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1.
ACS Chem Biol ; 18(9): 1909-1914, 2023 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-37561838

RESUMO

The natural product holomycin contains a unique cyclic ene-disulfide and exhibits broad-spectrum antimicrobial activities. Reduced holomycin chelates metal ions with a high affinity and disrupts metal homeostasis in the cell. To identify cellular metalloproteins inhibited by holomycin, reactive-cysteine profiling was performed using isotopic tandem orthogonal proteolysis-activity-based protein profiling (isoTOP-ABPP). This chemoproteomic analysis demonstrated that holomycin treatment increases the reactivity of metal-coordinating cysteine residues in several zinc-dependent and iron-sulfur cluster-dependent enzymes, including carbonic anhydrase II and fumarase A. We validated that holomycin inhibits fumarase A activity in bacterial cells and diminishes the presence of iron-sulfur clusters in fumarase A. Whole-proteome abundance analysis revealed that holomycin treatment induces zinc and iron starvation and cellular stress. This study suggests that holomycin inhibits bacterial growth by impairing the functions of multiple metalloenzymes and sets the stage for investigating the impact of metal-binding molecules on metalloproteomes by using chemoproteomics.


Assuntos
Antibacterianos , Metaloproteínas , Antibacterianos/farmacologia , Metaloproteínas/química , Metaloproteínas/metabolismo , Cisteína , Metais/química , Zinco , Ferro , Homeostase
2.
J Am Chem Soc ; 143(31): 12003-12013, 2021 08 11.
Artigo em Inglês | MEDLINE | ID: mdl-34342433

RESUMO

Hybrid antibiotics are an emerging antimicrobial strategy to overcome antibiotic resistance. The natural product thiomarinol A is a hybrid of two antibiotics: holothin, a dithiolopyrrolone (DTP), and marinolic acid, a close analogue of the drug mupirocin that is used to treat methicillin-resistant Staphylococcus aureus (MRSA). DTPs disrupt metal homeostasis by chelating metal ions in cells, whereas mupirocin targets the essential enzyme isoleucyl-tRNA synthetase (IleRS). Thiomarinol A is over 100-fold more potent than mupirocin against mupirocin-sensitive MRSA; however, its mode of action has been unknown. We show that thiomarinol A targets IleRS. A knockdown of the IleRS-encoding gene, ileS, exhibited sensitivity to a synthetic analogue of thiomarinol A in a chemical genomics screen. Thiomarinol A inhibits MRSA IleRS with a picomolar Ki and binds to IleRS with low femtomolar affinity, 1600 times more tightly than mupirocin. We find that thiomarinol A remains effective against high-level mupirocin-resistant MRSA and provide evidence to support a dual mode of action for thiomarinol A that may include both IleRS inhibition and metal chelation. We demonstrate that MRSA develops resistance to thiomarinol A to a substantially lesser degree than mupirocin and the potent activity of thiomarinol A requires hybridity between DTP and mupirocin. Our findings identify a mode of action of a natural hybrid antibiotic and demonstrate the potential of hybrid antibiotics to combat antibiotic resistance.


Assuntos
Antibacterianos/farmacologia , Inibidores Enzimáticos/farmacologia , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Mupirocina/análogos & derivados , Antibacterianos/química , Inibidores Enzimáticos/química , Isoleucina-tRNA Ligase/antagonistas & inibidores , Isoleucina-tRNA Ligase/metabolismo , Staphylococcus aureus Resistente à Meticilina/metabolismo , Testes de Sensibilidade Microbiana , Estrutura Molecular , Mupirocina/química , Mupirocina/farmacologia
3.
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
4.
Chembiochem ; 20(11): 1387-1393, 2019 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-30694017

RESUMO

The natural product pepticinnamin E potently inhibits protein farnesyl transferases and has potential applications in treating cancer and malaria. Pepticinnamin E contains a rare N-terminal cinnamoyl moiety as well as several nonproteinogenic amino acids, including the unusual 2-chloro-3-hydroxy-4-methoxy-N-methyl-L-phenylalanine. The biosynthesis of pepticinnamin E has remained uncharacterized because its original producing strain is no longer available. Here we identified a gene cluster (pcm) for this natural product in a new producer, Actinobacteria bacterium OK006, by means of a targeted rediscovery strategy. We demonstrated that the pcm cluster is responsible for the biosynthesis of pepticinnamin E, a nonribosomal peptide/polyketide hybrid. We also characterized a key O-methyltransferase that modifies 3,4-dihydroxy-l-phenylalanine. Our work has identified the gene cluster for pepticinnamins for the first time and sets the stage for elucidating the unique chemistry required for biosynthesis.


Assuntos
Actinobacteria , Oligopeptídeos , Actinobacteria/genética , Actinobacteria/metabolismo , Produtos Biológicos/química , Metiltransferases/química , Família Multigênica , Oligopeptídeos/biossíntese , Oligopeptídeos/genética
5.
Chem Res Toxicol ; 32(3): 400-404, 2019 03 18.
Artigo em Inglês | MEDLINE | ID: mdl-30523678

RESUMO

The dithiolopyrrolone (DTP) natural products contain a unique ene-disulfide that is essential for their antimicrobial and anticancer activities. The ene-disulfide in some DTPs is oxidized to a cyclic thiosulfonate, but it is unknown how the DTP thiosulfonates react with biomolecules. We studied the reactivity of the thiosulfonate derivative of the DTP holomycin, oxo-holomycin, and discovered a unique redox reaction: Oxo-holomycin is reduced to its parent disulfide, while oxidizing small molecule and protein thiols to disulfides. Our work reveals that the DTP core is a privileged scaffold that undergoes unusual redox chemistry. The redox chemistry of the DTP natural products may contribute to their mechanism of action.


