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1.
Nat Commun ; 9(1): 4147, 2018 10 08.
Artigo em Inglês | MEDLINE | ID: mdl-30297823

RESUMO

Rifamycin antibiotics (Rifs) target bacterial RNA polymerases (RNAPs) and are widely used to treat infections including tuberculosis. The utility of these compounds is threatened by the increasing incidence of resistance (RifR). As resistance mechanisms found in clinical settings may also occur in natural environments, here we postulated that bacteria could have evolved to produce rifamycin congeners active against clinically relevant resistance phenotypes. We survey soil metagenomes and identify a tailoring enzyme-rich family of gene clusters encoding biosynthesis of rifamycin congeners (kanglemycins, Kangs) with potent in vivo and in vitro activity against the most common clinically relevant RifR mutations. Our structural and mechanistic analyses reveal the basis for Kang inhibition of RifR RNAP. Unlike Rifs, Kangs function through a mechanism that includes interfering with 5'-initiating substrate binding. Our results suggest that examining soil microbiomes for new analogues of clinically used antibiotics may uncover metabolites capable of circumventing clinically important resistance mechanisms.


Assuntos
Farmacorresistência Bacteriana/efeitos dos fármacos , Mycobacterium tuberculosis/efeitos dos fármacos , Rifampina/farmacologia , Tuberculose/prevenção & controle , Aminobenzoatos/química , Antibióticos Antituberculose/biossíntese , Antibióticos Antituberculose/química , Antibióticos Antituberculose/farmacologia , Bactérias/genética , Bactérias/metabolismo , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , RNA Polimerases Dirigidas por DNA/antagonistas & inibidores , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Farmacorresistência Bacteriana/genética , Humanos , Hidroxibenzoatos/química , Metagenômica/métodos , Estrutura Molecular , Mutação , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/metabolismo , Rifampina/química , Rifampina/metabolismo , Rifamicinas/química , Rifamicinas/farmacologia , Microbiologia do Solo , Tuberculose/microbiologia
2.
Appl Microbiol Biotechnol ; 102(5): 2337-2350, 2018 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-29396588

RESUMO

We used a temperature differential assay with the opportunistic fungal pathogen Cryptococcus neoformans as a simple screening platform to detect small molecules with antifungal activity in natural product extracts. By screening of a collection extracts from two different strains of the coprophilous fungus, Amphichorda felina, we detected strong, temperature-dependent antifungal activity using a two-plate agar zone of inhibition assay at 25 and 37 °C. Bioassay-guided fractionation of the crude extract followed by liquid chromatography-mass spectrometry (LC-MS) and nuclear magnetic resonance spectroscopy (NMR) identified cyclosporin C (CsC) as the main component of the crude extract responsible for growth inhibition of C. neoformans at 37 °C. The presence of CsC was confirmed by comparison with a commercial standard. We sequenced the genome of A. felina to identify and annotate the CsC biosynthetic gene cluster. The only previously characterized gene cluster for the biosynthesis of similar compounds is that of the related immunosuppressant drug cyclosporine A (CsA). The CsA and CsC gene clusters share a high degree of synteny and sequence similarity. Amino acid changes in the adenylation domain of the CsC nonribosomal peptide synthase's sixth module may be responsible for the substitution of L-threonine compared to L-α-aminobutyric acid in the CsA peptide core. This screening strategy promises to yield additional antifungal natural products with a focused spectrum of antimicrobial activity.


Assuntos
Antifúngicos/farmacologia , Cryptococcus neoformans/efeitos dos fármacos , Ciclosporinas/farmacologia , Hypocreales/química , Antifúngicos/química , Antifúngicos/metabolismo , Cryptococcus neoformans/crescimento & desenvolvimento , Ciclosporinas/química , Ciclosporinas/metabolismo , Hypocreales/genética , Hypocreales/metabolismo , Temperatura Ambiente
3.
Nat Chem Biol ; 11(9): 625-31, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-26284661
4.
J Am Chem Soc ; 136(26): 9484-90, 2014 Jul 02.
Artigo em Inglês | MEDLINE | ID: mdl-24884988

RESUMO

Increasing evidence has shown that small-molecule chemistry in microbes (i.e., secondary metabolism) can modulate the microbe-host response in infection and pathogenicity. The bacterial disease melioidosis is conferred by the highly virulent, antibiotic-resistant pathogen Burkholderia pseudomallei (BP). Whereas some macromolecular structures have been shown to influence BP virulence (e.g., secretion systems, cellular capsule, pili), the role of the large cryptic secondary metabolome encoded within its genome has been largely unexplored for its importance to virulence. Herein we demonstrate that BP-encoded small-molecule biosynthesis is indispensible for in vivo BP pathogenicity. Promoter exchange experiments were used to induce high-level molecule production from two gene clusters (MPN and SYR) found to be essential for in vivo virulence. NMR structural characterization of these metabolites identified a new class of lipopeptide biosurfactants/biofilm modulators (the malleipeptins) and syrbactin-type proteasome inhibitors, both of which represent overlooked small-molecule virulence factors for BP. Disruption of Burkholderia virulence by inhibiting the biosynthesis of these small-molecule biosynthetic pathways may prove to be an effective strategy for developing novel melioidosis-specific therapeutics.


Assuntos
Burkholderia pseudomallei/metabolismo , Burkholderia pseudomallei/patogenicidade , Metabolismo Secundário , Fatores de Virulência/química , Fatores de Virulência/metabolismo , Animais , Burkholderia pseudomallei/química , Burkholderia pseudomallei/genética , Feminino , Genoma Bacteriano , Recombinação Homóloga , Lipopeptídeos/química , Lipopeptídeos/metabolismo , Lipopeptídeos/farmacologia , Lisina/análogos & derivados , Lisina/química , Lisina/metabolismo , Lisina/farmacologia , Melioidose/microbiologia , Camundongos Endogâmicos BALB C , Família Multigênica , Mutação , Peptídeos Cíclicos/química , Peptídeos Cíclicos/metabolismo , Peptídeos Cíclicos/farmacologia , Regiões Promotoras Genéticas , Fatores de Virulência/genética
5.
J Am Chem Soc ; 134(32): 13192-5, 2012 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-22765305

RESUMO

Sequenced bacterial genomes are routinely found to contain gene clusters that are predicted to encode metabolites not seen in fermentation-based studies. Pseudomallei group Burkholderia are emerging pathogens whose genomes are particularly rich in cryptic natural product biosynthetic gene clusters. We systematically probed the influence of the cryptic secondary metabolome on the virulence of these bacteria and found that disruption of the MAL gene cluster, which is natively silent in laboratory fermentation experiments and conserved across this group of pathogens, attenuates virulence in animal models. Using a promoter exchange strategy to activate the MAL cluster, we identified malleilactone, a polyketide synthase-derived cytotoxic siderophore encoded by this gene cluster. Small molecules targeting malleilactone biosynthesis either alone or in conjunction with antibiotics could prove useful as therapeutics to combat melioidosis and glanders.


Assuntos
Burkholderia pseudomallei/enzimologia , Burkholderia pseudomallei/genética , Lactonas/química , Metaboloma , Família Multigênica , Fatores de Virulência , Linhagem Celular , Ativação Enzimática , Humanos , Concentração Inibidora 50 , Modelos Biológicos , Policetídeo Sintases/genética , Policetídeo Sintases/metabolismo , Regiões Promotoras Genéticas , Fatores de Virulência/genética , Fatores de Virulência/metabolismo
6.
Org Lett ; 13(6): 1536-9, 2011 Mar 18.
Artigo em Inglês | MEDLINE | ID: mdl-21348454

RESUMO

Natural product gene clusters are often tightly regulated, resulting in gene cluster silencing in laboratory fermentation studies. The systematic overexpression of transcription factors (TFs) associated with biosynthetic gene clusters found in the genome of Burkholderia thailandensis E264 identified a set of TFs that, when overexpressed, alter the secondary metabolome of this bacterium. The isolation and characterization of burkholdacs A and B, two new acyldepsitripeptide histone deacetylase inhibitors produced by B. thailandensis overexpressing the TF bhcM, is reported.


Assuntos
Burkholderia/metabolismo , Depsipeptídeos/isolamento & purificação , Depsipeptídeos/farmacologia , Inibidores de Histona Desacetilases/isolamento & purificação , Inibidores de Histona Desacetilases/farmacologia , Burkholderia/química , Burkholderia/genética , Depsipeptídeos/química , Inibidores de Histona Desacetilases/química , Estrutura Molecular , Família Multigênica , Ressonância Magnética Nuclear Biomolecular , Peptídeos Cíclicos/química , Fatores de Transcrição/genética
7.
J Am Chem Soc ; 133(6): 1638-41, 2011 Feb 16.
Artigo em Inglês | MEDLINE | ID: mdl-21247113

RESUMO

Bacterial genome sequencing projects routinely uncover gene clusters that are predicted to encode the biosynthesis of uncharacterized small molecules. A subset of these cryptic genetic elements appears as individual operons. Here we investigate potential single-operon biosynthetic systems found in the genome of the pathogenic bacterium Burkholderia pseudomallei . Placing these operons under the control of an inducible promoter led to the production of seven new metabolites. Among the molecules we identified are inhibitors of type-4 phosphodiesterases, suggesting that previously cryptic biosynthetic operons may encode metabolites that could contribute to microbial virulence by disrupting host signaling pathways.


Assuntos
Burkholderia pseudomallei/genética , Burkholderia pseudomallei/metabolismo , Genoma Bacteriano/genética , Óperon/genética , Expressão Gênica , Células HeLa , Humanos
8.
ACS Chem Biol ; 5(3): 313-20, 2010 Mar 19.
Artigo em Inglês | MEDLINE | ID: mdl-20050613

RESUMO

Light-directed gene patterning methods have been described as a means to regulate gene expression in a spatially and temporally controlled manner. Several methods have been reported that use photocaged forms of small molecule effectors to control ligand-dependent transcription factors. Whereas these methods offer many advantages including high specificity and transient light-sensitivity, the free diffusion of the uncaged effector can limit both the magnitude and resolution of localized gene induction. Methods to date have been limited by the small fraction of irradiated cells that have expression levels significantly above uninduced background and have not been shown to affect a defined biological response. The tetracycline-dependent transactivator/transrepressor system, RetroTET-ART, combined with a photocaged form of doxycycline (NvOC-Dox) can be used to form photolithographic patterns of induced expression wherein up to 85% of the patterned cells show expression levels above uninduced regions. The efficiency and inducibility of the RetroTET-ART system allows one to quantitatively measure the limits of resolution and the relative induction levels mediated by a small molecule photocaged effector for the first time. Well-defined patterns of reporter genes were reproducibly formed within 6-36 h with feature sizes as small as 300 microm. After photo-patterning, NvOC-Dox can be rapidly removed, rendering cells photoinsensitive and allowing one to monitor GFP product formation in real time. Patterned co-expression of the cell surface ligand ephrin A5 on cell monolayers creates well-defined patterns that are sufficient to direct and segregate co-cultured cells via either attractive or repulsive signaling cues. The ability to direct the arrangement of cells on living cell monolayers through the action of light may serve as a model system for engineering artificial tissues.


Assuntos
Técnicas de Cocultura/métodos , Regulação da Expressão Gênica/efeitos da radiação , Luz , Células 3T3 , Animais , Proteínas de Fluorescência Verde/análise , Proteínas de Fluorescência Verde/genética , Camundongos
9.
Chembiochem ; 8(7): 799-803, 2007 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-17393546

RESUMO

Nuclear hormone receptors (NHRs) represent a diverse class of ligand-dependent transcriptional regulators. NHRs that have been rendered functionally inactive due to mutations that abrogate proper ligand binding can often be rescued by appropriately designed hormone analogues. The analogue-specific receptor-ligand pairs provide an ideal platform from which to develop new chemogenomic tools for the spatial and temporal control of gene expression. Here, we describe the synthesis and in vitro assessment of a photocaged VDR agonist specific to a mutant NHR that is associated with vitamin D-resistant rickets. The results provide insight into the utility of the agonist as a potential tool for photoinduced gene patterning.


Assuntos
Receptores de Calcitriol/agonistas , Espectroscopia de Ressonância Magnética , Espectrometria de Massas , Fotoquímica
10.
Curr Opin Chem Biol ; 11(1): 99-110, 2007 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-17188557

RESUMO

The nuclear hormone receptors are ligand-gated transcription factors that modulate gene expression by directly acting upon genomic DNA, and have been of profound interest across all biological disciplines. Recent advancements in this area have included the expansion of transgene activation through ligand-receptor engineering, drug development from structural design and the exploitation of innate ligand-specific associations towards developing novel conditional protein-based recombinant and diagnostic tools. These advancements come on the heels of exciting new modes of hormone action that challenge and expand upon the classic paradigms of hormone receptor function.


Assuntos
Receptores de Superfície Celular/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Esteroides/química , Esteroides/metabolismo , Animais , DNA/genética , DNA/metabolismo , Desenho de Drogas , Regulação da Expressão Gênica/genética , Regulação da Expressão Gênica/fisiologia , Humanos , Ligantes , Modelos Biológicos , Modelos Moleculares , Receptores de Superfície Celular/química , Receptores Citoplasmáticos e Nucleares/química , Proteínas Recombinantes , Fatores de Transcrição/genética , Fatores de Transcrição/fisiologia , Transgenes/genética , Transgenes/fisiologia
11.
Methods Mol Biol ; 335: 83-92, 2006.
Artigo em Inglês | MEDLINE | ID: mdl-16785622

