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1.
J Nat Prod ; 86(7): 1801-1814, 2023 07 28.
Artigo em Inglês | MEDLINE | ID: mdl-37463274

RESUMO

Discovery and structure elucidation of natural products available in infinitesimally small quantities are recognized challenge. This challenge is epitomized by the diphenazine class of molecules that contain three bridged stereocenters, several conformations, ring fusions, and multiple spatially isolated phenols. Because empirical NMR and spatial analyses using ROESY/NOESY were unsuccessful in tackling these challenges, we developed a computational pipeline to determine the relative and absolute configurations and phenol positions of diphenazines as inhibitors of eukaryotic translation initiation factor 4E (eIF4E) protein-protein interactions. In this pipeline, we incorporated ECD and GIAO NMR calculations coupled with a DP4+ probability measure, enabling the structure revision of phenazinolin D (4), izumiphenazine A (5), and baraphenazine G (7) and the structure characterization of two new diphenazines, baraphenazine H (3) and izumiphenazine E (6). Importantly, through these efforts, we demonstrate the feasibility of NMR/DP4+ analysis for the determination of phenol positions in phenazine-based molecules, further expanding the limits of computational methods for the structure elucidation of complex natural products.


Assuntos
Produtos Biológicos , Estrutura Molecular , Produtos Biológicos/química , Fenol , Espectroscopia de Ressonância Magnética
2.
Tetrahedron Lett ; 61(5)2020 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-32863451

RESUMO

Phosphopantetheine is a key structural element in biological acyl transfer reactions found embedded within coenzyme A (CoA). Phosphopantothenoylcysteine synthetase (PPCS) is responsible for installing a cysteamine group within phosphopantetheine. Therefore, it holds considerable potential as a drug target for developing new antimicrobials. In this study, we adapted a biochemical assay specific for bacterial PPCS to screen for inhibitors of CoA biosynthesis against a library of marine microbial derived natural product extracts (NPEs). Analysis of the NPE derived from Streptomyces blancoensis led to the isolation of novel antibiotics (10-12, and 14) from the adipostatin class of molecules. The most potent molecule (10) displayed in vitro activity with IC50= 0.93 µM, against S. pneumoniae PPCS. The whole cell antimicrobial assay against isolated molecules demonstrated their ability to penetrate bacterial cells and inhibit clinically relevant pathogenic strains. This establishes the validity of PPCS as a pertinent drug target, and the value of NPEs to provide new antibiotics.

3.
J Am Chem Soc ; 139(23): 7913-7920, 2017 06 14.
Artigo em Inglês | MEDLINE | ID: mdl-28525276

RESUMO

Polyketide synthases (PKSs) represent a powerful catalytic platform capable of effecting multiple carbon-carbon bond forming reactions and oxidation state adjustments. We explored the functionality of two terminal PKS modules that produce the 16-membered tylosin macrocycle, using them as biocatalysts in the chemoenzymatic synthesis of tylactone and its subsequent elaboration to complete the first total synthesis of the juvenimicin, M-4365, and rosamicin classes of macrolide antibiotics via late-stage diversification. Synthetic chemistry was employed to generate the tylactone hexaketide chain elongation intermediate that was accepted by the juvenimicin (Juv) ketosynthase of the penultimate JuvEIV PKS module. The hexaketide is processed through two complete modules (JuvEIV and JuvEV) in vitro, which catalyze elongation and functionalization of two ketide units followed by cyclization of the resulting octaketide into tylactone. After macrolactonization, a combination of in vivo glycosylation, selective in vitro cytochrome P450-mediated oxidation, and chemical oxidation was used to complete the scalable construction of a series of macrolide natural products in as few as 15 linear steps (21 total) with an overall yield of 4.6%.


