RESUMEN
Proprotein convertase substilisin-like/kexin type 9 (PCSK9) is a serine protease involved in a protein-protein interaction with the low-density lipoprotein (LDL) receptor that has both human genetic and clinical validation. Blocking this protein-protein interaction prevents LDL receptor degradation and thereby decreases LDL cholesterol levels. Our pursuit of small-molecule direct binders for this difficult to drug PPI target utilized affinity selection/mass spectrometry, which identified one confirmed hit compound. An X-ray crystal structure revealed that this compound was binding in an unprecedented allosteric pocket located between the catalytic and C-terminal domain. Optimization of this initial hit, using two distinct strategies, led to compounds with high binding affinity to PCSK9. Direct target engagement was demonstrated in the cell lysate with a cellular thermal shift assay. Finally, ligand-induced protein degradation was shown with a proteasome recruiting tag attached to the high-affinity allosteric ligand for PCSK9.
Asunto(s)
Descubrimiento de Drogas , Evaluación Preclínica de Medicamentos , Proproteína Convertasa 9/metabolismo , Proteolisis/efectos de los fármacos , Inhibidores de Serina Proteinasa/farmacología , Bibliotecas de Moléculas Pequeñas/farmacología , Humanos , Ligandos , Modelos Moleculares , Estructura Molecular , Inhibidores de Serina Proteinasa/química , Bibliotecas de Moléculas Pequeñas/químicaRESUMEN
PCSK9 plays a significant role in regulating low-density lipoprotein (LDL) cholesterol levels and has become an important drug target for treating hypercholesterolemia. Although a member of the serine protease family, PCSK9 only catalyzes a single reaction, the autocleavage of its prodomain. The maturation of the proprotein is an essential prerequisite for the secretion of PCSK9 to the extracellular space where it binds the LDL receptor and targets it for degradation. We have found that a construct of proPCSK9 where the C-terminal domain has been truncated has sufficient stability to be expressed and purified from Escherichia coli for the in vitro study of autoprocessing. Using automated Western analysis, we demonstrate that autoprocessing exhibits the anticipated first-order kinetics. A high-throughput time-resolved fluorescence resonance energy transfer assay for autocleavage has been developed using a PCSK9 monoclonal antibody that is sensitive to the conformational changes that occur upon maturation of the proprotein. Kinetic theory has been developed that describes the behavior of both reversible and irreversible inhibitors of autocleavage. The analysis of an irreversible lactone inhibitor validates the expected relationship between potency and the reaction end point. An orthogonal liquid chromatography-mass spectrometry assay has also been implemented for the confirmation of hits from the antibody-based assays.
Asunto(s)
Sistemas de Liberación de Medicamentos/métodos , Ensayos Analíticos de Alto Rendimiento/métodos , Hipercolesterolemia/tratamiento farmacológico , Proproteína Convertasa 9/química , Escherichia coli/genética , Transferencia Resonante de Energía de Fluorescencia/métodos , Células Hep G2 , Humanos , Hipercolesterolemia/genética , Cinética , Lactonas/antagonistas & inhibidores , Espectrometría de Masas/métodos , Inhibidores de PCSK9 , Proproteína Convertasa 9/genética , Conformación Proteica/efectos de los fármacos , Receptores de LDL/genéticaRESUMEN
The ever-increasing bacterial resistance to clinical antibiotics is making many drugs ineffective and creating significant treatment gaps. This can be only circumvented by the discovery of antibiotics with new mechanisms of action. We report here the identification of a new tetramic acid, ascosetin, from an Ascomycete using the Staphylococcus aureus fitness test screening method. The structure was elucidated by spectroscopic methods including 2D NMR and HRMS. Relative stereochemistry was determined by ROESY and absolute configuration was deduced by comparative CD spectroscopy. Ascosetin inhibited bacterial growth with 2-16 µg ml(-1) MIC values against Gram-positive strains including methicillin-resistant S. aureus. It also inhibited the growth of Haemophilus influenzae with a MIC value of 8 µg ml(-1). It inhibited DNA, RNA, protein and lipid synthesis with similar IC50 values, suggesting a lack of specificity; however, it produced neither bacterial membrane nor red blood cell lysis. It showed selectivity for bacterial growth inhibition compared with fungal but not mammalian cells. The isolation, structure and biological activity of ascosetin have been detailed here.