RESUMO

Exploring the properties of molecules that cleave DNA (i.e., enzymatic nucleases, chemical footprinting agents, and naturally occurring DNA cleaving antibiotics) has been an ongoing process with benefits extending toward both laboratory and clinical applications. Despite the progress that has been made toward understanding the mechanics of DNA cleavage, a simple and continuous assay for detecting DNA cleavage has been lacking. Herein, we describe the molecular break light assay, wherein a single oligo-nucleotide modified by a 5'-fluorophore-3'-quencher pair adopting a stem-loop structure with an appropriate DNA recognition site, provides for the rapid assaying of DNA cleavage with high sensitivity. Furthermore, the described methodology is highly convenient in that it is readily adaptable to common laboratory fluorometers and multi-well plate/ array systems, which may provide the basis for high-throughput screening of novel DNA cleaving agents. This assay may also be further extended to natural or "unnatural" transcription factor protection assay systems.


Assuntos
Enzimas de Restrição do DNA , DNA/análise , Corantes Fluorescentes , Técnicas de Sonda Molecular , Sondas Moleculares , Hibridização de Ácido Nucleico/métodos , DNA/metabolismo
12.
Curr Top Med Chem ; 5(4): 413-20, 2005.
Artigo em Inglês | MEDLINE | ID: mdl-15892683

RESUMO

In some instances, small molecules can restore function to proteins that are impaired by genetic mutations. There are now many examples where non-specific molecules or specific ligands can act as chemical chaperones to fold proteins or stabilize folded proteins harboring genetic mutations. In contrast a few recent examples have shown that functionally impaired proteins that are stably folded can be "functionally rescued" by appropriate small molecules. Compounds that can rescue functionally impaired proteins may provide new strategies for the treatment of genetic diseases such as rickets and resistance to thyroid hormone (RTH). In addition mutant-complementing analogs and substrates that act exclusively on mutant proteins are providing important tools for the study of complex biological systems that are controlled by molecules that have multiple cellular targets.


Assuntos
Doenças Genéticas Inatas/terapia , Engenharia de Proteínas , Animais , Teste de Complementação Genética , Humanos , Chaperonas Moleculares , Proteínas/genética , Proteínas/fisiologia
13.
Science ; 301(5639): 1537-41, 2003 Sep 12.
Artigo em Inglês | MEDLINE | ID: mdl-12970566

RESUMO

Antibiotic self-resistance mechanisms, which include drug elimination, drug modification, target modification, and drug sequestration, contribute substantially to the growing problem of antibiotic resistance among pathogenic bacteria. Enediynes are among the most potent naturally occurring antibiotics, yet the mechanism of resistance to these toxins has remained a mystery. We characterize an enediyne self-resistance protein that reveals a self-sacrificing paradigm for resistance to highly reactive antibiotics, and thus another opportunity for nonpathogenic or pathogenic bacteria to evade extremely potent small molecules.


Assuntos
Aminoglicosídeos , Antibacterianos/farmacologia , Antibióticos Antineoplásicos/farmacologia , Proteínas de Bactérias/metabolismo , Metaloproteínas/metabolismo , Antibacterianos/biossíntese , Antibacterianos/química , Antibióticos Antineoplásicos/biossíntese , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , DNA/metabolismo , Farmacorresistência Bacteriana , Enedi-Inos , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Metaloproteínas/química , Relação Estrutura-Atividade
14.
Org Lett ; 5(13): 2223-6, 2003 Jun 26.
Artigo em Inglês | MEDLINE | ID: mdl-12816414

RESUMO

In vitro glycorandomization (IVG) technology is dependent upon the ability to rapidly synthesize sugar phosphates. Compared with chemical synthesis, enzymatic (kinase) routes to sugar phosphates would be attractive for this application. This work focuses upon the development of a high-throughput colorimetric galactokinase (GalK) assay and its application toward probing the substrate specificity and kinetic parameters of Escherichia coli GalK. The demonstrated dinitrosalicylic assay should also be generally applicable to a variety of sugar-processing enzymes. [reaction: see text]


Assuntos
Escherichia coli/enzimologia , Galactoquinase/química , Galactoquinase/metabolismo , Colorimetria , Galactose/análogos & derivados , Galactose/metabolismo , Glicosídeos/química , Cinética , Salicilatos/química , Especificidade por Substrato
15.
Org Lett ; 5(6): 933-6, 2003 Mar 20.
Artigo em Inglês | MEDLINE | ID: mdl-12633109

RESUMO

[reaction: see text] In an effort to expand the scope of natural product in vitro glycorandomization (IVG), the substrate specificity of NovM was investigated. A test of four aglycon analogues and over 40 nucleotide sugars revealed NovM has a surprisingly stringent substrate specificity and provided only three new "unnatural" natural products. On the basis of the determined substrate specificity, an alternative to the sugar nucleotide biosynthetic dogma and a cautionary note for the general applicability of IVG are introduced.


