RESUMO
The anaerobic gut microbial pathway that converts choline into trimethylamine (TMA) is broadly linked to human disease. Here, we describe the discovery that betaine aldehyde inhibits TMA production from choline by human gut bacterial isolates and a complex gut community. In vitro assays and a crystal structure suggest betaine aldehyde targets the gut microbial enzyme choline TMA-lyase (CutC). In our system, we do not observe activity for the previously reported CutC inhibitor 3,3-dimethylbutanol (DMB). The workflow we establish for identifying and characterizing betaine aldehyde provides a framework for developing additional inhibitors of gut microbial choline metabolism, including therapeutic candidates.
Assuntos
Bactérias/efeitos dos fármacos , Bactérias/metabolismo , Colina/metabolismo , Microbioma Gastrointestinal/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/farmacologia , Anaerobiose/efeitos dos fármacos , Domínio Catalítico , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/química , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Modelos Moleculares , Bibliotecas de Moléculas Pequenas/químicaRESUMO
The essential mammalian enzyme O-GlcNAc Transferase (OGT) is uniquely responsible for transferring N-acetylglucosamine to over a thousand nuclear and cytoplasmic proteins, yet there is no known consensus sequence and it remains unclear how OGT recognizes its substrates. To address this question, we developed a protein microarray assay that chemoenzymatically labels de novo sites of glycosylation with biotin, allowing us to simultaneously assess OGT activity across >6000 human proteins. With this assay we examined the contribution to substrate selection of a conserved asparagine ladder within the lumen of OGT's superhelical tetratricopeptide repeat (TPR) domain. When five asparagines were mutated, OGT retained significant activity against short peptides, but showed limited limited glycosylation of protein substrates on the microarray. O-GlcNAcylation of protein substrates in cell extracts was also greatly attenuated. We conclude that OGT recognizes the majority of its substrates by binding them to the asparagine ladder in the TPR lumen proximal to the catalytic domain.
Assuntos
Asparagina/metabolismo , N-Acetilglucosaminiltransferases/metabolismo , Análise Serial de Proteínas , Proteínas/química , Proteínas/metabolismo , Repetições de Tetratricopeptídeos , Humanos , Especificidade por SubstratoRESUMO
Cophotolysis of noradamantyldiazirine with the phenanthride precursor of dichlorocarbene or phenylchlorodiazirine in pentane at room temperature produces noradamantylethylenes in 11% yield with slight diastereoselectivity. Cophotolysis of adamantyldiazirine with phenylchlorodiazirine in pentane at room temperature generates adamantylethylenes in 6% yield with no diastereoselectivity. (1)H NMR showed the reaction of noradamantyldiazirine + phenylchlorodiazirine to be independent of solvent, and the rate of noradamantyldiazirine consumption correlated with the rate of ethylene formation. Laser flash photolysis showed that reaction of phenylchlorocarbene + adamantene was independent of adamantene concentration. The reaction of phenylchlorocarbene + homoadamantene produces the ethylene products with k = 9.6 × 10(5) M(-1) s(-1). Calculations at the UB3LYP/6-31+G(d,p) and UM062X/6-31+G(d,p)//UB3LYP/6-31+G(d,p) levels show the formation of exocyclic ethylenes to proceed (a) on the singlet surface via stepwise addition of phenylchlorocarbene (PhCCl) to bridgehead alkenes adamantene and homoadamantene, respectively, producing an intermediate singlet diradical in each case, or (b) via addition of PhCCl to the diazo analogues of noradamantyl- and adamantyldiazirine. Preliminary direct dynamics calculations on adamantene + PhCCl show a high degree of recrossing (68%), indicative of a flat transition state surface. Overall, 9% of the total trajectories formed noradamantylethylene product, each proceeding via the computed singlet diradical.
Assuntos
Adamantano/análogos & derivados , Adamantano/química , Alcenos/química , Metano/análogos & derivados , Etilenos/química , Metano/química , Fotólise , Solventes/químicaRESUMO
Ferrostatin-1 (Fer-1) inhibits ferroptosis, a form of regulated, oxidative, nonapoptotic cell death. We found that Fer-1 inhibited cell death in cellular models of Huntington's disease (HD), periventricular leukomalacia (PVL), and kidney dysfunction; Fer-1 inhibited lipid peroxidation, but not mitochondrial reactive oxygen species formation or lysosomal membrane permeability. We developed a mechanistic model to explain the activity of Fer-1, which guided the development of ferrostatins with improved properties. These studies suggest numerous therapeutic uses for ferrostatins, and that lipid peroxidation mediates diverse disease phenotypes.
Assuntos
Cicloexilaminas/farmacologia , Doença de Huntington/tratamento farmacológico , Nefropatias/tratamento farmacológico , Leucomalácia Periventricular/tratamento farmacológico , Lipídeos de Membrana/metabolismo , Fenilenodiaminas/farmacologia , Morte Celular/efeitos dos fármacos , Cicloexilaminas/uso terapêutico , Doença de Huntington/metabolismo , Doença de Huntington/patologia , Nefropatias/metabolismo , Nefropatias/patologia , Leucomalácia Periventricular/metabolismo , Leucomalácia Periventricular/patologia , Peroxidação de Lipídeos/efeitos dos fármacos , Oxirredução/efeitos dos fármacos , Fenilenodiaminas/uso terapêuticoRESUMO
Alkane monooxygenase (AlkB) is a non-heme diiron enzyme that catalyzes the hydroxylation of alkanes. It is commonly found in alkanotrophic organisms that can live on alkanes as their sole source of carbon and energy. Activation of AlkB occurs via two-electron reduction of its diferric active site, which facilitates the binding, activation, and cleavage of molecular oxygen for insertion into an inert CH bond. Electrons are typically supplied by NADH via a rubredoxin reductase (AlkT) to a rubredoxin (AlkG) to AlkB, although alternative electron transfer partners have been observed. Here we report a family of AlkBs in which both electron transfer partners (a ferredoxin and a ferredoxin reductase) appear as an N-terminal gene fusion to the hydroxylase (ferr_ferrR_AlkB). This enzyme catalyzes the hydroxylation of medium chain alkanes (C6-C14), with a preference for C10-C12. It requires only NADH for activity. It is present in a number of bacteria that are known to be human pathogens. A survey of the genome neighborhoods in which is it found suggest it may be involved in alkane metabolism, perhaps facilitating growth of pathogens in non-host environments.
Assuntos
Alcanos/metabolismo , Citocromo P-450 CYP4A/metabolismo , Oxigenases de Função Mista/metabolismo , Oxigênio/metabolismo , Alcanos/química , Citocromo P-450 CYP4A/química , Transporte de Elétrons , Elétrons , Ferredoxinas/metabolismo , Humanos , Hidroxilação , Leptospira/metabolismo , Oxigenases de Função Mista/química , NADH NADPH Oxirredutases/metabolismo , Oxigênio/química , Pseudomonas aeruginosa/metabolismo , Rubredoxinas/metabolismoRESUMO
SMIFH2 is a small molecule inhibitor of the formin family of cytoskeletal regulators that was originally identified in a screen for suppression of actin polymerization induced by the mouse formin Diaphanous 1 (mDia1). Despite widespread use of this compound, it is unknown whether SMIFH2 inhibits all human formins. Additionally, the nature of protein/inhibitor interactions remains elusive. We assayed SMIFH2 against human formins representing six of the seven mammalian classes and found inhibitory activity against all formins tested. We synthesized a panel of SMIFH2 derivatives and found that, while many alterations disrupt SMIFH2 activity, substitution of an electron-donating methoxy group in place of the bromine along with halogenation of the furan ring increases potency by approximately five-fold. Similar to SMIFH2, the active derivatives are also pan-inhibitors for the formins tested. This result suggests that while potency can be improved, the goal of distinguishing between highly conserved FH2 domains may not be achievable using the SMIFH2 scaffold.