Assuntos
Antibacterianos/biossíntese , Macrolídeos/metabolismo , Policetídeo Sintases/metabolismo , Policetídeos/metabolismo , Tilosina/análogos & derivados , Antibacterianos/química , Antibacterianos/farmacologia , Biocatálise , Relação Dose-Resposta a Droga , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Positivas/efeitos dos fármacos , Macrolídeos/química , Macrolídeos/farmacologia , Testes de Sensibilidade Microbiana , Conformação Molecular , Policetídeo Sintases/química , Policetídeos/química , Policetídeos/farmacologia , Relação Estrutura-Atividade , Tilosina/biossíntese , Tilosina/química , Tilosina/farmacologia
4.
J Nat Prod ; 78(10): 2411-22, 2015 Oct 23.
Artigo em Inglês | MEDLINE | ID: mdl-26465675

RESUMO

Methods to identify the bioactive diversity within natural product extracts (NPEs) continue to evolve. NPEs constitute complex mixtures of chemical substances varying in structure, composition, and abundance. NPEs can therefore be challenging to evaluate efficiently with high-throughput screening approaches designed to test pure substances. Here we facilitate the rapid identification and prioritization of antimalarial NPEs using a pharmacologically driven, quantitative high-throughput-screening (qHTS) paradigm. In qHTS each NPE is tested across a concentration range from which sigmoidal response, efficacy, and apparent EC50s can be used to rank order NPEs for subsequent organism reculture, extraction, and fractionation. Using an NPE library derived from diverse marine microorganisms we observed potent antimalarial activity from two Streptomyces sp. extracts identified from thousands tested using qHTS. Seven compounds were isolated from two phylogenetically related Streptomyces species: Streptomyces ballenaensis collected from Costa Rica and Streptomyces bangulaensis collected from Papua New Guinea. Among them we identified actinoramides A and B, belonging to the unusually elaborated nonproteinogenic amino-acid-containing tetrapeptide series of natural products. In addition, we characterized a series of new compounds, including an artifact, 25-epi-actinoramide A, and actinoramides D, E, and F, which are closely related biosynthetic congeners of the previously reported metabolites.


Assuntos
Antimaláricos/isolamento & purificação , Antimaláricos/farmacologia , Produtos Biológicos/isolamento & purificação , Produtos Biológicos/farmacologia , Oligopeptídeos/isolamento & purificação , Oligopeptídeos/farmacologia , Streptomyces/química , Antimaláricos/química , Produtos Biológicos/química , Costa Rica , Sedimentos Geológicos/química , Biologia Marinha , Estrutura Molecular , Ressonância Magnética Nuclear Biomolecular , Oligopeptídeos/química , Papua Nova Guiné , Filogenia , Plasmodium falciparum/efeitos dos fármacos , Streptomyces/genética
5.
Mol Pharmacol ; 86(4): 406-16, 2014 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-25086086

RESUMO

Biochemical high-throughput screening is widely used in drug discovery, using a variety of small molecule libraries. However, broader screening strategies may be more beneficial to identify novel biologic mechanisms. In the current study we used a ß-galactosidase complementation method to screen a selection of microbial-derived pre-fractionated natural product extracts for those that increase regulator of G protein signaling 2 (RGS2) protein levels. RGS2 is a member of a large family of proteins that all regulate signaling through G protein-coupled receptors (GPCRs) by accelerating GTPase activity on active Gα as well as through other mechanisms. RGS2(-/-) mice are hypertensive, show increased anxiety, and are prone to heart failure. RGS2 has a very short protein half-life due to rapid proteasomal degradation, and we propose that enhancement of RGS2 protein levels could be a beneficial therapeutic strategy. Bioassay-guided fractionation of one of the hit strains yielded a pure compound, Indolactam V, a known protein kinase C (PKC) activator, which selectively increased RGS2 protein levels in a time- and concentration-dependent manner. Similar results were obtained with phorbol 12-myristate 13-acetate as well as activation of the Gq-coupled muscarinic M3 receptor. The effect on RGS2 protein levels was blocked by the nonselective PKC inhibitor Gö6983 (3-[1-[3-(dimethylamino)propyl]-5-methoxy-1H-indol-3-yl]-4-(1H-indol-3-yl)-1H-pyrrole-2,5-dione), the PKCß-selective inhibitor Ruboxastaurin, as well as small interfering RNA-mediated knockdown of PKCß. Indolactam V-mediated increases in RGS2 protein levels also had functional effects on GPCR signaling. This study provides important proof-of-concept for our screening strategy and could define a negative feedback mechanism in Gq/Phospholipase C signaling through RGS2 protein upregulation.


