Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 10 de 10
Filtrar
Mais filtros








Base de dados
Intervalo de ano de publicação
1.
J Am Chem Soc ; 146(9): 6114-6124, 2024 03 06.
Artigo em Inglês | MEDLINE | ID: mdl-38389455

RESUMO

Microorganisms are remarkable chemists capable of assembling complex molecular architectures that penetrate cells and bind biomolecular targets with exquisite selectivity. Consequently, microbial natural products have wide-ranging applications in medicine and agriculture. How the "blind watchmaker" of evolution creates skeletal diversity is a key question in natural products research. Comparative analysis of biosynthetic pathways to structurally related metabolites is an insightful approach to addressing this. Here, we report comparative biosynthetic investigations of gladiolin, a polyketide antibiotic from Burkholderia gladioli with promising activity against multidrug-resistant Mycobacterium tuberculosis, and etnangien, a structurally related antibiotic produced by Sorangium cellulosum. Although these metabolites have very similar macrolide cores, their C21 side chains differ significantly in both length and degree of saturation. Surprisingly, the trans-acyltransferase polyketide synthases (PKSs) that assemble these antibiotics are almost identical, raising intriguing questions about mechanisms underlying structural diversification in this important class of biosynthetic assembly line. In vitro reconstitution of key biosynthetic transformations using simplified substrate analogues, combined with gene deletion and complementation experiments, enabled us to elucidate the origin of all the structural differences in the C21 side chains of gladiolin and etnangien. The more saturated gladiolin side chain arises from a cis-acting enoylreductase (ER) domain in module 1 and in trans recruitment of a standalone ER to module 5 of the PKS. Remarkably, module 5 of the gladiolin PKS is intrinsically iterative in the absence of the standalone ER, accounting for the longer side chain in etnangien. These findings have important implications for biosynthetic engineering approaches to the creation of novel polyketide skeletons.


Assuntos
Produtos Biológicos , Imidazóis , Macrolídeos , Polienos , Policetídeos , Sulfonamidas , Tiofenos , Policetídeo Sintases/metabolismo , Aciltransferases , Antibacterianos , Policetídeos/metabolismo , Produtos Biológicos/metabolismo
2.
J Ind Microbiol Biotechnol ; 50(1)2023 Feb 17.
Artigo em Inglês | MEDLINE | ID: mdl-38052426

RESUMO

Microbial natural products are specialized metabolites that are sources of many bioactive compounds including antibiotics, antifungals, antiparasitics, anticancer agents, and probes of biology. The assembly of libraries of producers of natural products has traditionally been the province of the pharmaceutical industry. This sector has gathered significant historical collections of bacteria and fungi to identify new drug leads with outstanding outcomes-upwards of 60% of drug scaffolds originate from such libraries. Despite this success, the repeated rediscovery of known compounds and the resultant diminishing chemical novelty contributed to a pivot from this source of bioactive compounds toward more tractable synthetic compounds in the drug industry. The advent of advanced mass spectrometry tools, along with rapid whole genome sequencing and in silico identification of biosynthetic gene clusters that encode the machinery necessary for the synthesis of specialized metabolites, offers the opportunity to revisit microbial natural product libraries with renewed vigor. Assembling a suitable library of microbes and extracts for screening requires the investment of resources and the development of methods that have customarily been the proprietary purview of large pharmaceutical companies. Here, we report a perspective on our efforts to assemble a library of natural product-producing microbes and the establishment of methods to extract and fractionate bioactive compounds using resources available to most academic labs. We validate the library and approach through a series of screens for antimicrobial and cytotoxic agents. This work serves as a blueprint for establishing libraries of microbial natural product producers and bioactive extract fractions suitable for screens of bioactive compounds. ONE-SENTENCE SUMMARY: Natural products are key to discovery of novel antimicrobial agents: Here, we describe our experience and lessons learned in constructing a microbial natural product and pre-fractionated extract library.


Assuntos
Antineoplásicos , Produtos Biológicos , Produtos Biológicos/química , Biblioteca Gênica , Fungos/genética , Indústria Farmacêutica
3.
mBio ; : e0179123, 2023 Nov 28.
Artigo em Inglês | MEDLINE | ID: mdl-38014974

RESUMO

IMPORTANCE: Cfr is an antibiotic resistance enzyme that inhibits five clinically important antibiotic classes, is genetically mobile, and has a minimal fitness cost, making Cfr a serious threat to antibiotic efficacy. The significance of our work is in discovering molecules that inhibit Cfr-dependent methylation of the ribosome, thus protecting the efficacy of the PhLOPSA antibiotics. These molecules are the first reported inhibitors of Cfr-mediated ribosome methylation and, as such, will guide the further discovery of chemical scaffolds against Cfr-mediated antibiotic resistance. Our work acts as a foundation for further development of molecules that safeguard the PhLOPSA antibiotics from Cfr.