Assuntos
Actinas , Proteínas de Transporte , Tionas/farmacologia , Uracila/análogos & derivados , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Animais , Proteínas de Transporte/metabolismo , Citoesqueleto/metabolismo , Forminas , Humanos , Mamíferos/metabolismo , Camundongos , Estrutura Terciária de Proteína , Uracila/farmacologiaRESUMO
Metallothioneins (MTs) are a ubiquitous class of small metal-binding proteins involved in metal homeostasis and detoxification. While known for their high affinity for d10 metal ions, there is a surprising dearth of thermodynamic data on metals binding to MTs. In this study, Zn2+ and Cu+ binding to mammalian metallothionein-3 (MT-3) were quantified at pH 7.4 by isothermal titration calorimetry (ITC). Zn2+ binding was measured by chelation titrations of Zn7MT-3, while Cu+ binding was measured by Zn2+ displacement from Zn7MT-3 with competition from glutathione (GSH). Titrations in multiple buffers enabled a detailed analysis that yielded condition-independent values for the association constant (K) and the change in enthalpy (ΔH) and entropy (ΔS) for these metal ions binding to MT-3. Zn2+ was also chelated from the individual α and ß domains of MT-3 to quantify the thermodynamics of inter-domain interactions in metal binding. Comparative titrations of Zn7MT-2 with Cu+ revealed that both MT isoforms have similar Cu+ affinities and binding thermodynamics, indicating that ΔH and ΔS are determined primarily by the conserved Cys residues. Inductively coupled plasma mass spectrometry (ICP-MS) analysis and low temperature luminescence measurements of Cu-replete samples showed that both proteins form two Cu4 +-thiolate clusters when Cu+ displaces Zn2+ under physiological conditions. Comparison of the Zn2+ and Cu+ binding thermodynamics reveal that enthalpically-favoured Cu+, which forms Cu4 +-thiolate clusters, displaces the entropically-favoured Zn2+. These results provide a detailed thermodynamic analysis of d10 metal binding to these thiolate-rich proteins and quantitative support for, as well as molecular insight into, the role that MT-3 plays in the neuronal chemistry of copper.
RESUMO
The anaerobic conversion of choline to trimethylamine (TMA) by the human gut microbiota has been linked to multiple human diseases. The potential impact of this microbial metabolic activity on host health has inspired multiple efforts to identify small molecule inhibitors. Here, we use information about the structure and mechanism of the bacterial enzyme choline TMA-lyase (CutC) to develop a cyclic choline analog that inhibits the conversion of choline to TMA in bacterial whole cells and in a complex gut microbial community. In vitro biochemical assays and a crystal structure suggest that this analog is a competitive, mechanism-based inhibitor. This work demonstrates the utility of structure-based design to access inhibitors of radical enzymes from the human gut microbiota.
RESUMO
INTRODUCCIÓN: El cambio climático es un determinante estructural de la salud y una amenaza para la salud pública global. Por ello, la Asamblea Mundial de la Salud de 2008 llamó a elaborar planes de acción para minimizar los impactos sanitarios del cambio climático. El objetivo de este trabajo fue sistematizar el proceso de desarrollo de una política sanitaria en la materia, que se plasma en el Plan de Acción Nacional en Salud y Cambio Climático. MÉTODOS: Para el diseño de la política de salud y cambio climático se conformó una Mesa de Trabajo al interior del Ministerio de Salud de la Nación, integrada por diferentes áreas con competencias en la temática. Esta mesa fue la responsable de llevar a cabo un diagnóstico de situación para la priorización de los temas a incluir en un plan de acción que puesto a consideración de otras áreas del Estado Nacional a través del Grupo de Trabajo de Salud al interior del Gabinete Nacional de Cambio Climático y de la sociedad civil. RESULTADOS: La política sanitaria en materia de cambio climático cuenta con tres instrumentos: un programa nacional, una mesa de trabajo y un plan de acción. El plan está organizado en 7 ejes de intervención y 21 medidas. DISCUSIÓN: El Ministerio de Salud ha trabajado en Salud y Cambio Climático desde 2008, pero fue a partir de la conformación de la Mesa de Trabajo que se logró la transversalización de la temática.
Assuntos
Argentina , Mudança Climática , Colaboração Intersetorial , Política de SaúdeRESUMO
O-GlcNAc transferase (OGT) is an essential mammalian enzyme that regulates numerous cellular processes through the attachment of O-linked N-acetylglucosamine (O-GlcNAc) residues to nuclear and cytoplasmic proteins. Its targets include kinases, phosphatases, transcription factors, histones, and many other intracellular proteins. The biology of O-GlcNAc modification is still not well understood, and cell-permeable inhibitors of OGT are needed both as research tools and for validating OGT as a therapeutic target. Here, we report a small molecule OGT inhibitor, OSMI-1, developed from a high-throughput screening hit. It is cell-permeable and inhibits protein O-GlcNAcylation in several mammalian cell lines without qualitatively altering cell surface N- or O-linked glycans. The development of this molecule validates high-throughput screening approaches for the discovery of glycosyltransferase inhibitors, and further optimization of this scaffold may lead to yet more potent OGT inhibitors useful for studying OGT in animal models.