Assuntos
Antibacterianos/biossíntese , Novobiocina/biossíntese , Glicosídeos/química , Streptomyces , Especificidade por Substrato
16.
Chem Biol ; 9(12): 1305-14, 2002 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-12498883

RESUMO

Analogs of the glycopeptide antibiotics vancomycin and teicoplanin with alterations in one or both sugar moieties of the disaccharide have been prepared by tandem action of the vancomycin pathway glycosyltransferases GtfE and GtfD. All four regioisomers (2-, 3-, 4-, 6-) of TDP-deoxyglucoses and UDP/TDP-aminoglucoses were prepared, predominantly by action of D-glucopyranosyl-1-phosphate thymidylyltransferase, E(p). GtfE transferred the deoxyglucoses or aminoglucoses onto the 4-OH of 4-hydroxyphenylglycine of both the vancomycin and teicoplanin aglycone scaffolds. Kinetic analysis indicated the 2-, 3-, 4-, and 6-amino-glucoses were transferred by GtfE with only a 4- to 30-fold drop in k(cat) and no effect on K(m) compared to the native substrate, UDP/TDP-glucose, suggesting preparative utility. The next enzyme, GtfD, could utilize the variant glucosyl-peptides as substrates for transfer of L-4-epi-vancosamine. The aminosugar moieties in these variant glycopeptides introduce sites for acylation or reductive alkylation.


Assuntos
Técnicas de Química Combinatória , Glucose/análogos & derivados , Glucosiltransferases/metabolismo , Glicopeptídeos/biossíntese , Vancomicina/análogos & derivados , Antibacterianos/biossíntese , Antibacterianos/química , Glucose/metabolismo , Glicopeptídeos/química , Cinética , Estrutura Molecular , Especificidade por Substrato , Teicoplanina/análogos & derivados , Teicoplanina/biossíntese , Vancomicina/biossíntese
17.
Proc Natl Acad Sci U S A ; 99(21): 13397-402, 2002 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-12374866

RESUMO

In vitro "glycorandomization" is a chemoenzymatic approach for generating diverse libraries of glycosylated biomolecules based on natural product scaffolds. This technology makes use of engineered variants of specific enzymes affecting metabolite glycosylation, particularly nucleotidylyltransferases and glycosyltransferases. To expand the repertoire of UDP/dTDP sugars readily available for glycorandomization, we now report a structure-based engineering approach to increase the diversity of alpha-d-hexopyranosyl phosphates accepted by Salmonella enterica LT2 alpha-d-glucopyranosyl phosphate thymidylyltransferase (E(p)). This article highlights the design rationale, determined substrate specificity, and structural elucidation of three "designed" mutations, illustrating both the success and unexpected outcomes from this type of approach. In addition, a single amino acid substitution in the substrate-binding pocket (L89T) was found to significantly increase the set of alpha-d-hexopyranosyl phosphates accepted by E(p) to include alpha-d-allo-, alpha-d-altro-, and alpha-d-talopyranosyl phosphate. In aggregate, our results provide valuable blueprints for altering nucleotidylyltransferase specificity by design, which is the first step toward in vitro glycorandomization.


Assuntos
Nucleotidiltransferases/metabolismo , Nucleotídeos de Pirimidina/química , Substituição de Aminoácidos , Domínio Catalítico/genética , Cristalografia por Raios X , Desenho de Drogas , Escherichia coli/genética , Glicosilação , Modelos Moleculares , Mutagênese Sítio-Dirigida , Nucleotidiltransferases/genética , Engenharia de Proteínas , Nucleotídeos de Pirimidina/biossíntese , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Salmonella enterica/enzimologia , Salmonella enterica/genética , Especificidade por Substrato
19.
Angew Chem Int Ed Engl ; 40(8): 1502-1505, 2001 Apr 17.
Artigo em Inglês | MEDLINE | ID: mdl-29712364

RESUMO

The exploitation of a unique thymidylyltransferase (Ep ) allows the rapid syntheses of thymidine and uridine 5'-(aminodeoxy-α-D-hexopyranosyl diphosphates), 5'-(acetamidodeoxy-α-D-hexopyranosyl diphosphates), and even 5'-(aminodideoxy-α-D-hexopyranosyl diphosphates), which are amino analogues of the products from the native reaction of Ep .

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