RESUMEN
BACKGROUND: The prevalence of antibiotic resistance is increasing, and multidrug-resistant Pseudomonas aeruginosa has been identified as a serious threat to human health. The production of ß-lactamase is a key mechanism contributing to imipenem resistance in P. aeruginosa. Relebactam is a novel ß-lactamase inhibitor, active against class A and C ß-lactamases, that has been shown to restore imipenem susceptibility. In a series of studies, we assessed the interaction of relebactam with key mechanisms involved in carbapenem resistance in P. aeruginosa and to what extent relebactam might overcome imipenem non-susceptibility. RESULTS: Relebactam demonstrated no intrinsic antibacterial activity against P. aeruginosa, had no inoculum effect, and was not subject to efflux. Enzymology studies showed relebactam is a potent (overall inhibition constant: 27 nM), practically irreversible inhibitor of P. aeruginosa AmpC. Among P. aeruginosa clinical isolates from the SMART global surveillance program (2009, n = 993; 2011, n = 1702; 2015, n = 5953; 2016, n = 6165), imipenem susceptibility rates were 68.4% in 2009, 67.4% in 2011, 70.4% in 2015, and 67.3% in 2016. With the addition of 4 µg/mL relebactam, imipenem susceptibility rates increased to 87.6, 86.0, 91.7, and 89.8%, respectively. When all imipenem-non-susceptible isolates were pooled, the addition of 4 µg/mL relebactam reduced the mode imipenem minimum inhibitory concentration (MIC) 8-fold (from 16 µg/mL to 2 µg/mL) among all imipenem-non-susceptible isolates. Of 3747 imipenem-non-susceptible isolates that underwent molecular profiling, 1200 (32%) remained non-susceptible to the combination imipenem/relebactam (IMI/REL); 42% of these encoded class B metallo-ß-lactamases, 11% encoded a class A GES enzyme, and no class D enzymes were detected. No relationship was observed between alleles of the chromosomally-encoded P. aeruginosa AmpC and IMI/REL MIC. CONCLUSIONS: IMI/REL exhibited potential in the treatment of carbapenem-resistant P. aeruginosa infections, with the exception of isolates encoding class B, some GES alleles, and class D carbapenemases.
Asunto(s)
Compuestos de Azabiciclo/farmacología , Imipenem/farmacología , Pseudomonas aeruginosa/efectos de los fármacos , Antibacterianos/farmacología , Proteínas Bacterianas/efectos de los fármacos , Combinación de Medicamentos , Farmacorresistencia Bacteriana Múltiple/efectos de los fármacos , Humanos , Cinética , Pruebas de Sensibilidad Microbiana , Infecciones por Pseudomonas/microbiología , Pseudomonas aeruginosa/enzimología , beta-Lactamasas/efectos de los fármacosRESUMEN
5'-Adenosine monophosphate-activated protein kinase (AMPK) is a master regulator of energy homeostasis in eukaryotes. Despite three decades of investigation, the biological roles of AMPK and its potential as a drug target remain incompletely understood, largely because of a lack of optimized pharmacological tools. We developed MK-8722, a potent, direct, allosteric activator of all 12 mammalian AMPK complexes. In rodents and rhesus monkeys, MK-8722-mediated AMPK activation in skeletal muscle induced robust, durable, insulin-independent glucose uptake and glycogen synthesis, with resultant improvements in glycemia and no evidence of hypoglycemia. These effects translated across species, including diabetic rhesus monkeys, but manifested with concomitant cardiac hypertrophy and increased cardiac glycogen without apparent functional sequelae.