Asunto(s)
Antibacterianos/química , Antibacterianos/farmacología , Pirrolidinonas/química , Pirrolidinonas/farmacología , Antibacterianos/aislamiento & purificación , Ascomicetos/efectos de los fármacos , Haemophilus influenzae/efectos de los fármacos , Espectroscopía de Resonancia Magnética , Staphylococcus aureus Resistente a Meticilina/efectos de los fármacos , Pruebas de Sensibilidad Microbiana , Conformación Molecular , Pirrolidinonas/aislamiento & purificación , Staphylococcus aureus/efectos de los fármacosRESUMEN
Bacteria continue to evade existing antibiotics by acquiring resistance by various mechanisms, leading to loss of antibiotic effectiveness. To avoid an epidemic from infections of incurable drug-resistant bacteria, new antibiotics with new modes of action are desperately needed. Using a genome-wide mechanism of action-guided whole cell screening approach based on antisense Staphylococcus aureus fitness test technology, we report herein the discovery of altersolanol P (1), a new tetrahydroanthraquinone from an unknown fungus from the Hypocreales isolated from forest litter collected in Puerto Rico. The structure was elucidated by high-resolution mass spectrometry and 2D NMR spectroscopy. Relative stereochemistry was established by NOESY correlations, and absolute configuration was deduced by the application of MPA ester-based methodology. Observed (1)H and (13)C NMR shifts were well aligned with the corresponding chemical shifts predicted by DFT calculations. Altersolanol P exhibited Gram-positive antibacterial activity (MIC range 1-8 µg/mL) and inhibited the growth of Gram-negative Haemophilus influenzae (MIC 2 µg/mL). The isolation, structure elucidation, and antibacterial activity of altersolanol P are described.
Asunto(s)
Antraquinonas/aislamiento & purificación , Antraquinonas/farmacología , Antibacterianos/aislamiento & purificación , Antibacterianos/farmacología , Hypocreales/química , Staphylococcus aureus/efectos de los fármacos , Antraquinonas/química , Antibacterianos/química , Farmacorresistencia Bacteriana/efectos de los fármacos , Haemophilus influenzae/efectos de los fármacos , Estructura Molecular , Resonancia Magnética Nuclear Biomolecular , Puerto RicoRESUMEN
Natural products have been major sources of antibacterial agents and remain very promising. Frequent rediscoveries of known compounds hampers progress of new discoveries and demands development and utilization of new methods for rapid biological and chemical dereplication. This paper describes an efficient approach for discovery of new thiazolyl peptides by sensitive-resistant pair screening and dereplication in a time and cost-effective manner at industrial scale. A highly effective library-based dereplication of thiazolyl peptides by high resolution fourier transform liquid chromatography mass spectrometry (HRFTLCMS) has been developed, which can detect and dereplicate very low levels of thiazolyl peptides particularly when combined with miniaturized high-throughput 96-well solid-phase extraction separation, and as well can be automated. Combination of sensitive (susceptible)-resistant pair screening, diversified screening collection and miniaturized high-throughput SPE and HRFTLCMS techniques were applied for discovery of new thiazolyl peptides. The combined approach allowed for identification of over 24 thiazolyl peptides represented by three of the five structural subgroups, including three novel compounds. In addition, it is possible for the first time to mechanistically group three structural subgroups of over 24 thiazolyl peptides. Furthermore, these studies helped to understand natural frequency of distribution of these compounds and helped in discovery of new producing strains of many thiazolyl compounds.
Asunto(s)
Actinobacteria/metabolismo , Antibacterianos/farmacología , Productos Biológicos/farmacología , Péptidos Cíclicos/farmacología , Péptidos/farmacología , Tiazoles/farmacología , Antibacterianos/química , Antibacterianos/metabolismo , Productos Biológicos/química , Productos Biológicos/metabolismo , Cromatografía Liquida , Farmacorresistencia Bacteriana , Análisis de Fourier , Ensayos Analíticos de Alto Rendimiento/métodos , Espectrometría de Masas/métodos , Pruebas de Sensibilidad Microbiana/métodos , Péptidos/química , Péptidos/metabolismo , Tiazoles/química , Tiazoles/metabolismoRESUMEN
Phaeofungin (1), a new cyclic depsipeptide isolated from Phaeosphaeria sp., was discovered by application of reverse genetics technology, using the Candida albicans fitness test (CaFT). Phaeofungin is comprised of seven amino acids and a ß,γ-dihydroxy-γ-methylhexadecanoic acid arranged in a 25-membered cyclic depsipeptide. Five of the amino acids were assigned with d-configurations. The structure was elucidated by 2D-NMR and HRMS-MS analysis of the natural product and its hydrolyzed linear peptide. The absolute configuration of the amino acids was determined by Marfey's method by complete and partial hydrolysis of 1. The CaFT profile of the phaeofungin-containing extract overlapped with that of phomafungin (3), another structurally different cyclic lipodepsipeptide isolated from a Phoma sp. using the same approach. Comparative biological characterization further demonstrated that these two fungal lipodepsipeptides are functionally distinct. While phomafungin was potentiated by cyclosporin A (an inhibitor of the calcineurin pathway), phaeofungin was synergized with aureobasidin A (2) (an inhibitor of the sphingolipid biosynthesis) and to some extent caspofungin (an inhibitor of glucan synthase). Furthermore, phaeofungin caused ATP release in wild-type C. albicans strains but phomafungin did not. It showed modest antifungal activity against C. albicans (MIC 16-32 µg/mL) and better activity against Aspergillus fumigatus (MIC 8-16 µg/mL) and Trichophyton mentagrophytes (MIC 4 µg/mL). The linear peptide was inactive, suggesting that the macrocyclic depsipeptide ring is essential for target engagement and antifungal activity.
Asunto(s)
Antifúngicos/aislamiento & purificación , Antifúngicos/farmacología , Ascomicetos/química , Candida albicans/efectos de los fármacos , Depsipéptidos/aislamiento & purificación , Depsipéptidos/farmacología , Lipopéptidos/aislamiento & purificación , Lipopéptidos/farmacología , Antifúngicos/química , Candida albicans/genética , Caspofungina , Crassulaceae/microbiología , Depsipéptidos/química , Equinocandinas/química , Genoma , Lipopéptidos/química , Pruebas de Sensibilidad Microbiana , Estructura Molecular , Resonancia Magnética Nuclear Biomolecular , Hojas de la Planta/microbiología , Tallos de la Planta/microbiologíaRESUMEN
Emergence of bacterial resistance has eroded the effectiveness of many life saving antibiotics leading to an urgent need for new chemical classes of antibacterial agents. We have applied a Staphylococcus aureus fitness test strategy to natural products screening to meet this challenge. In this paper we report the discovery of kibdelomycin A, a demethylated congener of kibdelomycin, the representative of a novel class of antibiotics produced by a new strain of Kibdelosporangium. Kibdelomycin A is a potent inhibitor of DNA gyrase and topoisomerase IV, inhibits DNA synthesis and shows whole cell antibiotic activity, albeit, less potently than kibdelomycin. Kibdelomycin C-33 acetate and tetrahydro-bisdechloro derivatives of kibdelomycin were prepared which helped define a basic SAR of the family.
Asunto(s)
Aminoglicósidos/aislamiento & purificación , Aminoglicósidos/farmacología , Antibacterianos/química , Naftalenos/aislamiento & purificación , Naftalenos/farmacología , Actinomycetales/química , Antibacterianos/aislamiento & purificación , Antibacterianos/farmacología , Girasa de ADN/metabolismo , Topoisomerasa de ADN IV/antagonistas & inhibidores , Topoisomerasa de ADN IV/metabolismo , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/aislamiento & purificación , Inhibidores Enzimáticos/farmacología , Escherichia coli/efectos de los fármacos , Escherichia coli/enzimología , Espectroscopía de Resonancia Magnética , Pruebas de Sensibilidad Microbiana , Conformación Molecular , Staphylococcus aureus/efectos de los fármacos , Staphylococcus aureus/enzimología , Relación Estructura-Actividad , Inhibidores de Topoisomerasa IIRESUMEN
Drug-resistant bacteria continue to make many existing antibiotic classes ineffective. In order to avoid a future epidemic from drug-resistant bacterial infections, new antibiotics with new modes of action are needed. In an antibiotic screening program for new drug leads with new modes of action using antisense Staphylococcus aureus Fitness Test screening, we discovered a new tetramic acid, methiosetin, from a tropical sooty mold, Capnodium sp. The fungus also produced epicorazine A, a known antibiotic. The structure and relative configuration of methiosetin was elucidated by 2D NMR and ESIMS techniques. Methiosetin and epicorazine A showed weak to modest antibacterial activity against S. aureus and Haemophilus influenzae. The isolation, structure elucidation, and antibacterial activity of both compounds are described.
Asunto(s)
Antibacterianos/aislamiento & purificación , Ascomicetos/química , Pirrolidinonas/aislamiento & purificación , Pirrolidinonas/farmacología , Antibacterianos/química , Antibacterianos/farmacología , Guatemala , Haemophilus influenzae/efectos de los fármacos , Staphylococcus aureus Resistente a Meticilina/efectos de los fármacos , Pruebas de Sensibilidad Microbiana , Estructura Molecular , Piperazinas/aislamiento & purificación , Piperazinas/farmacología , Pirrolidinonas/químicaRESUMEN
Bacterial resistance to known therapeutics has led to an urgent need for new chemical classes of antibacterial agents. To address this we have applied a Staphylococcus aureus fitness test strategy to natural products screening. Here we report the discovery of kibdelomycin, a novel class of antibiotics produced by a new member of the genus Kibdelosporangium. Kibdelomycin exhibits broad-spectrum, gram-positive antibacterial activity and is a potent inhibitor of DNA synthesis. We demonstrate through chemical genetic fitness test profiling and biochemical enzyme assays that kibdelomycin is a structurally new class of bacterial type II topoisomerase inhibitor preferentially inhibiting the ATPase activity of DNA gyrase and topoisomerase IV. Kibdelomycin is thus the first truly novel bacterial type II topoisomerase inhibitor with potent antibacterial activity discovered from natural product sources in more than six decades.
Asunto(s)
Actinomycetales/química , Antibacterianos/química , Antibacterianos/farmacología , Pirroles/química , Pirroles/farmacología , Pirrolidinonas/química , Pirrolidinonas/farmacología , Staphylococcus aureus/efectos de los fármacos , Staphylococcus aureus/enzimología , Inhibidores de Topoisomerasa II/química , Inhibidores de Topoisomerasa II/farmacología , Antibacterianos/aislamiento & purificación , Girasa de ADN/metabolismo , Topoisomerasa de ADN IV/antagonistas & inhibidores , Topoisomerasa de ADN IV/metabolismo , Farmacorresistencia Bacteriana , Humanos , Pruebas de Sensibilidad Microbiana , Modelos Moleculares , Pirroles/aislamiento & purificación , Pirrolidinonas/aislamiento & purificación , Infecciones Estafilocócicas/tratamiento farmacológico , Staphylococcus aureus/genética , Inhibidores de Topoisomerasa II/aislamiento & purificaciónRESUMEN
In a whole-cell mechanism of action (MOA)-based screening strategy for discovery of antifungal agents, Candida albicans was used, followed by testing of active extracts in the C. albicans fitness test (CaFT), which provides insight into the mechanism of action. A fermentation extract of an undescribed species of Metulocladosporiella that inhibited proteasome activity in a C. albicans fitness test was identified. The chemical genomic profile of the extract contained hypersensitivity of heterozygous deletion strains (strains that had one of the genes of the diploid genes knocked down) of genes represented by multiple subunits of the 25S proteasome. Two structurally related peptide aldehydes, named fellutamides C and D, were isolated from the extract. Fellutamides were active against C. albicans and Aspergillus fumigatus with MICs ranging from 4 to 16 µg/mL and against fungal proteasome (IC50 0.2 µg/mL). Both compounds showed proteasome activity against human tumor cell lines, potently inhibiting the growth of PC-3 prostate carcinoma cells, but not A549 lung carcinoma cells. In PC-3 cells compound treatment produced a G2M cell cycle block and induced apoptosis. Preliminary SAR studies indicated that the aldehyde group is critical for the antifungal activity and that the two hydroxy groups are quantitatively important for potency.
