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1.
Cell Rep ; 42(3): 112199, 2023 03 28.
Artigo em Inglês | MEDLINE | ID: mdl-36870054

RESUMO

The DNA-alkylating metabolite tilimycin is a microbial genotoxin. Intestinal accumulation of tilimycin in individuals carrying til+ Klebsiella spp. causes apoptotic erosion of the epithelium and colitis. Renewal of the intestinal lining and response to injury requires the activities of stem cells located at the base of intestinal crypts. This study interrogates the consequences of tilimycin-induced DNA damage to cycling stem cells. We charted the spatial distribution and luminal quantities of til metabolites in Klebsiella-colonized mice in the context of a complex microbial community. Loss of marker gene G6pd function indicates genetic aberrations in colorectal stem cells that became stabilized in monoclonal mutant crypts. Mice colonized with tilimycin-producing Klebsiella displayed both higher frequencies of somatic mutation and more mutations per affected individual than animals carrying a non-producing mutant. Our findings imply that genotoxic til+ Klebsiella may drive somatic genetic change in the colon and increase disease susceptibility in human hosts.


Assuntos
Microbiota , Mutagênicos , Humanos , Camundongos , Animais , Mutagênicos/metabolismo , Colo/metabolismo , Mutação/genética , Células-Tronco , Mucosa Intestinal
2.
Nat Microbiol ; 7(11): 1834-1848, 2022 11.
Artigo em Inglês | MEDLINE | ID: mdl-36289400

RESUMO

Klebsiella spp. that secrete the DNA-alkylating enterotoxin tilimycin colonize the human intestinal tract. Numbers of toxigenic bacteria increase during antibiotic use, and the resulting accumulation of tilimycin in the intestinal lumen damages the epithelium via genetic instability and apoptosis. Here we examine the impact of this genotoxin on the gut ecosystem. 16S rRNA sequencing of faecal samples from mice colonized with Klebsiella oxytoca strains and mechanistic analyses show that tilimycin is a pro-mutagenic antibiotic affecting multiple phyla. Transient synthesis of tilimycin in the murine gut antagonized niche competitors, reduced microbial richness and altered taxonomic composition of the microbiota both during and following exposure. Moreover, tilimycin secretion increased rates of mutagenesis in co-resident opportunistic pathogens such as Klebsiella pneumoniae and Escherichia coli, as shown by de novo acquisition of antibiotic resistance. We conclude that tilimycin is a bacterial mutagen, and flares of genotoxic Klebsiella have the potential to drive the emergence of resistance, destabilize the gut microbiota and shape its evolutionary trajectory.


Assuntos
Enterotoxinas , Klebsiella , Animais , Humanos , Camundongos , Antibacterianos/farmacologia , Resistência Microbiana a Medicamentos , Ecossistema , Escherichia coli/genética , Klebsiella/genética , RNA Ribossômico 16S/genética , Microbioma Gastrointestinal
3.
J Am Chem Soc ; 144(14): 6237-6250, 2022 04 13.
Artigo em Inglês | MEDLINE | ID: mdl-35362954

RESUMO

Chronically elevated circulating fatty acid levels promote lipid accumulation in nonadipose tissues and cause lipotoxicity. Adipose triglyceride lipase (ATGL) critically determines the release of fatty acids from white adipose tissue, and accumulating evidence suggests that inactivation of ATGL has beneficial effects on lipotoxicity-driven disorders including insulin resistance, steatohepatitis, and heart disease, classifying ATGL as a promising drug target. Here, we report on the development and biological characterization of the first small-molecule inhibitor of human ATGL. This inhibitor, designated NG-497, selectively inactivates human and nonhuman primate ATGL but not structurally and functionally related lipid hydrolases. We demonstrate that NG-497 abolishes lipolysis in human adipocytes in a dose-dependent and reversible manner. The combined analysis of mouse- and human-selective inhibitors, chimeric ATGL proteins, and homology models revealed detailed insights into enzyme-inhibitor interactions. NG-497 binds ATGL within a hydrophobic cavity near the active site. Therein, three amino acid residues determine inhibitor efficacy and species selectivity and thus provide the molecular scaffold for selective inhibition.


Assuntos
Aciltransferases/antagonistas & inibidores , Adipócitos , Ácidos Graxos/metabolismo , Lipólise , Aciltransferases/metabolismo , Adipócitos/metabolismo , Animais , Humanos , Lipólise/fisiologia , Camundongos
4.
Chemistry ; 27(56): 14108-14120, 2021 Oct 07.
Artigo em Inglês | MEDLINE | ID: mdl-34314529

RESUMO

Dipeptidyl peptidase III (DPP3) is a ubiquitously expressed Zn-dependent protease, which plays an important role in regulating endogenous peptide hormones, such as enkephalins or angiotensins. In previous biophysical studies, it could be shown that substrate binding is driven by a large entropic contribution due to the release of water molecules from the closing binding cleft. Here, the design, synthesis and biophysical characterization of peptidomimetic inhibitors is reported, using for the first time an hydroxyethylene transition-state mimetic for a metalloprotease. Efficient routes for the synthesis of both stereoisomers of the pseudopeptide core were developed, which allowed the synthesis of peptidomimetic inhibitors mimicking the VVYPW-motif of tynorphin. The best inhibitors inhibit DPP3 in the low µM range. Biophysical characterization by means of ITC measurement and X-ray crystallography confirm the unusual entropy-driven mode of binding. Stability assays demonstrated the desired stability of these inhibitors, which efficiently inhibited DPP3 in mouse brain homogenate.


Assuntos
Peptidomiméticos , Animais , Dipeptidil Peptidases e Tripeptidil Peptidases , Entropia , Etilenos , Camundongos
5.
Talanta ; 222: 121677, 2021 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-33167283

RESUMO

Non-ribosomal peptides are one class of bacterial metabolites formed by gut microbiota. Intestinal resident Klebsiella oxytoca produces two pyrrolobenzodiazepines, tilivalline and tilimycin, via the same nonribosomal biosynthesis platform. These molecules cause human disease by genotoxic and tubulin inhibitory activities resulting in apoptosis of the intestinal epithelium, loss of barrier integrity and ultimately colitis. Here we report a fast, reliable, HPLC-HR-ESMS2 method for quantifying simultaneously the bacterial enterotoxins tilimycin and tilivalline in complex biological matrices. We synthesized and applied stable isotopically labeled internal standards for precise quantification of the metabolites. Sample preparation was optimized using clinical and laboratory specimens including serum, colonic fluid and stool. The developed method overcame the disadvantage of low selectivity by applying high resolution mass spectrometry in MS2 mode. High sensitivity and low interference from matrices were achieved and validated. We show that the approach is suitable for detection and quantification of the enterotoxic metabolites produced in vivo, in infected human or animal hosts, and in bacterial culture in vitro.


Assuntos
Benzodiazepinonas , Enterotoxinas , Animais , Toxinas Bacterianas , Benzodiazepinas , Cromatografia Líquida de Alta Pressão , Humanos , Pirróis
6.
Adv Synth Catal ; 362(2): 331-336, 2020 Jan 23.
Artigo em Inglês | MEDLINE | ID: mdl-32063821

RESUMO

The Pd-catalyzed S-allylation of thiols with stable allylcarbonate and allylacetate reagents offers several advantages over established reactions for the formation of thioethers. We could demonstrate that Pd/BIPHEPHOS is a catalyst system which allows the transition metal-catalyzed S-allylation of thiols with excellent n-regioselectivity. Mechanistic studies showed that this reaction is reversible under the applied reaction conditions. The excellent functional group tolerance of this transformation was demonstrated with a broad variety of thiol nucleophiles (18 examples) and allyl substrates (9 examples), and could even be applied for the late-stage diversification of cephalosporins, which might find application in the synthesis of new antibiotics.

7.
J Am Chem Soc ; 141(37): 14931-14937, 2019 09 18.
Artigo em Inglês | MEDLINE | ID: mdl-31469558

RESUMO

The prenylation of peptides and proteins is an important post-translational modification observed in vivo. We report that the Pd-catalyzed Tsuji-Trost allylation with a Pd/BIPHEPHOS catalyst system allows the allylation of Cys-containing peptides and proteins with complete chemoselectivity and high n/i regioselectivity. In contrast to recently established methods, which use non-native connections, the Pd-catalyzed prenylation produces the natural n-prenylthioether bond. In addition, a variety of biophysical probes such as affinity handles and fluorescent tags can be introduced into Cys-containing peptides and proteins. Furthermore, peptides containing two cysteine residues can be stapled or cyclized using homobifunctional allylic carbonate reagents.


Assuntos
Cisteína/química , Paládio/química , Peptídeos/química , Processamento de Proteína Pós-Traducional , Proteínas/química , Catálise , Prenilação
8.
Proc Natl Acad Sci U S A ; 116(9): 3774-3783, 2019 02 26.
Artigo em Inglês | MEDLINE | ID: mdl-30808763

RESUMO

Establishing causal links between bacterial metabolites and human intestinal disease is a significant challenge. This study reveals the molecular basis of antibiotic-associated hemorrhagic colitis (AAHC) caused by intestinal resident Klebsiella oxytoca Colitogenic strains produce the nonribosomal peptides tilivalline and tilimycin. Here, we verify that these enterotoxins are present in the human intestine during active colitis and determine their concentrations in a murine disease model. Although both toxins share a pyrrolobenzodiazepine structure, they have distinct molecular targets. Tilimycin acts as a genotoxin. Its interaction with DNA activates damage repair mechanisms in cultured cells and causes DNA strand breakage and an increased lesion burden in cecal enterocytes of colonized mice. In contrast, tilivalline binds tubulin and stabilizes microtubules leading to mitotic arrest. To our knowledge, this activity is unique for microbiota-derived metabolites of the human intestine. The capacity of both toxins to induce apoptosis in intestinal epithelial cells-a hallmark feature of AAHC-by independent modes of action, strengthens our proposal that these metabolites act collectively in the pathogenicity of colitis.


Assuntos
Enterocolite Pseudomembranosa/genética , Enterotoxinas/metabolismo , Interações entre Hospedeiro e Microrganismos/genética , Klebsiella oxytoca/genética , Animais , Benzodiazepinonas/metabolismo , Benzodiazepinonas/toxicidade , Dano ao DNA/efeitos dos fármacos , Enterocolite Pseudomembranosa/microbiologia , Enterocolite Pseudomembranosa/patologia , Enterotoxinas/biossíntese , Células Epiteliais/microbiologia , Células Epiteliais/patologia , Humanos , Intestinos/microbiologia , Intestinos/patologia , Infecções por Klebsiella/genética , Infecções por Klebsiella/microbiologia , Klebsiella oxytoca/metabolismo , Klebsiella oxytoca/patogenicidade , Camundongos , Microtúbulos/efeitos dos fármacos , Oxiquinolina/análogos & derivados , Oxiquinolina/metabolismo , Oxiquinolina/toxicidade , Peptídeos/metabolismo , Peptídeos/toxicidade
9.
Angew Chem Int Ed Engl ; 56(46): 14753-14757, 2017 11 13.
Artigo em Inglês | MEDLINE | ID: mdl-28977734

RESUMO

The nonribosomal enterotoxin tilivalline was the first naturally occurring pyrrolobenzodiazepine to be linked to disease in the human intestine. Since the producing organism Klebsiella oxytoca is part of the intestinal microbiota and the pyrrolobenzodiazepine causes the pathogenesis of colitis it is important to understand the biosynthesis and regulation of tilivalline activity. Here we report the biosynthesis of tilivalline and show that this nonribosomal peptide assembly pathway initially generates tilimycin, a simple pyrrolobenzodiazepine with cytotoxic properties. Tilivalline results from the non-enzymatic spontaneous reaction of tilimycin with biogenetically generated indole. Through a chemical total synthesis of tilimycin we could corroborate the predictions made about the biosynthesis. Production of two cytotoxic pyrrolobenzodiazepines with distinct functionalities by human gut resident Klebsiella oxytoca has important implications for intestinal disease.


Assuntos
Benzodiazepinas/metabolismo , Produtos Biológicos/metabolismo , Pirróis/metabolismo , Klebsiella oxytoca/metabolismo
10.
Org Biomol Chem ; 13(42): 10456-60, 2015 Nov 14.
Artigo em Inglês | MEDLINE | ID: mdl-26421937

RESUMO

N-Protected amino acids can be easily converted into chiral α-amino aldehydes in a one-pot reaction by activation with CDI followed by reduction with DIBAL-H. This method delivers Boc-, Cbz- and Fmoc-protected amino aldehydes from proteinogenic amino acids in very good isolated yields and complete stereointegrity.


Assuntos
Aldeídos/síntese química , Aminoácidos/química , Imidazóis/química , Compostos Organometálicos/química , Aldeídos/química , Oxirredução , Fatores de Tempo
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