Asunto(s)
Proteínas Quinasas Activadas por AMP/metabolismo , Cardiomegalia/inducido químicamente , Glucosa/metabolismo , Homeostasis/efectos de los fármacos , Imidazoles/farmacología , Piridinas/farmacología , Animales , Bencimidazoles , Glucemia/efectos de los fármacos , Ayuno , Glucógeno/metabolismo , Hipoglucemia/inducido químicamente , Imidazoles/efectos adversos , Imidazoles/química , Insulina/farmacología , Macaca mulatta , Masculino , Ratones , Ratones Endogámicos C57BL , Músculo Esquelético/efectos de los fármacos , Músculo Esquelético/metabolismo , Piridinas/efectos adversos , Piridinas/químicaRESUMEN
Resistance to existing classes of antibiotics drives the need for discovery of novel compounds with unique mechanisms of action. Nargenicin A1, a natural product with limited antibacterial spectrum, was rediscovered in a whole-cell antisense assay. Macromolecular labeling in both Staphylococcus aureus and an Escherichia coli tolC efflux mutant revealed selective inhibition of DNA replication not due to gyrase or topoisomerase IV inhibition. S. aureus nargenicin-resistant mutants were selected at a frequency of â¼1 × 10(-9), and whole-genome resequencing found a single base-pair change in the dnaE gene, a homolog of the E. coli holoenzyme α subunit. A DnaE single-enzyme assay was exquisitely sensitive to inhibition by nargenicin, and other in vitro characterization studies corroborated DnaE as the target. Medicinal chemistry efforts may expand the spectrum of this novel mechanism antibiotic.
Asunto(s)
ADN Polimerasa III/genética , Descubrimiento de Drogas , Antibacterianos/química , Antibacterianos/metabolismo , Antibacterianos/farmacología , Replicación del ADN/efectos de los fármacos , ADN Polimerasa Dirigida por ADN/metabolismo , Farmacorresistencia Bacteriana/genética , Escherichia coli/efectos de los fármacos , Concentración 50 Inhibidora , Lactonas/química , Lactonas/metabolismo , Lactonas/farmacología , Mutación , Inhibidores de la Síntesis del Ácido Nucleico/química , Inhibidores de la Síntesis del Ácido Nucleico/farmacología , Staphylococcus aureus/efectos de los fármacosRESUMEN
The function of a particular protein is dependent upon its localization and milieu. The ability to track the "fate" of a protein is a valuable tool to elucidate its function. We present the use of HaloTag technology to study the localization and fate of human Proprotein Convertase Subtilisin-like Kexin type 9 (PCSK9).The role of PCSK9 in the regulation of circulating low density lipoprotein-cholesterol (LDL-c) levels is ascribed to binding of circulating PCSK9 to the LDL receptor (LDLR) and subsequent lysosomal degradation of LDLR. However, hints in the literature indicate that intracellular PCSK9 may act on the LDLR, possibly during processing of newly synthesized protein. To address this question, the source and fate of intracellular PCSK9 requires further investigation.We applied HaloTag technology to distinguish the source of intracellular PCSK9 and showed that newly synthesized intracellular PCSK9 has unique localization from the PCSK9 after re-uptake. This suggests different functions of PCSK9 while interacting with the LDLR.
RESUMEN
Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a promising therapeutic target for treating coronary heart disease. We report a novel antibody 1B20 that binds to PCSK9 with sub-nanomolar affinity and antagonizes PCSK9 function in-vitro. In CETP/LDLR-hemi mice two successive doses of 1B20, administered 14 days apart at 3 or 10 mpk, induced dose dependent reductions in LDL-cholesterol (≥ 25% for 7-14 days) that correlated well with the extent of PCSK9 occupancy by the antibody. In addition, 1B20 induces increases in total plasma antibody-bound PCSK9 levels and decreases in liver mRNA levels of SREBP-regulated genes PCSK9 and LDLR, with a time course that parallels decreases in plasma LDL-cholesterol (LDL-C). Consistent with this observation in mice, in statin-responsive human primary hepatocytes, 1B20 lowers PCSK9 and LDLR mRNA levels and raises serum steady-state levels of antibody-bound PCSK9. In addition, mRNA levels of several SREBP regulated genes involved in cholesterol and fatty-acid synthesis including ACSS2, FDPS, IDI1, MVD, HMGCR, and CYP51A1 were decreased significantly with antibody treatment of primary human hepatocytes. In rhesus monkeys, subcutaneous (SC) dosing of 1B20 dose-dependently induces robust LDL-C lowering (maximal ~70%), which is correlated with increases in target engagement and total antibody-bound PCSK9 levels. Importantly, a combination of 1B20 and Simvastatin in dyslipidemic rhesus monkeys reduced LDL-C more than either agent alone, consistent with a mechanism of action that predicts additive effects of anti-PCSK9 agents with statins. Our results suggest that antibodies targeting PCSK9 could provide patients powerful LDL lowering efficacy on top of statins, and lower cardiovascular risk.