4.
Nat Chem Biol ; 18(12): 1410-1416, 2022 12.
Artigo em Inglês | MEDLINE | ID: mdl-36109649

RESUMO

Modular polyketide synthases (PKSs) are biosynthetic assembly lines that construct structurally diverse natural products with wide-ranging applications in medicine and agriculture. Various mechanisms contribute to structural diversification during PKS-mediated chain assembly, including dehydratase (DH) domain-mediated elimination of water from R and S-configured 3-hydroxy-thioesters to introduce E- and Z-configured carbon-carbon double bonds, respectively. Here we report the discovery of a DH domain variant that catalyzes the sequential elimination of two molecules of water from a (3R, 5S)-3,5-dihydroxy thioester during polyketide chain assembly, introducing a conjugated E,Z-diene into various modular PKS products. We show that the reaction proceeds via a (2E, 5S)-2-enoyl-5-hydroxy-thioester intermediate and involves an additional universally conserved histidine residue that is absent from the active site of most conventional DH domains. These findings expand the diverse range of chemistries mediated by DH-like domains in modular PKSs, highlighting the catalytic versatility of the double hotdog fold.


Assuntos
Policetídeo Sintases , Policetídeos , Policetídeo Sintases/metabolismo , Polienos , Hidroliases/genética , Hidroliases/metabolismo , Água , Carbono , Especificidade por Substrato
5.
Nat Microbiol ; 7(3): 451-462, 2022 03.
Artigo em Inglês | MEDLINE | ID: mdl-35246663

RESUMO

The caseinolytic protease (ClpP) is part of a highly conserved proteolytic complex whose disruption can lead to antibacterial activity but for which few specific inhibitors have been discovered. Specialized metabolites produced by bacteria have been shaped by evolution for specific functions, making them a potential source of selective ClpP inhibitors. Here, we describe a target-directed genome mining strategy for discovering ClpP-interacting compounds by searching for biosynthetic gene clusters that contain duplicated copies of ClpP as putative antibiotic resistance genes. We identify a widespread family of ClpP-associated clusters that are known to produce pyrrolizidine alkaloids but whose connection to ClpP has never been made. We show that previously characterized molecules do not affect ClpP function but are shunt metabolites derived from the genuine product of these gene clusters, a reactive covalent ClpP inhibitor. Focusing on one such cryptic gene cluster from Streptomyces cattleya, we identify the relevant inhibitor, which we name clipibicyclene, and show that it potently and selectively inactivates ClpP. Finally, we solve the crystal structure of clipibicyclene-modified Escherichia coli ClpP. Clipibicyclene's discovery reveals the authentic function of a family of natural products whose specificity for ClpP and abundance in nature illuminate the role of eco-evolutionary forces during bacterial competition.


Assuntos
Endopeptidase Clp , Inibidores de Proteases , Antibacterianos/química , Antibacterianos/farmacologia , Resistência Microbiana a Medicamentos , Endopeptidase Clp/química , Endopeptidase Clp/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Genes Bacterianos/genética , Família Multigênica , Peptídeo Hidrolases/metabolismo , Inibidores de Proteases/farmacologia
6.
ACS Omega ; 7(5): 4170-4184, 2022 Feb 08.
Artigo em Inglês | MEDLINE | ID: mdl-35155911

RESUMO

The aminopolycarboxylic acid aspergillomarasmine A (AMA) is a natural Zn2+ metallophore and inhibitor of metallo-ß-lactamases (MBLs) which reverses ß-lactam resistance. The first crystal structure of an AMA coordination complex is reported and reveals a pentadentate ligand with distorted octahedral geometry. We report the solid-phase synthesis of 23 novel analogs of AMA involving structural diversification of each subunit (l-Asp, l-APA1, and l-APA2). Inhibitory activity was evaluated in vitro using five strains of Escherichia coli producing globally prevalent MBLs. Further in vitro assessment was performed with purified recombinant enzymes and intracellular accumulation studies. Highly constrained structure-activity relationships were demonstrated, but three analogs revealed favorable characteristics where either Zn2+ affinity or the binding mode to MBLs were improved. This study identifies compounds that can further be developed to produce more potent and broader-spectrum MBL inhibitors with improved pharmacodynamic/pharmacokinetic properties.

