic acid derived from the shikimate pathway. We show here that the first committed step in its formation is hydrolysis of chorismate to form (4R,5R)-4,5-dihydroxycyclohexa-1,5-dienecarboxylic acid. This chorismatase activity is encoded by fkbO in the FK506 and FK520 biosynthetic gene clusters, and by rapK in the rapamycin gene cluster of Streptomyces hygroscopicus. Purified recombinant FkbO (from FK520) efficiently catalyzed the chorismatase reaction in vitro, as judged by HPLC-MS and NMR analysis. Complementation using fkbO from either the FK506 or the FK520 gene cluster of a strain of S. hygroscopicus specifically deleted in rapK (BIOT-4010) restored rapamycin production, as did supplementation with (4R,5R)-4,5-dihydroxycyclohexa-1,5-dienecarboxylic acid. Although BIOT-4010 produced no rapamycin, it did produce low levels of BC325, a rapamycin analog containing a 3-hydroxybenzoate starter unit. This led us to identify the rapK homolog hyg5 as encoding a chorismatase/3-hydroxybenzoate synthase. Similar enzymes in other bacteria include the product of the bra8 gene from the pathway to the terpenoid natural product brasilicardin. Expression of either hyg5 or bra8 in BIOT-4010 led to increased levels of BC325. Also, purified Hyg5 catalyzed the predicted conversion of chorismate into 3-hydroxybenzoate. FkbO, RapK, Hyg5, and Bra8 are thus founder members of a previously unrecognized family of enzymes acting on chorismate.