Asunto(s)
Anticuerpos Monoclonales/uso terapéutico , Anticolesterolemiantes/uso terapéutico , LDL-Colesterol/sangre , Regulación de la Expresión Génica/efectos de los fármacos , Hepatocitos/efectos de los fármacos , Inmunización Pasiva , Síndrome Metabólico/terapia , Proproteína Convertasas/antagonistas & inhibidores , Proproteína Convertasas/inmunología , Serina Endopeptidasas/inmunología , Simvastatina/uso terapéutico , Proteínas de Unión a los Elementos Reguladores de Esteroles/fisiología , Animales , Anticuerpos Monoclonales/farmacología , Afinidad de Anticuerpos , Anticolesterolemiantes/administración & dosificación , Células Cultivadas/efectos de los fármacos , Células Cultivadas/metabolismo , Proteínas de Transferencia de Ésteres de Colesterol/genética , Proteínas de Transferencia de Ésteres de Colesterol/metabolismo , Perfilación de la Expresión Génica , Células Hep G2/efectos de los fármacos , Células Hep G2/metabolismo , Hepatocitos/metabolismo , Humanos , Metabolismo de los Lípidos/genética , Hígado/efectos de los fármacos , Hígado/metabolismo , Macaca mulatta , Síndrome Metabólico/tratamiento farmacológico , Síndrome Metabólico/genética , Ratones , Ratones Transgénicos , Proproteína Convertasa 9 , Proproteína Convertasas/biosíntesis , Proproteína Convertasas/genética , ARN Mensajero/metabolismo , Receptores de LDL/biosíntesis , Receptores de LDL/genética , Proteínas Recombinantes/metabolismo , Serina Endopeptidasas/biosíntesis , Serina Endopeptidasas/genética , Simvastatina/administración & dosificaciónRESUMEN
The preparation and characterization of a series of thiophenyl oxime phosphonate beta-lactamase inhibitors is described. A number of these analogs were potent and selective inhibitors of class C beta-lactamases from Pseudomonas aeruginosa and Enterobacter cloacae. Compounds 3b and 7 reduced the MIC of imipenem against an AmpC expressing strain of imipenem-resistant P. aeruginosa. A number of the title compounds retained micromolar potency against the class D OXA-40 beta-lactamase from Acinetobacter baumannii and at high concentrations compound 3b was shown to reduce the MIC of imipenem against a highly imipenem-resistant strain of A. baumanii expressing the OXA-40 beta-lactamase. In mice compound 3b exhibited phamacokinetics similar to imipenem.
Asunto(s)
Acinetobacter baumannii/efectos de los fármacos , Antibacterianos/química , Inhibidores Enzimáticos/química , Organofosfonatos/química , Oximas/química , Pseudomonas aeruginosa/efectos de los fármacos , Inhibidores de beta-Lactamasas , Antibacterianos/síntesis química , Antibacterianos/farmacología , Farmacorresistencia Bacteriana , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/farmacología , Imipenem/farmacología , Pruebas de Sensibilidad Microbiana , Organofosfonatos/síntesis química , Organofosfonatos/farmacología , Oximas/síntesis química , Oximas/farmacología , Tiofenos/química , beta-Lactamasas/metabolismoRESUMEN
Proprotein convertase subtilisin-like/kexin type 9 (PCSK9) regulates LDL cholesterol levels by inhibiting LDL receptor (LDLr)-mediated cellular LDL uptake. We have identified a fragment antigen-binding (Fab) 1D05 which binds PCSK9 with nanomolar affinity. The fully human antibody 1D05-IgG2 completely blocks the inhibitory effects of wild-type PCSK9 and two gain-of-function human PCSK9 mutants, S127R and D374Y. The crystal structure of 1D05-Fab bound to PCSK9 reveals that 1D05-Fab binds to an epitope on the PCSK9 catalytic domain which includes the entire LDLr EGF(A) binding site. Notably, the 1D05-Fab CDR-H3 and CDR-H2 loops structurally mimic the EGF(A) domain of LDLr. In a transgenic mouse model (CETP/LDLr-hemi), in which plasma lipid and PCSK9 profiles are comparable to those of humans, 1D05-IgG2 reduces plasma LDL cholesterol to 40% and raises hepatic LDLr protein levels approximately fivefold. Similarly, in healthy rhesus monkeys, 1D05-IgG2 effectively reduced LDL cholesterol 20%-50% for over 2 weeks, despite its relatively short terminal half-life (t(1/2) = 3.2 days). Importantly, the decrease in circulating LDL cholesterol corresponds closely to the reduction in free PCSK9 levels. Together these results clearly demonstrate that the LDL-lowering effect of the neutralizing anti-PCSK9 1D05-IgG2 antibody is mediated by reducing the amount of PCSK9 that can bind to the LDLr.
Asunto(s)
LDL-Colesterol/sangre , Fragmentos Fab de Inmunoglobulinas/farmacología , Receptores de LDL/química , Serina Endopeptidasas/inmunología , Animales , Anticuerpos Monoclonales/metabolismo , Sitios de Unión , Proteínas de Transferencia de Ésteres de Colesterol/metabolismo , Fluoroinmunoensayo , Humanos , Fragmentos Fab de Inmunoglobulinas/química , Inmunoglobulina G/inmunología , Inmunoglobulina G/metabolismo , Macaca mulatta , Masculino , Ratones , Ratones Transgénicos , Proproteína Convertasa 9 , Proproteína Convertasas , Receptores de LDL/metabolismo , Serina Endopeptidasas/químicaRESUMEN
PCSK9 binds to the low density lipoprotein receptor (LDLR) and leads to LDLR degradation and inhibition of plasma LDL cholesterol clearance. Consequently, the role of PCSK9 in modulating circulating LDL makes it a promising therapeutic target for treating hypercholesterolemia and coronary heart disease. Although the C-terminal domain of PCSK9 is not involved in LDLR binding, the location of several naturally occurring mutations within this region suggests that it has an important role for PCSK9 function. Using a phage display library, we identified an anti-PCSK9 Fab (fragment antigen binding), 1G08, with subnanomolar affinity for PCSK9. In an assay measuring LDL uptake in HEK293 and HepG2 cells, 1G08 Fab reduced 50% the PCSK9-dependent inhibitory effects on LDL uptake. Importantly, we found that 1G08 did not affect the PCSK9-LDLR interaction but inhibited the internalization of PCSK9 in these cells. Furthermore, proteolysis and site-directed mutagenesis studies demonstrated that 1G08 Fab binds a region of beta-strands encompassing Arg-549, Arg-580, Arg-582, Glu-607, Lys-609, and Glu-612 in the PCSK9 C-terminal domain. Consistent with these results, 1G08 fails to bind PCSK9DeltaC, a truncated form of PCSK9 lacking the C-terminal domain. Additional studies revealed that lack of the C-terminal domain compromised the ability of PCSK9 to internalize into cells, and to inhibit LDL uptake. Together, the present study demonstrate that the PCSK9 C-terminal domain contribute to its inhibition of LDLR function mainly through its role in the cellular uptake of PCSK9 and LDLR complex. 1G08 Fab represents a useful new tool for delineating the mechanism of PCSK9 uptake and LDLR degradation.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Fragmentos Fab de Inmunoglobulinas/farmacología , Lipoproteínas LDL/metabolismo , Receptores de LDL/metabolismo , Serina Endopeptidasas/metabolismo , Sustitución de Aminoácidos , Anticuerpos Monoclonales/genética , Anticuerpos Monoclonales/metabolismo , Células Hep G2 , Humanos , Hipercolesterolemia/tratamiento farmacológico , Hipercolesterolemia/genética , Hipercolesterolemia/inmunología , Hipercolesterolemia/metabolismo , Fragmentos Fab de Inmunoglobulinas/genética , Fragmentos Fab de Inmunoglobulinas/inmunología , Lipoproteínas LDL/genética , Lipoproteínas LDL/inmunología , Mutagénesis Sitio-Dirigida , Proproteína Convertasa 9 , Proproteína Convertasas , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Receptores de LDL/genética , Receptores de LDL/inmunología , Serina Endopeptidasas/genética , Serina Endopeptidasas/inmunologíaRESUMEN
Leukotrienes (LTs) are signaling molecules derived from arachidonic acid that initiate and amplify innate and adaptive immunity. In turn, how their synthesis is organized on the nuclear envelope of myeloid cells in response to extracellular signals is not understood. We define the supramolecular architecture of LT synthesis by identifying the activation-dependent assembly of novel multiprotein complexes on the outer and inner nuclear membranes of mast cells. These complexes are centered on the integral membrane protein 5-Lipoxygenase-Activating Protein, which we identify as a scaffold protein for 5-Lipoxygenase, the initial enzyme of LT synthesis. We also identify these complexes in mouse neutrophils isolated from inflamed joints. Our studies reveal the macromolecular organization of LT synthesis.
Asunto(s)
Araquidonato 5-Lipooxigenasa/metabolismo , Proteínas Portadoras/metabolismo , Leucotrienos/biosíntesis , Proteínas de la Membrana/metabolismo , Complejos Multiproteicos/análisis , Membrana Nuclear/metabolismo , Proteínas Activadoras de la 5-Lipooxigenasa , Animales , Artritis/enzimología , Artritis/metabolismo , Proteínas de la Membrana/análisis , Ratones , Células Mieloides/química , Células Mieloides/metabolismo , Neutrófilos/química , Neutrófilos/metabolismo , Membrana Nuclear/químicaRESUMEN
To discover antifungal treatments that possess the desired characteristics of broad spectrum activity, a strong safety profile, and oral bioavailability, new discovery strategies must be implemented to identify structural classes of molecules capable of combating these microorganisms. One such technique that has been implemented is the Candida albicans Fitness Test, a whole cell screening platform capable of delineating the mechanism of action of compounds that demonstrate activity against the clinically relevant pathogenic fungus, C. albicans. Screening crude natural product extracts with this technology has resulted in the identification of a novel family of antifungal natural products, named the parnafungins, which inhibit the enzyme polyadenosine polymerase (PAP), a key component of the mRNA cleavage and polyadenylation complex. Owing to the rapid interconversion of the structural and stereoisomers of the parnafungins at neutral pH, the determination of the structural isomer with the highest affinity for PAP with standard biochemical assays has not been possible. Herein, we present an application of affinity-selection/mass spectrometry (AS-MS) to determine that the "straight" parnafungin structural isomer (parnafungin A) binds preferentially to PAP compared to the "bent" structural isomer (parnafungin B).
Asunto(s)
Oxazolidinonas/química , Oxazolidinonas/metabolismo , Polinucleotido Adenililtransferasa/metabolismo , Productos Biológicos/química , Productos Biológicos/metabolismo , Cromatografía Liquida , Hongos/enzimología , Humanos , Isomerismo , Ligandos , Espectrometría de Masas , Oxazolidinonas/análisisRESUMEN
Natural products provide an unparalleled source of chemical scaffolds with diverse biological activities and have profoundly impacted antimicrobial drug discovery. To further explore the full potential of their chemical diversity, we survey natural products for antifungal, target-specific inhibitors by using a chemical-genetic approach adapted to the human fungal pathogen Candida albicans and demonstrate that natural-product fermentation extracts can be mechanistically annotated according to heterozygote strain responses. Applying this approach, we report the discovery and characterization of a natural product, parnafungin, which we demonstrate, by both biochemical and genetic means, to inhibit poly(A) polymerase. Parnafungin displays potent and broad spectrum activity against diverse, clinically relevant fungal pathogens and reduces fungal burden in a murine model of disseminated candidiasis. Thus, mechanism-of-action determination of crude fermentation extracts by chemical-genetic profiling brings a powerful strategy to natural-product-based drug discovery.
Asunto(s)
Antifúngicos/farmacología , Antifúngicos/uso terapéutico , Productos Biológicos/farmacología , Productos Biológicos/uso terapéutico , Candida albicans/efectos de los fármacos , Candida albicans/genética , Evaluación Preclínica de Medicamentos/métodos , Polinucleotido Adenililtransferasa/antagonistas & inhibidores , Alelos , Secuencia de Aminoácidos , Animales , Antifúngicos/química , Antifúngicos/aislamiento & purificación , Aspergillus fumigatus/efectos de los fármacos , Aspergillus fumigatus/crecimiento & desarrollo , Aspergillus fumigatus/metabolismo , Productos Biológicos/química , Productos Biológicos/aislamiento & purificación , Candida albicans/metabolismo , Candidiasis/tratamiento farmacológico , Candidiasis/metabolismo , Mezclas Complejas/farmacología , Desoxiadenosinas/metabolismo , Desoxiadenosinas/farmacología , Farmacorresistencia Fúngica , Fermentación , Heterocigoto , Ratones , Pruebas de Sensibilidad Microbiana , Datos de Secuencia Molecular , Mutación , Poliadenilación/efectos de los fármacos , Polinucleotido Adenililtransferasa/genética , Polinucleotido Adenililtransferasa/metabolismo , ARN Mensajero/metabolismo , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/metabolismo , Resultado del TratamientoRESUMEN
The nuclear membrane protein 5-lipoxygenase-activating protein (FLAP) plays an essential role in leukotriene synthesis. Recombinant full-length human FLAP with a C-terminal hexahistidine tag has been expressed and purified from the cytoplasmic membrane of Escherichia coli. Diffraction-quality crystals of FLAP in complex with leukotriene-synthesis inhibitor MK-591 and with an iodinated analogue of MK-591 have been grown using the sitting-drop vapor-diffusion method. The crystals exhibit tetragonal symmetry (P42(1)2) and diffracted to a resolution limit of 4 A.
Asunto(s)
Proteínas Portadoras/química , Proteínas Portadoras/metabolismo , Expresión Génica , Leucotrienos/biosíntesis , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Proteínas Activadoras de la 5-Lipooxigenasa , Proteínas Portadoras/genética , Proteínas Portadoras/aislamiento & purificación , Cristalización , Cristalografía por Rayos X , Humanos , Proteínas de la Membrana/genética , Proteínas de la Membrana/aislamiento & purificación , Estructura MolecularRESUMEN
Leukotrienes are proinflammatory products of arachidonic acid oxidation by 5-lipoxygenase that have been shown to be involved in respiratory and cardiovascular diseases. The integral membrane protein FLAP is essential for leukotriene biosynthesis. We describe the x-ray crystal structures of human FLAP in complex with two leukotriene biosynthesis inhibitors at 4.0 and 4.2 angstrom resolution, respectively. The structures show that inhibitors bind in membrane-embedded pockets of FLAP, which suggests how these inhibitors prevent arachidonic acid from binding to FLAP and subsequently being transferred to 5-lipoxygenase, thereby preventing leukotriene biosynthesis. This structural information provides a platform for the development of therapeutics for respiratory and cardiovascular diseases.
Asunto(s)
Proteínas Portadoras/química , Indoles/química , Proteínas de la Membrana/química , Quinolinas/química , Proteínas Activadoras de la 5-Lipooxigenasa , Araquidonato 5-Lipooxigenasa/metabolismo , Ácido Araquidónico/metabolismo , Sitios de Unión , Proteínas Portadoras/antagonistas & inhibidores , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Dominio Catalítico , Cristalografía por Rayos X , Citosol/química , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Indoles/metabolismo , Indoles/farmacología , Proteínas de la Membrana/antagonistas & inhibidores , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Modelos Moleculares , Mutagénesis , Membrana Nuclear/química , Conformación Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Subunidades de Proteína/química , Quinolinas/metabolismo , Quinolinas/farmacologíaRESUMEN
The m7GpppN cap at the 5' end of eukaryotic mRNAs is important for transcript stability and translation. Three enzymatic activities that generate the mRNA cap include an RNA 5'-triphosphatase, an RNA guanylyltransferase, and an RNA (guanine-7-) -methyltransferase. The physical organization of the genes encoding these enzymes differs between mammalian cells and yeast, fungi, or viruses. The catalytic mechanism used by the RNA triphosphatases of mammalian cells also differs from that used by the yeast, fungal, or viral enzymes. These structural and functional differences suggest that inhibitors of mRNA capping might be useful antifungal or antiviral agents. The authors describe several whole-cell yeast-based assays developed to identify and characterize inhibitors of fungal mRNA capping. They also report the identification and characterization of the natural product sinefungin in the assays. Their characterization of this S-adenosylmethionine analog suggests that it inhibits mRNA cap methyltransferases and exhibits approximately 5- to 10-fold specificity for the yeast ABD1 and fungal CCM1 enzymes over the human Hcm1 enzyme expressed in yeast cells.
Asunto(s)
Adenosina/análogos & derivados , Inhibidores Enzimáticos/análisis , Nucleotidiltransferasas/antagonistas & inhibidores , Saccharomyces cerevisiae/genética , Adenosina/química , Secuencia de Aminoácidos , Genes Reporteros , Datos de Secuencia Molecular , Nucleotidiltransferasas/química , Nucleotidiltransferasas/genética , Plásmidos , Homología de Secuencia de AminoácidoRESUMEN
We have developed assays for the binding of nucleotide and protein substrates to p38alpha protein kinase based on time-resolved Forster resonance energy transfer. p38alpha was biotinylated by addition of a sequence that targets biotin to a single lysine when coexpressed with biotin ligase in Escherichia coli, allowing formation of a complex between a streptavidin "LANCE" europium chelate conjugate and p38alpha. When this reagent was combined with M39AF, a p38 inhibitor containing a fluorescent moiety whose excitation wavelengths match the emission wavelengths of the europium chelate, a change in ratio of light emitted at 665 nm/615 nm is detected. Less than 100pM complex was detected with a signal/background ratio of >30-fold. The complex exhibits slow, tight binding kinetics where the apparent K(d) decreases with a relaxation time of 21 min at 125 pM biotin-p38alpha. Preincubating inhibitors or ATP with biotin-p38alpha and adding M39AF as a competitor yielded IC(50)s consistent with those measured by enzyme assay for the activated form of biotin-p38alpha. The same technique was also used to measure affinity of inhibitors for the unphosphorylated and catalytically inactive form of biotin-p38alpha. To measure affinity of p38alpha for its protein substrate MK2, we incubated biotin-p38alpha with a glutathione S-transferase MK2 fusion protein. Detection of the complex after incubation with streptavidin-allophycocyanin and a LANCE-conjugated anti-GST allowed measurement of affinity of MK2 for biotin-p38alpha and detection of 0.5 nM p38alpha.MK2 complex with signal/background ratio >5-fold. Competition with unbiotinylated p38alpha yielded an IC(50) value of 5 nM. Activation of either p38alpha or MK2 had no effect on the measured K(d). M39AF was found to bind in a ternary complex with p38alpha.MK2 with lower affinity than that observed in the binary complex with p38alpha alone.
Asunto(s)
Transferencia Resonante de Energía de Fluorescencia , Colorantes Fluorescentes/química , Nucleótidos/química , Proteínas Quinasas p38 Activadas por Mitógenos/química , Animales , Transferencia Resonante de Energía de Fluorescencia/métodos , Colorantes Fluorescentes/metabolismo , Humanos , Nucleótidos/metabolismo , Unión Proteica , Proteínas Quinasas p38 Activadas por Mitógenos/genéticaRESUMEN
Cell signaling leading to the formation of leukotriene (LT)C(4) requires the localization of the four key biosynthetic enzymes on the outer nuclear membrane and endoplasmic reticulum. Whether any macromolecular organization of these proteins exists is unknown. By using fluorescence lifetime imaging microscopy and biochemical analysis, we demonstrate the presence of two distinct multimeric complexes that regulate the formation of LTs in RBL-2H3 cells. One complex consists of multimers of LTC(4) synthase and the 5-lipoxygenase activating protein (FLAP). The second complex consists of multimers of FLAP. Surprisingly, all LTC(4) synthase was found to be in association with FLAP. The results indicate that the formation of LTC(4) and LTB(4) may be determined by the compartmentalization of biosynthetic enzymes in discrete molecular complexes.
Asunto(s)
Leucotrieno C4/biosíntesis , Proteínas Activadoras de la 5-Lipooxigenasa , Northern Blotting , Proteínas Portadoras/metabolismo , Línea Celular , Membrana Celular/metabolismo , Transferencia de Energía , Humanos , Leucotrieno C4/metabolismo , Proteínas de la Membrana/metabolismo , Microscopía FluorescenteRESUMEN
A fluorescence resonance energy transfer assay has been developed for monitoring Bacillus anthracis lethal factor (LF) protease activity. A fluorogenic 16-mer peptide based on the known LF protease substrate MEK1 was synthesized and found to be cleaved by the enzyme at the anticipated site. Extension of this work to a fluorogenic 19-mer peptide, derived, in part, from a consensus sequence of known LF protease targets, produced a much better substrate, cleaving approximately 100 times more efficiently. This peptide sequence was modified further on resin to incorporate donor/quencher pairs to generate substrates for use in fluorescence resonance energy transfer-based appearance assays. All peptides cleaved at similar rates with signal/background ranging from 9-16 at 100% turnover. One of these substrates, denoted (Cou)Consensus(K(QSY-35)GG)-NH(2), was selected for additional assay optimization. A plate-based assay requiring only low nanomolar levels of enzyme was developed for screening and inhibitor characterization.