Assuntos
Produtos Biológicos/farmacologia , Indóis/farmacologia , Lactamas/farmacologia , Proteína Quinase C beta/efeitos dos fármacos , Proteínas RGS/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Regulação para Cima , Actinobacteria/química , Animais , Células HEK293 , Ensaios de Triagem em Larga Escala , Humanos , Maleimidas/farmacologia , Miócitos de Músculo Liso/efeitos dos fármacos , Fenótipo , Proteína Quinase C beta/antagonistas & inibidores , Proteína Quinase C beta/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Proteínas RGS/genética , Ratos , Receptor Muscarínico M3/agonistas , Acetato de Tetradecanoilforbol/farmacologia
6.
J Am Chem Soc ; 136(4): 1579-86, 2014 Jan 29.
Artigo em Inglês | MEDLINE | ID: mdl-24401083

RESUMO

Siderophores are high-affinity iron chelators produced by microorganisms and frequently contribute to the virulence of human pathogens. Targeted inhibition of the biosynthesis of siderophores staphyloferrin B of Staphylococcus aureus and petrobactin of Bacillus anthracis hold considerable potential as a single or combined treatment for methicillin-resistant S. aureus (MRSA) and anthrax infection, respectively. The biosynthetic pathways for both siderophores involve a nonribosomal peptide synthetase independent siderophore (NIS) synthetase, including SbnE in staphyloferrin B and AsbA in petrobactin. In this study, we developed a biochemical assay specific for NIS synthetases to screen for inhibitors of SbnE and AsbA against a library of marine microbial-derived natural product extracts (NPEs). Analysis of the NPE derived from Streptomyces tempisquensis led to the isolation of the novel antibiotics baulamycins A (BmcA, 6) and B (BmcB, 7). BmcA and BmcB displayed in vitro activity with IC50 values of 4.8 µM and 19 µM against SbnE and 180 µM and 200 µM against AsbA, respectively. Kinetic analysis showed that the compounds function as reversible competitive enzyme inhibitors. Liquid culture studies with S. aureus , B. anthracis , E. coli , and several other bacterial pathogens demonstrated the capacity of these natural products to penetrate bacterial barriers and inhibit growth of both Gram-positive and Gram-negative species. These studies provide proof-of-concept that natural product inhibitors targeting siderophore virulence factors can provide access to novel broad-spectrum antibiotics, which may serve as important leads for the development of potent anti-infective agents.


Assuntos
Antibacterianos/farmacologia , Bacillus anthracis/efeitos dos fármacos , Produtos Biológicos/farmacologia , Daunorrubicina/análogos & derivados , Escherichia coli/efeitos dos fármacos , Sideróforos/antagonistas & inibidores , Staphylococcus aureus/efeitos dos fármacos , Antibacterianos/química , Antibacterianos/isolamento & purificação , Bacillus anthracis/química , Bacillus anthracis/metabolismo , Produtos Biológicos/química , Produtos Biológicos/isolamento & purificação , Daunorrubicina/síntese química , Daunorrubicina/química , Daunorrubicina/farmacologia , Relação Dose-Resposta a Droga , Ensaios de Triagem em Larga Escala , Testes de Sensibilidade Microbiana , Conformação Molecular , Sideróforos/biossíntese , Staphylococcus aureus/química , Staphylococcus aureus/metabolismo , Relação Estrutura-Atividade
7.
Nat Commun ; 15(1): 5356, 2024 Jun 25.
Artigo em Inglês | MEDLINE | ID: mdl-38918378

RESUMO

Type 1 polyketides are a major class of natural products used as antiviral, antibiotic, antifungal, antiparasitic, immunosuppressive, and antitumor drugs. Analysis of public microbial genomes leads to the discovery of over sixty thousand type 1 polyketide gene clusters. However, the molecular products of only about a hundred of these clusters are characterized, leaving most metabolites unknown. Characterizing polyketides relies on bioactivity-guided purification, which is expensive and time-consuming. To address this, we present Seq2PKS, a machine learning algorithm that predicts chemical structures derived from Type 1 polyketide synthases. Seq2PKS predicts numerous putative structures for each gene cluster to enhance accuracy. The correct structure is identified using a variable mass spectral database search. Benchmarks show that Seq2PKS outperforms existing methods. Applying Seq2PKS to Actinobacteria datasets, we discover biosynthetic gene clusters for monazomycin, oasomycin A, and 2-aminobenzamide-actiphenol.


Assuntos
Espectrometria de Massas , Família Multigênica , Policetídeo Sintases , Policetídeos , Policetídeos/metabolismo , Policetídeos/química , Policetídeo Sintases/genética , Policetídeo Sintases/metabolismo , Espectrometria de Massas/métodos , Mineração de Dados/métodos , Aprendizado de Máquina , Actinobacteria/genética , Actinobacteria/metabolismo , Genoma Bacteriano , Algoritmos , Produtos Biológicos/química , Produtos Biológicos/metabolismo
8.
Mycologia ; 115(1): 1-15, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36541902

RESUMO

Gyromitrin (acetaldehyde N-methyl-N-formylhydrazone) and its homologs are deadly mycotoxins produced most infamously by the lorchel (also known as false morel) Gyromitra esculenta, which is paradoxically consumed as a delicacy in some parts of the world. There is much speculation about the presence of gyromitrin in other species of the lorchel family (Discinaceae), but no studies have broadly assessed its distribution. Given the history of poisonings associated with the consumption of G. esculenta and G. ambigua, we hypothesized that gyromitrin evolved in the last common ancestor of these taxa and would be present in their descendants with adaptive loss of function in the nested truffle clade, Hydnotrya. To test this hypothesis, we developed a sensitive analytical derivatization method for the detection of gyromitrin using 2,4-dinitrobenzaldehyde as the derivatization reagent. In total, we analyzed 66 specimens for the presence of gyromitrin over 105 tests. Moreover, we sequenced the nuc rDNA internal transcribed spacer region ITS1-5.8S-ITS2 (ITS barcode) and nuc 28S rDNA to assist in species identification and to infer a supporting phylogenetic tree. We detected gyromitrin in all tested specimens from the G. esculenta group as well as G. leucoxantha. This distribution is consistent with a model of rapid evolution coupled with horizontal transfer, which is typical for secondary metabolites. We clarified that gyromitrin production in Discinaceae is both discontinuous and more limited than previously thought. Further research is required to elucidate the gyromitrin biosynthesis gene cluster and its evolutionary history in lorchels.


Assuntos
Acetaldeído , Filogenia , Cromatografia Líquida de Alta Pressão , DNA Ribossômico/genética , DNA Espaçador Ribossômico/genética
9.
Angew Chem Int Ed Engl ; 51(45): 11258-62, 2012 Nov 05.
Artigo em Inglês | MEDLINE | ID: mdl-23042634

RESUMO

Capturing a coactivator, naturally: the natural products sekikaic acid and lobaric acid, isolated after a high-throughput screen of a structurally diverse extract collection, effectively target the dynamic binding interfaces of the GACKIX domain of the coactivator CBP/p300. These molecules are the most effective inhibitors of the GACKIX domain yet described and are uniquely selective for this domain.


Assuntos
Depsídeos/química , Lactonas/química , Salicilatos/química , Fatores de Transcrição de p300-CBP/química , Depsídeos/metabolismo , Lactonas/metabolismo , Modelos Moleculares , Simulação de Dinâmica Molecular , Estrutura Terciária de Proteína , Salicilatos/metabolismo , Fatores de Transcrição de p300-CBP/antagonistas & inibidores , Fatores de Transcrição de p300-CBP/genética , Fatores de Transcrição de p300-CBP/metabolismo
10.
ACS Med Chem Lett ; 12(6): 878-886, 2021 Jun 10.
Artigo em Inglês | MEDLINE | ID: mdl-34141065

RESUMO

MicroRNAs (miRNAs) are a family of small noncoding RNAs that regulate gene expression. Due to their important activity in the fine-tuning of protein translation, abnormal expression of miRNAs has been linked to many human diseases, making the targeting of miRNAs attractive as a novel therapeutic strategy. Accordingly, researchers have been heavily engaged in the discovery of small molecule modulators of miRNAs. With an interest in the identification of new chemical space for targeting miRNAs, we developed a high-throughput screening (HTS) technology, catalytic enzyme-linked click chemistry assay (cat-ELCCA), aimed at the discovery of small molecule ligands for pre-miR-21, a miRNA that is frequently overexpressed in human cancers. From our HTS campaign, we found that natural products, a source of many impactful human medicines, may be a promising source of potential pre-miR-21-selective maturation inhibitors. Herein we describe our first efforts in natural product inhibitor discovery leading to the identification of a depsipeptide class of natural products as RNA-binding inhibitors of Dicer-mediated miRNA processing.

11.
Chem Commun (Camb) ; 53(95): 12778-12781, 2017 Nov 28.
Artigo em Inglês | MEDLINE | ID: mdl-29139494
12.
Nat Commun ; 7: 10710, 2016 Feb 16.
Artigo em Inglês | MEDLINE | ID: mdl-26880271

RESUMO

Pathogenic microorganisms often have the ability to attach to a surface, building a complex matrix where they colonize to form a biofilm. This cellular superstructure can display increased resistance to antibiotics and cause serious, persistent health problems in humans. Here we describe a high-throughput in vitro screen to identify inhibitors of Acinetobacter baumannii biofilms using a library of natural product extracts derived from marine microbes. Analysis of extracts derived from Streptomyces gandocaensis results in the discovery of three peptidic metabolites (cahuitamycins A-C), with cahuitamycin C being the most effective inhibitor (IC50=14.5 µM). Biosynthesis of cahuitamycin C proceeds via a convergent biosynthetic pathway, with one of the steps apparently being catalysed by an unlinked gene encoding a 6-methylsalicylate synthase. Efforts to assess starter unit diversification through selective mutasynthesis lead to production of unnatural analogues cahuitamycins D and E of increased potency (IC50=8.4 and 10.5 µM).


Assuntos
Acinetobacter baumannii/efeitos dos fármacos , Antibacterianos/farmacologia , Proteínas de Bactérias/farmacologia , Biofilmes/efeitos dos fármacos , Oligopeptídeos/farmacologia , Acinetobacter baumannii/fisiologia , Antibacterianos/biossíntese , Proteínas de Bactérias/biossíntese , Vias Biossintéticas , Ensaios de Triagem em Larga Escala , Oligopeptídeos/biossíntese , Streptomyces
13.
J Biomol Screen ; 20(5): 673-80, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-25550354

RESUMO

Alphaviruses are a prominent class of reemergent pathogens due to their globally expanding ranges, potential for lethality, and possible use as bioweapons. The absence of effective treatments for alphaviruses highlights the need for innovative strategies to identify antiviral agents. Primary screens that use noninfectious self-replicating RNAs, termed replicons, have been used to identify potential antiviral compounds for alphaviruses. Only inhibitors of viral genome replication, however, will be identified using replicons, which excludes many other druggable steps in the viral life cycle. To address this limitation, we developed a western equine encephalitis virus pseudoinfectious particle system that reproduces several crucial viral life cycle steps in addition to genome replication. We used this system to screen a library containing ~26,000 extracts derived from marine microbes, and we identified multiple bacterial strains that produce compounds with potential antiviral activity. We subsequently used pseudoinfectious particle and replicon assays in parallel to counterscreen candidate extracts, and followed antiviral activity during biochemical fractionation and purification to differentiate between inhibitors of viral entry and genome replication. This novel process led to the isolation of a known alphavirus entry inhibitor, bafilomycin, thereby validating the approach for the screening and identification of potential antiviral compounds.


Assuntos
Alphavirus/efeitos dos fármacos , Alphavirus/fisiologia , Antivirais/farmacologia , Produtos Biológicos/farmacologia , Descoberta de Drogas/métodos , Animais , Antivirais/química , Produtos Biológicos/química , Linhagem Celular , Relação Dose-Resposta a Droga , Ensaios de Triagem em Larga Escala , Humanos , Concentração Inibidora 50 , Testes de Sensibilidade Microbiana/métodos , Reprodutibilidade dos Testes , Bibliotecas de Moléculas Pequenas , Replicação Viral/efeitos dos fármacos
14.
Chem Biol Drug Des ; 86(6): 1331-8, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26147927

RESUMO

Novel antimicrobials that effectively inhibit bacterial growth are essential to fight the growing threat of antibiotic resistance. A promising target is the bacterial ribosome, a 2.5 MDa organelle susceptible to several biorthogonal modes of action used by different classes of antibiotics. To promote the discovery of unique inhibitors, we have miniaturized a coupled transcription/translation assay using E. coli and applied it to screen a natural product library of ~30 000 extracts. We significantly reduced the scale of the assay to 2 µL in a 1536-well plate format and decreased the effective concentration of costly reagents. The improved assay returned 1327 hits (4.6% hit rate) with %CV and Z' values of 8.5% and 0.74, respectively. This assay represents a significant advance in molecular screening, both in miniaturization and its application to a natural product extract library, and we intend to apply it to a broad array of pathogenic microbes in the search for novel anti-infective agents.


Assuntos
Produtos Biológicos/farmacologia , Avaliação Pré-Clínica de Medicamentos/métodos , Ensaios de Triagem em Larga Escala/métodos , Anti-Infecciosos/farmacologia , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Luciferases/genética , Miniaturização/métodos , Biossíntese de Proteínas/efeitos dos fármacos , Ribossomos/efeitos dos fármacos , Ribossomos/genética , Bibliotecas de Moléculas Pequenas , Transcrição Gênica/efeitos dos fármacos
15.
ACS Med Chem Lett ; 6(8): 877-81, 2015 Aug 13.
Artigo em Inglês | MEDLINE | ID: mdl-26288688

RESUMO

As part of the International Cooperative Biodiversity Groups (ICBG) Program, we were interested in identifying biologically active unfolded protein response (UPR) inducing compounds from marine microorganisms isolated from Costa Rican biota. With this aim in mind we have now generated more than 33,000 unique prefractionated natural product extracts from marine and terrestrial organisms that have been submitted to the Center of Chemical Genomics (CCG) at the University of Michigan for high throughput screening (HTS). An effective complementary cell-based assay to identify novel modulators of UPR signaling was used for screening extracts. Active fractions were iteratively subjected to reverse-phase HPLC chromatographic analysis, and together with lobophorin A, B, E, and F (1-4), three new lobophorin congeners, designated as CR1 (5), CR2 (6), and CR3 (7) were isolated. Herein, we report that secondary assays revealed that the new lobophorins induced UPR-associated gene expression, inhibited oral squamous cell carcinoma cell growth, and led to UPR-dependent cell death in murine embryonic fibroblast (MEF) cells.

16.
Chem Biol Drug Des ; 83(4): 440-9, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24636344

RESUMO

Proteins with expanded polyglutamine (polyQ) segments cause a number of fatal neurodegenerative disorders, including Huntington's disease (HD). Previous high-throughput screens in cellular and biochemical models of HD have revealed compounds that mitigate polyQ aggregation and proteotoxicity, providing insight into the mechanisms of disease and leads for potential therapeutics. However, the structural diversity of natural products has not yet been fully mobilized toward these goals. Here, we have screened a collection of ~11 000 natural product extracts for the ability to recover the slow growth of ΔProQ103-expressing yeast cells in 384-well plates (Z' ~ 0.7, CV ~ 8%). This screen identified actinomycin D as a strong inhibitor of polyQ aggregation and proteotoxicity at nanomolar concentrations (~50-500 ng/mL). We found that a low dose of actinomycin D increased the levels of the heat-shock proteins Hsp104, Hsp70 and Hsp26 and enhanced binding of Hsp70 to the polyQ in yeast. Actinomycin also suppressed aggregation of polyQ in mammalian cells, suggesting a conserved mechanism. These results establish natural products as a rich source of compounds with interesting mechanisms of action against polyQ disorders.


Assuntos
Produtos Biológicos/química , Ensaios de Triagem em Larga Escala , Modelos Biológicos , Peptídeos/genética , Animais , Produtos Biológicos/análise , Dactinomicina/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Células PC12 , Peptídeos/química , Agregação Patológica de Proteínas/tratamento farmacológico , Ratos , Saccharomyces cerevisiae
17.
PLoS One ; 8(12): e82318, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24349254

RESUMO

Natural products provide a vast array of chemical structures to explore in the discovery of new medicines. Although secondary metabolites produced by microbes have been developed to treat a variety of diseases, including bacterial and fungal infections, to date there has been limited investigation of natural products with antiviral activity. In this report, we used a phenotypic cell-based replicon assay coupled with an iterative biochemical fractionation process to identify, purify, and characterize antiviral compounds produced by marine microbes. We isolated a compound from Streptomyces kaviengensis, a novel actinomycetes isolated from marine sediments obtained off the coast of New Ireland, Papua New Guinea, which we identified as antimycin A1a. This compound displays potent activity against western equine encephalitis virus in cultured cells with half-maximal inhibitory concentrations of less than 4 nM and a selectivity index of greater than 550. Our efforts also revealed that several antimycin A analogues display antiviral activity, and mechanism of action studies confirmed that these Streptomyces-derived secondary metabolites function by inhibiting the cellular mitochondrial electron transport chain, thereby suppressing de novo pyrimidine synthesis. Furthermore, we found that antimycin A functions as a broad spectrum agent with activity against a wide range of RNA viruses in cultured cells, including members of the Togaviridae, Flaviviridae, Bunyaviridae, Picornaviridae, and Paramyxoviridae families. Finally, we demonstrate that antimycin A reduces central nervous system viral titers, improves clinical disease severity, and enhances survival in mice given a lethal challenge with western equine encephalitis virus. Our results provide conclusive validation for using natural product resources derived from marine microbes as source material for antiviral drug discovery, and they indicate that host mitochondrial electron transport is a viable target for the continued development of broadly active antiviral compounds.


Assuntos
Actinobacteria/química , Antivirais/farmacologia , Sedimentos Geológicos/microbiologia , Animais , Antimicina A/química , Antimicina A/farmacologia , Antimicina A/uso terapêutico , Antivirais/química , Antivirais/isolamento & purificação , Produtos Biológicos/química , Produtos Biológicos/isolamento & purificação , Produtos Biológicos/farmacologia , Linhagem Celular , Sistema Nervoso Central/efeitos dos fármacos , Sistema Nervoso Central/patologia , Sistema Nervoso Central/virologia , Fracionamento Químico , Transporte de Elétrons/efeitos dos fármacos , Vírus da Encefalite/efeitos dos fármacos , Encefalite por Arbovirus/tratamento farmacológico , Encefalite por Arbovirus/patologia , Encefalite por Arbovirus/virologia , Ensaios de Triagem em Larga Escala , Humanos , Camundongos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , RNA Viral/metabolismo , Padrões de Referência , Reprodutibilidade dos Testes , Streptomyces/química , Análise de Sobrevida , Transcrição Gênica/efeitos dos fármacos
18.
Chem Biol ; 18(11): 1442-52, 2011 Nov 23.
Artigo em Inglês | MEDLINE | ID: mdl-22118678

RESUMO

The chemical diversity of nature has tremendous potential for the discovery of molecular probes and medicinal agents. However, sensitivity of HTS assays to interfering components of crude extracts derived from plants, and macro- and microorganisms has curtailed their use in lead discovery. Here, we describe a process for leveraging the concentration-response curves obtained from quantitative HTS to improve the initial selection of "actives" from a library of partially fractionated natural product extracts derived from marine actinomycetes and fungi. By using pharmacological activity, the first-pass CRC paradigm improves the probability that labor-intensive subsequent steps of reculturing, extraction, and bioassay-guided isolation of active component(s) target the most promising strains and growth conditions. We illustrate how this process identified a family of fungal metabolites as potent inhibitors of firefly luciferase, subsequently resolved in molecular detail by X-ray crystallography.


Assuntos
Inibidores Enzimáticos/química , Lactonas/química , Luciferases/antagonistas & inibidores , Aspergillus/química , Sítios de Ligação , Cristalografia por Raios X , Bases de Dados de Proteínas , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Ensaios de Triagem em Larga Escala , Lactonas/isolamento & purificação , Lactonas/farmacologia , Luciferases/metabolismo , Espectroscopia de Ressonância Magnética , Conformação Molecular , Estrutura Terciária de Proteína
19.
J Biomol Screen ; 16(8): 825-35, 2011 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-21844328

RESUMO

Despite advances toward understanding the prevention and treatment of many cancers, patients who suffer from oral squamous cell carcinoma (OSCC) confront a survival rate that has remained unimproved for more than 2 decades, indicating our ability to treat them pharmacologically has reached a plateau. In an ongoing effort to improve the clinical outlook for this disease, we previously reported that an essential component of the mechanism by which the proteasome inhibitor bortezomib (PS-341, Velcade) induced apoptosis in OSCC required the activation of a terminal unfolded protein response (UPR). Predicated on these studies, the authors hypothesized that high-throughput screening (HTS) of large diverse chemical libraries might identify more potent or selective small-molecule activators of the apoptotic arm of the UPR to control or kill OSCC. They have developed complementary cell-based assays using stably transfected CHO-K1 cell lines that individually assess the PERK/eIF2α/CHOP (apoptotic) or the IRE1/XBP1 (adaptive) UPR subpathways. An 66 K compound collection was screened at the University of Michigan Center for Chemical Genomics that included a unique library of prefractionated natural product extracts. The mycotoxin methoxycitrinin was isolated from a natural extract and found to selectively activate the CHOP-luciferase reporter at 80 µM. A series of citrinin derivatives was isolated from these extracts, including a unique congener that has not been previously described. In an effort to identify more potent compounds, the authors examined the ability of citrinin and the structurally related mycotoxins ochratoxin A and patulin to activate the UPR. Strikingly, it was found that patulin at 2.5 to 10 µM induced a terminal UPR in a panel of OSCC cells that was characterized by an increase in CHOP, GADD34, and ATF3 gene expression and XBP1 splicing. A luminescent caspase assay and the induction of several BH3-only genes indicated that patulin could induce apoptosis in OSCC cells. These data support the use of this complementary HTS strategy to identify novel modulators of UPR signaling and tumor cell death.


Assuntos
Antineoplásicos/farmacologia , Carcinoma de Células Escamosas/tratamento farmacológico , Avaliação Pré-Clínica de Medicamentos/métodos , Ensaios de Triagem em Larga Escala , Neoplasias Bucais/tratamento farmacológico , Micotoxinas/farmacologia , Animais , Apoptose , Ácidos Borônicos/farmacologia , Bortezomib , Células CHO , Carcinoma de Células Escamosas/patologia , Caspases/genética , Caspases/metabolismo , Proliferação de Células/efeitos dos fármacos , Cricetinae , Fator de Iniciação 2 em Eucariotos/genética , Fator de Iniciação 2 em Eucariotos/metabolismo , Genes Reporter , Humanos , Luciferases/análise , Neoplasias Bucais/patologia , Pirazinas/farmacologia , Transdução de Sinais , Fator de Transcrição CHOP/genética , Fator de Transcrição CHOP/metabolismo , Transdução Genética , Resposta a Proteínas não Dobradas/efeitos dos fármacos , eIF-2 Quinase/genética , eIF-2 Quinase/metabolismo
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