Asunto(s)
Antifúngicos , Ascomicetos/química , Candida albicans/efectos de los fármacos , Antifúngicos/química , Antifúngicos/aislamiento & purificación , Antifúngicos/farmacología , Fase G2/efectos de los fármacos , Humanos , Masculino , Estructura Molecular , Oligopéptidos/química , Oligopéptidos/aislamiento & purificación , Oligopéptidos/farmacología , Relación Estructura-ActividadRESUMEN
Platensimycin (1a) and platencin (2) are inhibitors of FabF and FabF/H bacterial fatty acid synthase. The discovery of natural congeners is an approach that can render a better understanding of the structure-function relationships of complex natural products. The isolation and structure elucidation of nine new congeners (11-20) of platensimycin and platencin are described from a fermentation broth of Streptomyces platensis. These hydroxylated congeners are likely derived by cytochrome P450 oxidation of the terpenoid units post-cyclization. Polar groups in the terpenoid portion of the molecule produce negative interactions with the hydrophobic pocket of FabF, resulting in poor activities. However, the discovery of these compounds serves an important purpose, not only to understand structure-function relationships, which cannot be easily accessed by chemical modification, but also to provide access to compounds that could be used for structural identification/confirmation of the oxidative trace metabolites produced in vivo during animal experiments.
Asunto(s)
Adamantano/química , Aminobenzoatos/química , Aminofenoles/química , Anilidas/química , Compuestos Policíclicos/química , Streptomyces/química , Adamantano/aislamiento & purificación , Adamantano/farmacología , Aminobenzoatos/aislamiento & purificación , Aminobenzoatos/farmacología , Aminofenoles/aislamiento & purificación , Aminofenoles/farmacología , Anilidas/aislamiento & purificación , Anilidas/farmacología , Antibacterianos/química , Antibacterianos/aislamiento & purificación , Antibacterianos/farmacología , Sistema Enzimático del Citocromo P-450/metabolismo , Acido Graso Sintasa Tipo II/antagonistas & inhibidores , Staphylococcus aureus Resistente a Meticilina/efectos de los fármacos , Pruebas de Sensibilidad Microbiana , Estructura Molecular , Compuestos Policíclicos/aislamiento & purificación , Compuestos Policíclicos/farmacología , Estereoisomerismo , Relación Estructura-ActividadRESUMEN
Bacterial resistance to antibiotics, particularly to multiple antibiotics, is becoming a cause for significant concern. The only really viable course of action to counter this is to discover new antibiotics with novel modes of action. We have recently implemented a new antisense-based chemical genetic screening technology to accomplish this goal. The discovery and antibacterial activity of coelomycin, a fully substituted 2,6-dioxo pyrazine, illustrates the application of the Staphylococcus aureus fitness test strategy to natural products discovery.
Asunto(s)
Antibacterianos/aislamiento & purificación , Antibacterianos/farmacología , Ascomicetos/metabolismo , Pirazinas/aislamiento & purificación , Pirazinas/farmacología , Staphylococcus aureus/efectos de los fármacos , Staphylococcus aureus/fisiología , Antibacterianos/química , Ascomicetos/aislamiento & purificación , Evaluación Preclínica de Medicamentos/métodos , Humanos , Juniperus/microbiología , Espectroscopía de Resonancia Magnética , Estructura Molecular , Pirazinas/químicaRESUMEN
Five diastereomeric polyketide glycosides, roselipins 3A-3E (1-5), have been isolated from the acetone extract of Clonostachys candelabrum on the basis of their positive anthelmintic activity. The structures of these compounds were elucidated by comparison of their NMR and MS data to those of previously reported roselipins and related structures, and were confirmed by 2D-NMR spectral analysis. Known compounds linoleic acid (6) and aurantiogliocladin (7) were also isolated as active anthelmintic components, although much less potent than the roselipins.
Asunto(s)
Antihelmínticos/química , Antihelmínticos/farmacología , Hypocreales/química , Animales , Bioensayo , Ciego/parasitología , Cromatografía Líquida de Alta Presión , Indicadores y Reactivos , Espectroscopía de Resonancia Magnética , Espectrometría de Masas , Ratones , Infecciones por Nematodos/tratamiento farmacológico , Infecciones por Nematodos/parasitología , Nematospiroides dubius/efectos de los fármacos , Espectrometría de Masa por Ionización de Electrospray , Espectrofotometría Ultravioleta , Estómago/parasitología , Relación Estructura-ActividadRESUMEN
Natural products continue to serve as one of the best sources for discovery of antibacterial agents as exemplified by the recent discoveries of platensimycin and platencin. Chemical modifications as well as discovery of congeners are the main sources for gaining knowledge of structure-activity relationship of natural products. Screening for congeners in the extracts of the fermentation broths of Streptomyces platensis led to the isolation of platencin A(1), a hydroxy congener of platencin. The hydroxylation of the tricyclic enone moiety negatively affected the antibacterial activity and appears to be consistent with the hydrophobic binding pocket of the FabF. Isolation, structure, enzyme-bound structure and activity of platencin A(1) and two other congeners have been described.
Asunto(s)
Adamantano/análogos & derivados , Aminobenzoatos/química , Antibacterianos/química , Streptomyces/química , Adamantano/química , Adamantano/aislamiento & purificación , Adamantano/farmacología , Aminobenzoatos/aislamiento & purificación , Aminobenzoatos/farmacología , Aminofenoles/química , Aminofenoles/aislamiento & purificación , Aminofenoles/farmacología , Anilidas/química , Anilidas/farmacología , Antibacterianos/aislamiento & purificación , Antibacterianos/farmacología , Cristalografía por Rayos X , Conformación Molecular , Compuestos Policíclicos/química , Compuestos Policíclicos/aislamiento & purificación , Compuestos Policíclicos/farmacología , Relación Estructura-ActividadRESUMEN
Treatment of drug-resistant bacteria is a significant unmet medical need. This challenge can be met only by the discovery and development of new antibiotics. Antisense technology is one of the newest discovery tools that provides enhanced sensitivity for detection of antibacterials, and has led to the discovery of a number of interesting new antibacterial natural products. Continued utilization of this technology led to the discovery of three new bicyclic lactones, glabramycins A-C, from a Neosartorya glabra strain. Glabramycin C showed strong antibiotic activity against Streptococcus pneumoniae (MIC 2 microg ml(-1)) and modest antibiotic activity against Staphylococcus aureus (MIC 16 microg ml(-1)). The isolation, structure, relative configuration and antibacterial activity, and plausible biogenesis of these compounds have been discussed.
Asunto(s)
Antibacterianos/química , Antibacterianos/farmacología , Ascomicetos/metabolismo , Caprilatos/química , Caprilatos/farmacología , ADN sin Sentido/genética , Lactonas/química , Lactonas/farmacología , Ascomicetos/genética , Bacterias/efectos de los fármacos , ADN de Hongos/genética , Fermentación , Espectroscopía de Resonancia Magnética , Pruebas de Sensibilidad Microbiana , Conformación Molecular , Inhibidores de la Síntesis del Ácido Nucleico/farmacología , Inhibidores de la Síntesis de la Proteína/farmacología , Microbiología del Suelo , Staphylococcus aureus/efectos de los fármacosRESUMEN
Thiazolyl peptides are a class of highly rigid trimacrocyclic compounds consisting of varying but large numbers of thiazole rings. The need for new antibacterial agents to treat infections caused by resistant bacteria prompted a reinvestigation of this class, leading to the previous isolation of thiazolyl peptides, namely, thiazomycin (5) and thiazomycin A (6), congeners of nocathiacins (1-4). Continued chemical screening led to the isolation of six new thiazolyl peptide congeners (8-13), of which three had truncated structures lacking an indole residue. From these, compound 8 showed activity similar to thiazomycin. Two compounds (9 and 10) showed intermediate activities, and the three truncated compounds (11-13) were essentially inactive. The discovery of the truncated compounds revealed the minimal structural requirements for activity and suggested probable biosynthetic pathways for more advanced compounds. The isolation, structure elucidation, antibacterial activity, and proposed biogenesis of thiazomycins are herein described.
Asunto(s)
Actinomycetales/química , Antibacterianos , Péptidos Cíclicos/aislamiento & purificación , Péptidos , Tiazoles/aislamiento & purificación , Antibacterianos/química , Antibacterianos/aislamiento & purificación , Antibacterianos/farmacología , Técnicas Químicas Combinatorias , Pruebas de Sensibilidad Microbiana , Estructura Molecular , Péptidos/química , Péptidos/aislamiento & purificación , Péptidos/farmacología , Péptidos Cíclicos/química , Péptidos Cíclicos/farmacología , Estereoisomerismo , Tiazoles/química , Tiazoles/farmacologíaRESUMEN
Sordarins are a class of natural antifungal agents which act by specifically inhibiting fungal protein synthesis through their interaction with the elongation factor 2, EF2. A number of natural sordarins produced by diverse fungi of different classes have been reported in the literature. We have run an exhaustive search of sordarin-producing fungi using two different approaches consecutively, the first one being a differential sensitivity screen using a sordarin-resistant mutant yeast strain run in parallel with a wild type strain, and the second one an empiric screen against Candida albicans followed by early detection of sordarins by LC-MS analysis. Using these two strategies we have detected as many as 22 new strains producing a number of different sordarin analogues, either known (sordarin, xylarin, zofimarin) or novel (isozofimarin and 4'-O-demethyl sordarin). Sordarin and xylarin were the most frequently found compounds in the class. The producing strains were subjected to sequencing of the ITS region to determine their phylogenetic affinities. All the strains were shown to belong to the Xylariales, being distributed across three families in this order, the Xylariaceae, the Amphisphaeriaceae, and the Diatrypaceae. Despite being screened in large numbers, we did not find sordarin production in any other fungal group, including those orders where sordarin producing fungi are known to exist (i.e., Sordariales, Eurotiales, and Microascales), suggesting that the production of sordarin is a trait more frequently associated to members of the Xylariales than to any other fungal order.
Asunto(s)
Antifúngicos/metabolismo , Hongos/metabolismo , Indenos/metabolismo , Antifúngicos/química , Antifúngicos/farmacología , Transporte Biológico , Candida albicans/efectos de los fármacos , Hongos/química , Hongos/clasificación , Hongos/genética , Indenos/química , Indenos/farmacología , Datos de Secuencia Molecular , FilogeniaRESUMEN
Protein synthesis inhibition is a highly successful target for developing clinically effective and safe antibiotics. There are several targets within the ribosomal machinery, and small ribosomal protein S4 (RPSD) is one of the newer targets. Screening of microbial extracts using antisense-sensitized rpsD Staphylococcus aureus strain led to isolation of okilactomycin and four new congeners from Streptomyces scabrisporus. The major compound, okilactomycin, was the most active, with a minimum detection concentration of 3-12 microg ml(-1) against antisense assay, and showed an MIC of 4-16 microg ml(-1) against Gram-positive bacteria, including S. aureus. The congeners were significantly less active in all assays, and all compounds showed a slight preferential inhibition of RNA synthesis over DNA and protein synthesis. Antisense technology, due to increased sensitivity, continues to yield new, even though weakly active, antibiotics.
Asunto(s)
Oligonucleótidos Antisentido/farmacología , Proteínas Ribosómicas/efectos de los fármacos , Proteínas Ribosómicas/genética , Streptomyces/genética , Streptomyces/metabolismo , Bacterias/efectos de los fármacos , Cromatografía Líquida de Alta Presión , Fermentación , Bacterias Grampositivas/efectos de los fármacos , Lactonas/química , Lactonas/aislamiento & purificación , Espectroscopía de Resonancia Magnética , Espectrometría de Masas , Pruebas de Sensibilidad Microbiana , Inhibidores de la Síntesis del Ácido Nucleico/farmacología , Filogenia , Inhibidores de la Síntesis de la Proteína/farmacología , ARN Ribosómico 16S/biosíntesis , ARN Ribosómico 16S/genética , Espectrofotometría Infrarroja , Espectrofotometría Ultravioleta , Staphylococcus aureus/efectos de los fármacos , Staphylococcus aureus/metabolismo , Streptomyces/clasificaciónRESUMEN
A glycosylated tetramic acid, virgineone (1), was isolated from saprotrophic Lachnum virgineum. The antifungal activity of the fermentation extract of L. virgineum was characterized in the Candida albicans fitness test as distinguishable from other natural products tested. Bioassay-guided fractionation yielded 1, a tyrosine-derived tetramic acid with a C-22 oxygenated chain and a beta-mannose. It displayed broad-spectrum antifungal activity against Candida spp. and Aspergillus fumigatus with a MIC of 4 and 16 microg/mL, respectively. Virgineone was also identified in a number of Lachnum strains collected from diverse geographies and habitats.