Assuntos
Dissulfetos/química , Lactamas/química , Pirrolidinonas/química , Compostos de Sulfidrila/química , Ácidos Sulfônicos/química , Estrutura Molecular , Oxirredução
6.
Proc Natl Acad Sci U S A ; 114(10): 2717-2722, 2017 03 07.
Artigo em Inglês | MEDLINE | ID: mdl-28209778

RESUMO

Natural products harbor unique and complex structures that provide valuable antibiotic scaffolds. With an increase in antibiotic resistance, natural products once again hold promise for new antimicrobial therapies, especially those with unique scaffolds that have been overlooked due to a lack of understanding of how they function. Dithiolopyrrolones (DTPs) are an underexplored class of disulfide-containing natural products, which exhibit potent antimicrobial activities against multidrug-resistant pathogens. DTPs were thought to target RNA polymerase, but conflicting observations leave the mechanisms elusive. Using a chemical genomics screen in Escherichia coli, we uncover a mode of action for DTPs-the disruption of metal homeostasis. We show that holomycin, a prototypical DTP, is reductively activated, and reduced holomycin chelates zinc with high affinity. Examination of reduced holomycin against zinc-dependent metalloenzymes revealed that it inhibits E. coli class II fructose bisphosphate aldolase, but not RNA polymerase. Reduced holomycin also strongly inhibits metallo-ß-lactamases in vitro, major contributors to clinical carbapenem resistance, by removing active site zinc. These results indicate that holomycin is an intracellular metal-chelating antibiotic that inhibits a subset of metalloenzymes and that RNA polymerase is unlikely to be the primary target. Our work establishes a link between the chemical structures of DTPs and their antimicrobial action; the ene-dithiol group of DTPs enables high-affinity metal binding as a central mechanism to inhibit metabolic processes. Our study also validates the use of chemical genomics in characterizing modes of actions of antibiotics and emphasizes the potential of metal-chelating natural products in antimicrobial therapy.


Assuntos
Escherichia coli/efeitos dos fármacos , Lactamas/farmacologia , Pirróis/química , Tolueno/análogos & derivados , Antibacterianos/química , Antibacterianos/uso terapêutico , Produtos Biológicos/química , Produtos Biológicos/uso terapêutico , Domínio Catalítico/efeitos dos fármacos , RNA Polimerases Dirigidas por DNA/antagonistas & inibidores , RNA Polimerases Dirigidas por DNA/genética , Dissulfetos/química , Dissulfetos/uso terapêutico , Farmacorresistência Bacteriana/genética , Escherichia coli/genética , Genômica , Homeostase/efeitos dos fármacos , Humanos , Lactamas/química , Metaloproteínas/antagonistas & inibidores , Metaloproteínas/genética , Metais/química , Pirróis/uso terapêutico , Tolueno/química , Tolueno/uso terapêutico , Zinco/metabolismo , beta-Lactamases/efeitos dos fármacos , beta-Lactamases/genética
7.
Curr Top Med Chem ; 16(15): 1695-704, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-26456469

RESUMO

Microbes are important producers of natural products, which have played key roles in understanding biology and treating disease. However, the full potential of microbes to produce natural products has yet to be realized; the overwhelming majority of natural product gene clusters encoded in microbial genomes remain "cryptic", and have not been expressed or characterized. In contrast to the fast-growing number of genomic sequences and bioinformatic tools, methods to connect these genes to natural product molecules are still limited, creating a bottleneck in genome-mining efforts to discover novel natural products. Here we review developing technologies that leverage the power of homologous recombination to directly capture natural product gene clusters and express them in model hosts for isolation and structural characterization. Although direct capture is still in its early stages of development, it has been successfully utilized in several different classes of natural products. These early successes will be reviewed, and the methods will be compared and contrasted with existing traditional technologies. Lastly, we will discuss the opportunities for the development of direct capture in other organisms, and possibilities to integrate direct capture with emerging genome-editing techniques to accelerate future study of natural products.


Assuntos
Produtos Biológicos/metabolismo , Descoberta de Drogas , Genômica , Produtos Biológicos/química , Biologia Computacional , Humanos
8.
J Am Chem Soc ; 137(10): 3494-7, 2015 Mar 18.
Artigo em Inglês | MEDLINE | ID: mdl-25742119

RESUMO

Thiocillins from Bacillus cereus ATCC 14579 are members of the well-known thiazolyl peptide class of natural product antibiotics, the biosynthesis of which has recently been shown to proceed via post-translational modification of ribosomally encoded precursor peptides. It has long been hypothesized that the final step of thiazolyl peptide biosynthesis involves a formal [4 + 2] cycloaddition between two dehydroalanines, a unique transformation that had eluded enzymatic characterization. Here we demonstrate that TclM, a single enzyme from the thiocillin biosynthetic pathway, catalyzes this transformation. To facilitate characterization of this new class of enzyme, we have developed a combined chemical and biological route to the complex peptide substrate, relying on chemical synthesis of a modified C-terminal fragment and coupling to a 38-residue leader peptide by means of native chemical ligation (NCL). This strategy, combined with active enzyme, provides a new chemoenzymatic route to this promising class of antibiotics.


Assuntos
Biocatálise , Produtos Biológicos/síntese química , Enzimas/metabolismo , Peptídeos/síntese química , Sequência de Aminoácidos , Produtos Biológicos/química , Reação de Cicloadição , Dados de Sequência Molecular , Peptídeos/química
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