7.
Chem Rev ; 121(6): 3464-3494, 2021 03 24.
Artigo em Inglês | MEDLINE | ID: mdl-33606500

RESUMO

The use of life-saving antibiotics has long been plagued by the ability of pathogenic bacteria to acquire and develop an array of antibiotic resistance mechanisms. The sum of these resistance mechanisms, the antibiotic resistome, is a formidable threat to antibiotic discovery, development, and use. The study and understanding of the molecular mechanisms in the resistome provide the basis for traditional approaches to combat resistance, including semisynthetic modification of naturally occurring antibiotic scaffolds, the development of adjuvant therapies that overcome resistance mechanisms, and the total synthesis of new antibiotics and their analogues. Using two major classes of antibiotics, the aminoglycosides and tetracyclines as case studies, we review the success and limitations of these strategies when used to combat the many forms of resistance that have emerged toward natural product-based antibiotics specifically. Furthermore, we discuss the use of the resistome as a guide for the genomics-driven discovery of novel antimicrobials, which are essential to combat the growing number of emerging pathogens that are resistant to even the newest approved therapies.


Assuntos
Aminoglicosídeos/farmacologia , Antibacterianos/química , Produtos Biológicos/química , Tetraciclinas/farmacologia , Aminoglicosídeos/metabolismo , Animais , Antibacterianos/farmacologia , Descoberta de Drogas , Resistência Microbiana a Medicamentos , Humanos , Estrutura Molecular , Inibidores de Proteínas Quinases/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Transdução de Sinais , Relação Estrutura-Atividade , Tetraciclinas/metabolismo , beta-Lactamas/metabolismo , beta-Lactamas/farmacologia
8.
Nat Chem ; 11(10): 906-912, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31548673

RESUMO

Polyketide synthases assemble diverse natural products with numerous important applications. The thioester intermediates in polyketide assembly are covalently tethered to acyl carrier protein domains of the synthase. Several mechanisms for polyketide chain release are known, contributing to natural product structural diversification. Here, we report a dual transacylation mechanism for chain release from the enacyloxin polyketide synthase, which assembles an antibiotic with promising activity against Acinetobacter baumannii. A non-elongating ketosynthase domain transfers the polyketide chain from the final acyl carrier protein domain of the synthase to a separate carrier protein, and a non-ribosomal peptide synthetase condensation domain condenses it with (1S,3R,4S)-3,4-dihydroxycyclohexane carboxylic acid. Molecular dissection of this process reveals that non-elongating ketosynthase domain-mediated transacylation circumvents the inability of the condensation domain to recognize the acyl carrier protein domain. Several 3,4-dihydroxycyclohexane carboxylic acid analogues can be employed for chain release, suggesting a promising strategy for producing enacyloxin analogues.


Assuntos
Antibacterianos/biossíntese , Polienos/metabolismo , Policetídeo Sintases/metabolismo , Acinetobacter baumannii/efeitos dos fármacos , Acilação , Antibacterianos/química , Antibacterianos/farmacologia , Testes de Sensibilidade Microbiana , Estrutura Molecular , Polienos/química , Polienos/farmacologia
9.
Chem Sci ; 10(21): 5489-5494, 2019 Jun 07.
Artigo em Inglês | MEDLINE | ID: mdl-31293732

RESUMO

Burkholderia is a multi-talented genus of Gram-negative bacteria, which in recent years has become increasingly recognised as a promising source of bioactive natural products. Metabolite profiling of Burkholderia gladioli BCC0238 showed that it produces the asymmetric lipopeptidiolide antibiotic icosalide A1, originally isolated from a fungus. Comparative bioinformatics analysis of several genome-sequenced B. gladioli isolates identified a gene encoding a nonribosomal peptide synthase (NRPS) with an unusual architecture that was predicted to be responsible for icosalide biosynthesis. Inactivation of this gene in B. gladioli BCC0238 abolished icosalide production. PCR analysis and sequencing of total DNA from the original fungal icosalide A1 producer revealed it has a B. gladioli strain associated with it that harbours an NRPS with an identical architecture to that responsible for icosalide A1 assembly in B. gladioli BCC0238. Sequence analysis of the icosalide NRPS indicated that it contains two chain-initiating condensation (CI) domains. One of these is appended to the N-terminus of module 1 - a common architecture for NRPSs involved in lipopeptide assembly. The other is embedded in module 3, immediately downstream of a putative chain-elongating condensation domain. Analysis of the reactions catalysed by a tridomain construct from module 3 of the NRPS using intact protein mass spectrometry showed that the embedded CI domain initiates assembly of a second lipopeptide chain, providing key insights into the mechanism for asymmetric diolide assembly.

10.
Org Biomol Chem ; 15(44): 9372-9378, 2017 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-29090723

RESUMO

A simple 1H and 13C NMR spectrometric analysis is demonstrated that permits differentiation of isoleucine and allo-isoleucine residues by inspection of the chemical shift and coupling constants of the signals associated with the proton and carbon at the α-stereocentre. This is applied to the estimation of epimerisation during metal-free N-arylation and peptide coupling reactions.


Assuntos
Isoleucina/química , Espectroscopia de Ressonância Magnética , Peptídeos/química , Estereoisomerismo
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA