Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 22
Filtrar
1.
Cell ; 180(5): 862-877.e22, 2020 03 05.
Artigo em Inglês | MEDLINE | ID: mdl-32142679

RESUMO

Using untargeted metabolomics (n = 1,162 subjects), the plasma metabolite (m/z = 265.1188) phenylacetylglutamine (PAGln) was discovered and then shown in an independent cohort (n = 4,000 subjects) to be associated with cardiovascular disease (CVD) and incident major adverse cardiovascular events (myocardial infarction, stroke, or death). A gut microbiota-derived metabolite, PAGln, was shown to enhance platelet activation-related phenotypes and thrombosis potential in whole blood, isolated platelets, and animal models of arterial injury. Functional and genetic engineering studies with human commensals, coupled with microbial colonization of germ-free mice, showed the microbial porA gene facilitates dietary phenylalanine conversion into phenylacetic acid, with subsequent host generation of PAGln and phenylacetylglycine (PAGly) fostering platelet responsiveness and thrombosis potential. Both gain- and loss-of-function studies employing genetic and pharmacological tools reveal PAGln mediates cellular events through G-protein coupled receptors, including α2A, α2B, and ß2-adrenergic receptors. PAGln thus represents a new CVD-promoting gut microbiota-dependent metabolite that signals via adrenergic receptors.


Assuntos
Doenças Cardiovasculares/sangue , Microbioma Gastrointestinal/genética , Glutamina/análogos & derivados , Trombose/metabolismo , Animais , Artérias/lesões , Artérias/metabolismo , Artérias/microbiologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Plaquetas/metabolismo , Plaquetas/microbiologia , Doenças Cardiovasculares/genética , Doenças Cardiovasculares/microbiologia , Doenças Cardiovasculares/patologia , Morte Súbita Cardíaca/patologia , Glutamina/sangue , Glutamina/genética , Humanos , Masculino , Metaboloma/genética , Metabolômica/métodos , Camundongos , Infarto do Miocárdio/sangue , Infarto do Miocárdio/microbiologia , Ativação Plaquetária/genética , Receptores Adrenérgicos alfa/sangue , Receptores Adrenérgicos alfa/genética , Receptores Adrenérgicos beta/sangue , Receptores Adrenérgicos beta/genética , Fatores de Risco , Acidente Vascular Cerebral/sangue , Acidente Vascular Cerebral/microbiologia , Acidente Vascular Cerebral/patologia , Trombose/genética , Trombose/microbiologia , Trombose/patologia
2.
Cell ; 168(3): 517-526.e18, 2017 01 26.
Artigo em Inglês | MEDLINE | ID: mdl-28111075

RESUMO

The gut microbiota modulate host biology in numerous ways, but little is known about the molecular mediators of these interactions. Previously, we found a widely distributed family of nonribosomal peptide synthetase gene clusters in gut bacteria. Here, by expressing a subset of these clusters in Escherichia coli or Bacillus subtilis, we show that they encode pyrazinones and dihydropyrazinones. At least one of the 47 clusters is present in 88% of the National Institutes of Health Human Microbiome Project (NIH HMP) stool samples, and they are transcribed under conditions of host colonization. We present evidence that the active form of these molecules is the initially released peptide aldehyde, which bears potent protease inhibitory activity and selectively targets a subset of cathepsins in human cell proteomes. Our findings show that an approach combining bioinformatics, synthetic biology, and heterologous gene cluster expression can rapidly expand our knowledge of the metabolic potential of the microbiota while avoiding the challenges of cultivating fastidious commensals.


Assuntos
Bactérias/metabolismo , Microbioma Gastrointestinal , Microbiota , Peptídeo Sintases/metabolismo , Pirazinas/metabolismo , Animais , Bacillus subtilis/genética , Bactérias/classificação , Bactérias/genética , Escherichia coli/genética , Fezes/microbiologia , Humanos , Peptídeo Sintases/genética , Filogenia
3.
Nature ; 602(7898): 647-653, 2022 02.
Artigo em Inglês | MEDLINE | ID: mdl-35165440

RESUMO

Integration of sensory and molecular inputs from the environment shapes animal behaviour. A major site of exposure to environmental molecules is the gastrointestinal tract, in which dietary components are chemically transformed by the microbiota1 and gut-derived metabolites are disseminated to all organs, including the brain2. In mice, the gut microbiota impacts behaviour3, modulates neurotransmitter production in the gut and brain4,5, and influences brain development and myelination patterns6,7. The mechanisms that mediate the gut-brain interactions remain poorly defined, although they broadly involve humoral or neuronal connections. We previously reported that the levels of the microbial metabolite 4-ethylphenyl sulfate (4EPS) were increased in a mouse model of atypical neurodevelopment8. Here we identified biosynthetic genes from the gut microbiome that mediate the conversion of dietary tyrosine to 4-ethylphenol (4EP), and bioengineered gut bacteria to selectively produce 4EPS in mice. 4EPS entered the brain and was associated with changes in region-specific activity and functional connectivity. Gene expression signatures revealed altered oligodendrocyte function in the brain, and 4EPS impaired oligodendrocyte maturation in mice and decreased oligodendrocyte-neuron interactions in ex vivo brain cultures. Mice colonized with 4EP-producing bacteria exhibited reduced myelination of neuronal axons. Altered myelination dynamics in the brain have been associated with behavioural outcomes7,9-14. Accordingly, we observed that mice exposed to 4EPS displayed anxiety-like behaviours, and pharmacological treatments that promote oligodendrocyte differentiation prevented the behavioural effects of 4EPS. These findings reveal that a gut-derived molecule influences complex behaviours in mice through effects on oligodendrocyte function and myelin patterning in the brain.


Assuntos
Ansiedade , Microbioma Gastrointestinal , Microbiota , Animais , Ansiedade/metabolismo , Bactérias , Encéfalo/metabolismo , Microbioma Gastrointestinal/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Microbiota/fisiologia , Bainha de Mielina , Fenóis/metabolismo
4.
Nature ; 582(7813): 566-570, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32555455

RESUMO

The gut microbiota synthesize hundreds of molecules, many of which influence host physiology. Among the most abundant metabolites are the secondary bile acids deoxycholic acid (DCA) and lithocholic acid (LCA), which accumulate at concentrations of around 500 µM and are known to block the growth of Clostridium difficile1, promote hepatocellular carcinoma2 and modulate host metabolism via the G-protein-coupled receptor TGR5 (ref. 3). More broadly, DCA, LCA and their derivatives are major components of the recirculating pool of bile acids4; the size and composition of this pool are a target of therapies for primary biliary cholangitis and nonalcoholic steatohepatitis. Nonetheless, despite the clear impact of DCA and LCA on host physiology, an incomplete knowledge of their biosynthetic genes and a lack of genetic tools to enable modification of their native microbial producers limit our ability to modulate secondary bile acid levels in the host. Here we complete the pathway to DCA and LCA by assigning and characterizing enzymes for each of the steps in its reductive arm, revealing a strategy in which the A-B rings of the steroid core are transiently converted into an electron acceptor for two reductive steps carried out by Fe-S flavoenzymes. Using anaerobic in vitro reconstitution, we establish that a set of six enzymes is necessary and sufficient for the eight-step conversion of cholic acid to DCA. We then engineer the pathway into Clostridium sporogenes, conferring production of DCA and LCA on a nonproducing commensal and demonstrating that a microbiome-derived pathway can be expressed and controlled heterologously. These data establish a complete pathway to two central components of the bile acid pool.


Assuntos
Ácidos e Sais Biliares/química , Ácidos e Sais Biliares/metabolismo , Microbioma Gastrointestinal/genética , Microbioma Gastrointestinal/fisiologia , Hidroxilação/genética , Redes e Vias Metabólicas/genética , Animais , Clostridium/enzimologia , Clostridium/genética , Clostridium/metabolismo , Ácido Desoxicólico/química , Ácido Desoxicólico/metabolismo , Ácido Litocólico/química , Ácido Litocólico/metabolismo , Masculino , Engenharia Metabólica , Camundongos , Óperon/genética , Simbiose
5.
J Biol Chem ; 290(22): 13710-24, 2015 May 29.
Artigo em Inglês | MEDLINE | ID: mdl-25855790

RESUMO

A-500359s, A-503083s, and A-102395 are capuramycin-type nucleoside antibiotics that were discovered using a screen to identify inhibitors of bacterial translocase I, an essential enzyme in peptidoglycan cell wall biosynthesis. Like the parent capuramycin, A-500359s and A-503083s consist of three structural components: a uridine-5'-carboxamide (CarU), a rare unsaturated hexuronic acid, and an aminocaprolactam, the last of which is substituted by an unusual arylamine-containing polyamide in A-102395. The biosynthetic gene clusters for A-500359s and A-503083s have been reported, and two genes encoding a putative non-heme Fe(II)-dependent α-ketoglutarate:UMP dioxygenase and an l-Thr:uridine-5'-aldehyde transaldolase were uncovered, suggesting that C-C bond formation during assembly of the high carbon (C6) sugar backbone of CarU proceeds from the precursors UMP and l-Thr to form 5'-C-glycyluridine (C7) as a biosynthetic intermediate. Here, isotopic enrichment studies with the producer of A-503083s were used to indeed establish l-Thr as the direct source of the carboxamide of CarU. With this knowledge, the A-102395 gene cluster was subsequently cloned and characterized. A genetic system in the A-102395-producing strain was developed, permitting the inactivation of several genes, including those encoding the dioxygenase (cpr19) and transaldolase (cpr25), which abolished the production of A-102395, thus confirming their role in biosynthesis. Heterologous production of recombinant Cpr19 and CapK, the transaldolase homolog involved in A-503083 biosynthesis, confirmed their expected function. Finally, a phosphotransferase (Cpr17) conferring self-resistance was functionally characterized. The results provide the opportunity to use comparative genomics along with in vivo and in vitro approaches to probe the biosynthetic mechanism of these intriguing structures.


Assuntos
Aminoglicosídeos/biossíntese , Aminoglicosídeos/genética , Antibacterianos/biossíntese , Farmacorresistência Bacteriana , Família Multigênica , Uridina/análogos & derivados , Uridina/química , Aminoglicosídeos/química , Antibacterianos/química , Sequência de Bases , Desenho de Fármacos , Escherichia coli/metabolismo , Heme/química , Cinética , Espectroscopia de Ressonância Magnética , Dados de Sequência Molecular , Fases de Leitura Aberta , Fosforilação , Reação em Cadeia da Polimerase , Ligação Proteica , Proteínas Recombinantes/química , Streptomyces/metabolismo , Treonina/química , Transaldolase/metabolismo , Uridina/biossíntese , Uridina Monofosfato/química
6.
Chembiochem ; 17(9): 804-10, 2016 05 03.
Artigo em Inglês | MEDLINE | ID: mdl-26840634

RESUMO

Capuramycins are one of several known classes of natural products that contain an l-Lys-derived l-α-amino-ɛ-caprolactam (l-ACL) unit. The α-amino group of l-ACL in a capuramycin is linked to an unsaturated hexuronic acid component through an amide bond that was previously shown to originate by an ATP-independent enzymatic route. With the aid of a combined in vivo and in vitro approach, a predicted tridomain nonribosomal peptide synthetase CapU is functionally characterized here as the ATP-dependent amide-bond-forming catalyst responsible for the biosynthesis of the remaining amide bond present in l-ACL. The results are consistent with the adenylation domain of CapU as the essential catalytic component for l-Lys activation and thioesterification of the adjacent thiolation domain. However, in contrast to expectations, lactamization does not require any additional domains or proteins and is likely a nonenzymatic event. The results set the stage for examining whether a similar NRPS-mediated mechanism is employed in the biosynthesis of other l-ACL-containing natural products and, just as intriguingly, how spontaneous lactamization is avoided in the numerous NRPS-derived peptides that contain an unmodified l-Lys residue.


Assuntos
Aminoglicosídeos/biossíntese , Lisina/metabolismo , Peptídeo Sintases/metabolismo , Aminoglicosídeos/química , Caprolactama/química , Caprolactama/metabolismo , Cromatografia Líquida de Alta Pressão , Peptídeo Sintases/genética , Streptomyces/enzimologia , Streptomyces/genética
7.
Org Biomol Chem ; 14(16): 3956-62, 2016 Apr 28.
Artigo em Inglês | MEDLINE | ID: mdl-27050157

RESUMO

Using the ATP-independent transacylase CapW required for the biosynthesis of capuramycin-type antibiotics, we developed a biocatalytic approach for the synthesis of 43 analogues via a one-step aminolysis reaction from a methyl ester precursor as an acyl donor and various nonnative amines as acyl acceptors. Further examination of the donor substrate scope for CapW revealed that this enzyme can also catalyze a direct transamidation reaction using the major capuramycin congener as a semisynthetic precursor. Biological activity tests revealed that a few of the new capuramycin analogues have significantly improved antibiotic activity against Mycobacterium smegmatis MC2 155 and Mycobacterium tuberculosis H37Rv. Furthermore, most of the analogues are able to be covalently modified by the phosphotransferase CapP/Cpr17 involved in self resistance, providing critical insight for future studies regarding clinical development of the capuramycin antimycobacterial antibiotics.


Assuntos
Aciltransferases/metabolismo , Aminoglicosídeos/química , Aminoglicosídeos/farmacologia , Biocatálise , Testes de Sensibilidade Microbiana , Mycobacterium smegmatis/efeitos dos fármacos , Especificidade por Substrato
8.
Angew Chem Int Ed Engl ; 52(44): 11607-11, 2013 Oct 25.
Artigo em Inglês | MEDLINE | ID: mdl-24014169

RESUMO

Rise and shine: Using a gene-targeting approach aimed at identifying potential L-threonine:uridine-5'-transaldolases that catalyze the formation of (5'S,6'S)-C-glycyluridine, a new bacterial translocase I inhibitor was discovered from an actinomycete following fermentation optimization.


Assuntos
Actinobacteria/metabolismo , Antibacterianos/química , Inibidores Enzimáticos/química , Marcação de Genes/métodos , Transaldolase/química , Inibidores Enzimáticos/farmacologia , Transaldolase/farmacologia
9.
Cell Host Microbe ; 31(1): 18-32.e9, 2023 01 11.
Artigo em Inglês | MEDLINE | ID: mdl-36549300

RESUMO

Recent studies show gut microbiota-dependent metabolism of dietary phenylalanine into phenylacetic acid (PAA) is critical in phenylacetylglutamine (PAGln) production, a metabolite linked to atherosclerotic cardiovascular disease (ASCVD). Accordingly, microbial enzymes involved in this transformation are of interest. Using genetic manipulation in selected microbes and monocolonization experiments in gnotobiotic mice, we identify two distinct gut microbial pathways for PAA formation; one is catalyzed by phenylpyruvate:ferredoxin oxidoreductase (PPFOR) and the other by phenylpyruvate decarboxylase (PPDC). PPFOR and PPDC play key roles in gut bacterial PAA production via oxidative and non-oxidative phenylpyruvate decarboxylation, respectively. Metagenomic analyses revealed a significantly higher abundance of both pathways in gut microbiomes of ASCVD patients compared with controls. The present studies show a role for these two divergent microbial catalytic strategies in the meta-organismal production of PAGln. Given the numerous links between PAGln and ASCVD, these findings will assist future efforts to therapeutically target PAGln formation in vivo.


Assuntos
Doenças Cardiovasculares , Microbioma Gastrointestinal , Camundongos , Animais , Glutamina
10.
mBio ; : e0133123, 2023 Nov 10.
Artigo em Inglês | MEDLINE | ID: mdl-37947418

RESUMO

p-Cresol sulfate (pCS) and indoxyl sulfate (IS), gut microbiome-derived metabolites, are traditionally associated with cardiovascular disease (CVD) risks in the setting of impaired kidney function. While pharmacologic provision of pCS or IS can promote pro-thrombotic phenotypes, neither the microbial enzymes involved nor direct gut microbial production have been linked to CVD. Untargeted metabolomics was performed on a discovery cohort (n = 1,149) with relatively preserved kidney function, followed by stable isotope-dilution mass spectrometry quantification of pCS and IS in an independent validation cohort (n = 3,954). Genetic engineering of human commensals to produce p-cresol and indole gain-of-function and loss-of-function mutants, followed by colonization of germ-free mice, and studies on host thrombosis were performed. Systemic pCS and IS levels were independently associated with all-cause mortality. Both in vitro and within colonized germ-free mice p-cresol productions were recapitulated by collaboration of two organisms: a Bacteroides strain that converts tyrosine to 4-hydroxyphenylacetate, and a Clostridium strain that decarboxylates 4-hydroxyphenylacetate to p-cresol. We then engineered a single organism, Bacteroides thetaiotaomicron, to produce p-cresol, indole, or both metabolites. Colonizing germ-free mice with engineered strains, we show the gut microbial genes for p-cresol (hpdBCA) and indole (tryptophanase) are sufficient to confer a pro-thrombotic phenotype in vivo. Moreover, human fecal metagenomics analyses show that abundances of hpdBCA and tryptophanase are associated with CVD. These studies show that pCS and IS, two abundant microbiome-derived metabolites, play a broader potential role in CVD than was previously known. They also suggest that therapeutic targeting of gut microbial p-cresol- and indole-producing pathways represent rational targets for CVD.IMPORTANCEAlterations in gut microbial composition and function have been linked to numerous diseases. Identifying microbial pathways responsible for producing molecules that adversely impact the host is an important first step in the development of therapeutic interventions. Here, we first use large-scale clinical observations to link blood levels of defined microbial products to cardiovascular disease risks. Notably, the previously identified uremic toxins p-cresol sulfate and indoxyl sulfate were shown to predict 5-year mortality risks. After identifying the microbes and microbial enzymes involved in the generation of these uremic toxins, we used bioengineering technologies coupled with colonization of germ-free mice to show that the gut microbial genes that generate p-cresol and indole are sufficient to confer p-cresol sulfate and indoxyl sulfate formation, and a pro-thrombotic phenotype in vivo. The findings and tools developed serve as a critical step in both the study and targeting of these gut microbial pathways in vivo.

11.
J Biol Chem ; 286(10): 7885-7892, 2011 Mar 11.
Artigo em Inglês | MEDLINE | ID: mdl-21216959

RESUMO

Fe(II)- and α-ketoglutarate (α-KG)-dependent dioxygenases are a large and diverse superfamily of mononuclear, non-heme enzymes that perform a variety of oxidative transformations typically coupling oxidative decarboxylation of α-KG with hydroxylation of a prime substrate. The biosynthetic gene clusters for several nucleoside antibiotics that contain a modified uridine component, including the lipopeptidyl nucleoside A-90289 from Streptomyces sp. SANK 60405, have recently been reported, revealing a shared open reading frame with sequence similarity to proteins annotated as α-KG:taurine dioxygenases (TauD), a well characterized member of this dioxygenase superfamily. We now provide in vitro data to support the functional assignment of LipL, the putative TauD enzyme from the A-90289 gene cluster, as a non-heme, Fe(II)-dependent α-KG:UMP dioxygenase that produces uridine-5'-aldehyde to initiate the biosynthesis of the modified uridine component of A-90289. The activity of LipL is shown to be dependent on Fe(II), α-KG, and O(2), stimulated by ascorbic acid, and inhibited by several divalent metals. In the absence of the prime substrate UMP, LipL is able to catalyze oxidative decarboxylation of α-KG, although at a significantly reduced rate. The steady-state kinetic parameters using optimized conditions were determined to be K(m)(α-KG) = 7.5 µM, K(m)(UMP) = 14 µM, and k(cat) ≈ 80 min(-1). The discovery of this new activity not only sets the stage to explore the mechanism of LipL and related dioxygenases further but also has critical implications for delineating the biosynthetic pathway of several related nucleoside antibiotics.


Assuntos
Azepinas/metabolismo , Proteínas de Bactérias/metabolismo , Oxigenases de Função Mista/metabolismo , Streptomyces/enzimologia , Uracila/biossíntese , Azepinas/química , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Catálise , Escherichia coli/enzimologia , Escherichia coli/genética , Ferro/química , Ferro/metabolismo , Ácidos Cetoglutáricos/química , Ácidos Cetoglutáricos/metabolismo , Oxigenases de Função Mista/química , Oxigenases de Função Mista/genética , Família Multigênica/fisiologia , Oxigênio/química , Oxigênio/metabolismo , Uracila/análogos & derivados , Uracila/química
12.
Nat Chem Biol ; 6(8): 581-6, 2010 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-20562876

RESUMO

A-503083 B, a capuramycin-type antibiotic, contains an L-aminocaprolactam and an unsaturated hexuronic acid that are linked via an amide bond. A putative class C beta-lactamase (CapW) was identified within the biosynthetic gene cluster that-in contrast to the expected beta-lactamase activity-catalyzed an amide-ester exchange reaction to eliminate the L-aminocaprolactam with concomitant generation of a small but significant amount of the glyceryl ester derivative of A-503083 B, suggesting a potential role for an ester intermediate in the biosynthesis of capuramycins. A carboxyl methyltransferase, CapS, was subsequently demonstrated to function as an S-adenosylmethionine-dependent carboxyl methyltransferase to form the methyl ester derivative of A-503083 B. In the presence of free L-aminocaprolactam, CapW efficiently converts the methyl ester to A-503083 B, thereby generating a new amide bond. This ATP-independent amide bond formation using methyl esterification followed by an ester-amide exchange reaction represents an alternative to known strategies of amide bond formation.


Assuntos
Trifosfato de Adenosina/fisiologia , Amidas/metabolismo , Antibacterianos/biossíntese , Streptomyces/genética , Uridina/análogos & derivados , Azepinas , Ácidos Carboxílicos/química , Catálise , Clonagem Molecular , Análise Mutacional de DNA , DNA Bacteriano/genética , Ésteres/metabolismo , Biblioteca Gênica , Hidrólise , Cinética , Lisina/metabolismo , Família Multigênica , Proteína O-Metiltransferase/metabolismo , Streptomyces/metabolismo , Uridina/biossíntese , Uridina/genética , beta-Lactamases/biossíntese , beta-Lactamases/genética
13.
Clin Cancer Res ; 28(2): 390-403, 2022 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-34921025

RESUMO

PURPOSE: EGFR-tyrosine kinase inhibitor (TKI) is a standard first-line therapy for activated EGFR-mutated non-small cell lung cancer (NSCLC). Treatment options for patients with acquired EGFR-TKI resistance are limited. HER3 mediates EGFR-TKI resistance. Clinical trials of the HER3-targeting antibody-drug conjugate patritumab deruxtecan (HER3-DXd) demonstrated its anticancer activity in EGFR-mutated NSCLC; however, the mechanisms that regulate HER3 expression are unknown. This study was conducted with the aim to clarify the mechanisms underlying HER3 regulation in EGFR-mutated NSCLC tumors and explored the strategy for enhancing the anticancer activity of HER3-DXd in EGFR-mutated NSCLC. EXPERIMENTAL DESIGN: Paired tumor samples were obtained from 48 patients with EGFR-mutated NSCLC treated with EGFR-TKI(s). HER3 expression was immunohistochemically quantified with H-score, and genomic alteration and transcriptomic signature were tested in tumors from pretreatment to post-EGFR-TKI resistance acquisition. The anticancer efficacy of HER3-DXd and osimertinib was evaluated in EGFR-mutated NSCLC cells. RESULTS: We showed augmented HER3 expression in EGFR-mutated tumors with acquired EGFR-TKI resistance compared with paired pretreatment samples. RNA sequencing revealed that repressed PI3K/AKT/mTOR signaling was associated with HER3 augmentation, especially in tumors from patients who received continuous EGFR-TKI therapy. An in vitro study also showed that EGFR-TKI increased HER3 expression, repressed AKT phosphorylation in multiple EGFR-mutated cancers, and enhanced the anticancer activity of HER3-DXd. CONCLUSIONS: Our findings help clarify the mechanisms of HER3 regulation in EGFR-mutated NSCLC tumors and highlight a rationale for combination therapy with HER3-DXd and EGFR-TKI in EGFR-mutated NSCLC.


Assuntos
Anticorpos Monoclonais Humanizados , Camptotecina , Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Receptor ErbB-3 , Anticorpos Monoclonais Humanizados/uso terapêutico , Camptotecina/análogos & derivados , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/genética , Receptores ErbB , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Mutação , Fosfatidilinositol 3-Quinases/genética , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Proto-Oncogênicas c-akt/genética , Receptor ErbB-3/antagonistas & inibidores , Receptor ErbB-3/genética , Receptor ErbB-3/metabolismo
14.
J Biol Chem ; 285(17): 12899-905, 2010 Apr 23.
Artigo em Inglês | MEDLINE | ID: mdl-20202936

RESUMO

Capuramycin-related compounds, including A-500359s and A-503083s, are nucleoside antibiotics that inhibit the enzyme bacterial translocase I involved in peptidoglycan cell wall biosynthesis. Within the biosynthetic gene cluster for the A-500359s exists a gene encoding a putative aminoglycoside 3-phosphotransferase that was previously demonstrated to be highly expressed during the production of A-500359s and confers selective resistance to capuramycins when expressed in heterologous hosts. A similar gene (capP) was identified within the biosynthetic gene cluster for the A-503083s, and CapP is now shown to similarly confer selective resistance to capuramycins. Recombinant CapP was produced and purified from Escherichia coli, and the function of CapP is established as an ATP-dependent capuramycin phosphotransferase that regio-specifically transfers the gamma-phosphate to the 3''-hydroxyl of the unsaturated hexuronic acid moiety of A-503083 B. Kinetic analysis with the three major A-503083 congeners suggests that CapP preferentially phosphorylates A-503083s containing an aminocaprolactam moiety attached to the hexuronic acid, and bi-substrate kinetic analysis was consistent with CapP employing a sequential kinetic mechanism similar to most known aminoglycoside 3-phosphotransferases. The purified CapP product lost its antibiotic activity against Mycobacterium smegmatis, and this loss in bioactivity is primarily due to a 272-fold increase in the IC(50) in the bacterial translocase I-catalyzed reaction. The results establish CapP-mediated phosphorylation as a mechanism of resistance to capuramycins and now set the stage to explore this strategy of resistance as a potential mechanism inherent to pathogens and provide the impetus for preparing second generation analogues as a preemptive strike to such resistance strategies.


Assuntos
Trifosfato de Adenosina/química , Aminoglicosídeos/química , Antibacterianos/química , Proteínas de Bactérias/química , Farmacorresistência Bacteriana/fisiologia , Canamicina Quinase/química , Mycobacterium smegmatis/enzimologia , Trifosfato de Adenosina/metabolismo , Aminoglicosídeos/farmacologia , Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Clonagem Molecular , Farmacorresistência Bacteriana/efeitos dos fármacos , Canamicina Quinase/genética , Canamicina Quinase/metabolismo , Cinética , Mycobacterium smegmatis/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
15.
Biochim Biophys Acta Proteins Proteom ; 1867(1): 22-27, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30392561

RESUMO

Phenotype-based drug discovery is a key strategy for small molecule drug screening, and the molecular target identification of small molecules, termed "target deconvolution," is critical albeit challenging. In this review, we classify approaches for target deconvolution, including both direct and indirect approaches, summarize their underlying principles, and provide examples of current chemical proteomics strategies including affinity purification using compound-immobilized beads, photoaffinity labeling (PAL), cellular thermal shift assay (CETSA), and activity-based protein profiling (ABPP). Because there is no single best target deconvolution strategy, it is important to carefully select a strategy on the basis of the test compound characteristics.


Assuntos
Descoberta de Drogas/métodos , Avaliação Pré-Clínica de Medicamentos/métodos , Proteômica/métodos , Animais , Descoberta de Drogas/tendências , Avaliação Pré-Clínica de Medicamentos/tendências , Humanos , Fenótipo , Marcadores de Fotoafinidade , Bibliotecas de Moléculas Pequenas
17.
Medchemcomm ; 4(1): 239-243, 2013 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-23476724

RESUMO

Muraminomicin is a lipopeptidyl nucleoside antibiotic produced by Streptosporangium amethystogenes SANK 60709. Similar to several members of this antibiotic family such as A-90289 and muraymycin, the structure of muraminomicin consists of a disaccharide comprised of two modified ribofuranose units linked by an O-ß(1 → 5) glycosidic bond; however, muraminomicin holds the distinction in that both ribose units are 2-deoxy sugars. The biosynthetic gene cluster of muraminomicin has been identified, cloned and sequenced, and bioinformatic analysis revealed a minimum of 24 open reading frames putatively involved in the biosynthesis, resistance, and regulation of muraminomicin. Fives enzymes are likely involved in the assembly and attachment of the 2,5-dideoxy-5-aminoribose saccharide unit, and two are now functionally assigned and characterized: Mra20, a 5'-amino-2',5'-dideoxyuridine phosphorylase and Mra23, a UTP:5-amino-2,5-dideoxy-α-D-ribose-1-phosphate uridylyltransferase. The cumulative results are consistent with the incorporation of the ribosyl appendage of muraminomicin via the archetypical sugar biosynthetic pathway that parallels A-90289 biosynthesis, and the specificity for this appendage is dictated primarily by the two characterized enzymes.

18.
J Antibiot (Tokyo) ; 64(7): 495-501, 2011 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-21587261

RESUMO

During the course of screening for translocase I inhibitors, the new liposidomycin-related compounds, A-90289 A and B, were isolated from a culture broth of Streptomyces sp. SANK 60405. The structural elucidations were carried out by NMR and high-resolution mass spectral analyses, and they were classified as members of the liponucleoside antibiotics group with a sulfate group at the C-2' position. A-90289 A and B inhibited bacterial translocase I with IC(50) values of 36.5 ng ml(-1) and 33.8 ng ml(-1), respectively.


Assuntos
Antibacterianos/farmacologia , Azepinas/farmacologia , Streptomyces/metabolismo , Transferases (Outros Grupos de Fosfato Substituídos)/antagonistas & inibidores , Uracila/análogos & derivados , Azepinas/isolamento & purificação , Concentração Inibidora 50 , Espectroscopia de Ressonância Magnética , Espectrometria de Massas , Uracila/isolamento & purificação , Uracila/farmacologia
19.
J Antibiot (Tokyo) ; 62(6): 325-32, 2009 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-19478828

RESUMO

A-500359s, produced by Streptomyces griseus SANK60196, are inhibitors of bacterial phospho-N-acetylmuramyl-pentapeptide translocase. They are composed of three distinct moieties: a 5'-carbamoyl uridine, an unsaturated hexuronic acid and an aminocaprolactam. Two contiguous cosmids covering a 65-kb region of DNA and encoding 38 open reading frames (ORFs) putatively involved in the biosynthesis of A-500359s were identified. Reverse transcriptase PCR showed that most of the 38 ORFs are highly expressed during A-500359s production, but mutants that do not produce A-500359s did not express these same ORFs. Furthermore, orf21, encoding a putative aminoglycoside 3'-phosphotransferase, was heterologously expressed in Escherichia coli and Streptomyces albus, yielding strains having selective resistance against A-500359B, suggesting that ORF21 phosphorylates the unsaturated hexuronic acid as a mechanism of self-resistance to A-500359s. In total, the data suggest that the cloned region is involved in the resistance, regulation and biosynthesis of A-500359s.


Assuntos
Antibacterianos/biossíntese , Streptomyces griseus/genética , Uridina/análogos & derivados , Antibacterianos/farmacologia , Azepinas/farmacologia , DNA Bacteriano/biossíntese , DNA Bacteriano/genética , Farmacorresistência Bacteriana/genética , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Biblioteca Gênica , Genes Bacterianos/genética , Vetores Genéticos , Família Multigênica , Fases de Leitura Aberta , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Uridina/biossíntese , Uridina/farmacologia
20.
J Biol Chem ; 283(20): 13983-91, 2008 May 16.
Artigo em Inglês | MEDLINE | ID: mdl-18364359

RESUMO

Type III polyketide synthases (PKSs) found in plants, fungi, and bacteria synthesize a variety of aromatic polyketides. A Gram-positive, filamentous bacterium Streptomyces griseus contained an srs operon, in which srsA encoded a type III PKS, srsB encoded a methyltransferase, and srsC encoded a flavoprotein hydroxylase. Consistent with this annotation, overexpression of the srs genes in a heterologous host, Streptomyces lividans, showed that SrsA was a type III PKS responsible for synthesis of phenolic lipids, alkylresorcinols, and alkylpyrones, SrsB was a methyltransferase acting on the phenolic lipids to yield alkylresorcinol methyl ethers, and SrsC was a hydroxylase acting on the alkylresorcinol methyl ethers. In vitro SrsA reaction showed that SrsA synthesized alkylresorcinols from acyl-CoAs of various chain lengths as a starter substrate, one molecule of methylmalonyl-CoA, and two molecules of malonyl-CoA. SrsA was thus unique in that it incorporated the extender substrates in a strictly controlled order of malonyl-CoA, malonyl-CoA, and methylmalonyl-CoA to produce alkylresorcinols. An srsA mutant, which produced no phenolic lipids, was highly sensitive to beta-lactam antibiotics, such as penicillin G and cephalexin. Together with the fact that the alkylresorcinols were fractionated mainly in the cell wall fraction, this observation suggests that the phenolic lipids, perhaps associated with the cytoplasmic membrane because of their amphiphilic property, affect the characteristic and rigidity of the cytoplasmic membrane/peptidoglycan of a variety of bacteria. An srs-like operon is found widely among Gram-positive and -negative bacteria, indicating wide distribution of the phenolic lipids.


Assuntos
Farmacorresistência Bacteriana , Lipídeos/química , Penicilinas/farmacologia , Fenol/química , Policetídeo Sintases/química , Streptomyces griseus/química , Cefalexina/química , Regulação Bacteriana da Expressão Gênica , Malonil Coenzima A/química , Modelos Biológicos , Modelos Químicos , Mutação , Penicilinas/química , Fenóis/química , Fenótipo , Frações Subcelulares/metabolismo
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA