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1.
J Am Chem Soc ; 137(7): 2412-5, 2015 Feb 25.
Artículo en Inglés | MEDLINE | ID: mdl-25658416

RESUMEN

We introduce a stabilized diazo group as a reporter for chemical biology. ManDiaz, which is a diazo derivative of N-acetylmannosamine, is found to endure cellular metabolism and label the surface of a mammalian cell. There its diazo group can undergo a 1,3-dipolar cycloaddition with a strained alkyne, providing a signal comparable to that from the azido congener, ManNAz. The chemoselectivity of diazo and alkynyl groups enables dual labeling of cells that is not possible with azido and alkynyl groups. Thus, the diazo group, which is approximately half the size of an azido group, provides unique opportunities for orthogonal labeling of cellular components.


Asunto(s)
Compuestos Azo/química , Compuestos Azo/metabolismo , Alquinos/metabolismo , Animales , Células CHO , Cricetinae , Cricetulus , Hexosaminas/química , Especificidad por Sustrato
2.
Org Biomol Chem ; 12(43): 8598-602, 2014 Nov 21.
Artículo en Inglés | MEDLINE | ID: mdl-25266373

RESUMEN

Organocatalysts derived from diethylenetriamine effect the rapid isomerization of non-native protein disulfide bonds to native ones. These catalysts contain a pendant hydrophobic moiety to encourage interaction with the non-native state, and two thiol groups with low pKa values that form a disulfide bond with a high E°' value.


Asunto(s)
Disulfuros/química , Poliaminas/química , Proteína Disulfuro Isomerasas/química , Ribonucleasa Pancreática/química , Bibliotecas de Moléculas Pequeñas/síntesis química , Animales , Catálisis , Bovinos , Isomerismo , Cinética , Imitación Molecular , Oxidación-Reducción , Páncreas/química , Páncreas/enzimología , Conformación Proteica , Pliegue de Proteína
3.
Autism ; 23(8): 2096-2111, 2019 11.
Artículo en Inglés | MEDLINE | ID: mdl-31027422

RESUMEN

Three phase 2 trials were conducted to assess the efficacy and long-term safety of weight-based memantine extended release (ER) treatment in children with autism spectrum disorder. MEM-MD-91, a 50-week open-label trial, identified memantine extended-release treatment responders for enrollment into MEM-MD-68, a 12-week randomized, double-blind, placebo-controlled withdrawal trial. MEM-MD-69 was an open-label extension trial in which participants from MEM-MD-68, MEM-MD-91, and open-label trial MEM-MD-67 were treated ⩽48 weeks with memantine extended release. In MEM-MD-91, 517 (59.6%) participants were confirmed Social Responsiveness Scale responders at week 12; mean Social Responsiveness Scale total raw scores improved two to three times a minimal clinically important difference of 10 points. In MEM-MD-68, there was no difference between memantine and placebo on the primary efficacy parameter, the proportion of patients with a loss of therapeutic response (defined as ⩾10-point increase from baseline in Social Responsiveness Scale total raw score). MEM-MD-69 exploratory analyses revealed mean standard deviation improvement in Social Responsiveness Scale total raw score of 32.4 (26.4) from baseline of the first lead-in study. No new safety concerns were evident. While the a priori-defined efficacy results of the double-blind trial were not achieved, the considerable improvements in mean Social Responsiveness Scale scores from baseline in the open-label trials were presumed to be clinically important.


Asunto(s)
Trastorno del Espectro Autista/tratamiento farmacológico , Antagonistas de Aminoácidos Excitadores/uso terapéutico , Memantina/uso terapéutico , Conducta Social , Trastorno del Espectro Autista/fisiopatología , Trastorno del Espectro Autista/psicología , Niño , Preparaciones de Acción Retardada , Método Doble Ciego , Terminación Anticipada de los Ensayos Clínicos , Femenino , Fiebre/inducido químicamente , Cefalea/inducido químicamente , Humanos , Genio Irritable , Masculino , Nasofaringitis/inducido químicamente , Resultado del Tratamiento
4.
ACS Chem Biol ; 11(2): 319-23, 2016 Feb 19.
Artículo en Inglés | MEDLINE | ID: mdl-26629587

RESUMEN

The use of exogenous proteins as intracellular probes and therapeutic agents is in its infancy. A major hurdle has been the delivery of native proteins to an intracellular site of action. Herein, we report on a compact delivery vehicle that employs the intrinsic affinity of boronic acids for the carbohydrates that coat the surface of mammalian cells. In the vehicle, benzoxaborole is linked to protein amino groups via a "trimethyl lock." Immolation of this linker is triggered by cellular esterases, releasing native protein. Efficacy is demonstrated by enhanced delivery of green fluorescent protein and a cytotoxic ribonuclease into mammalian cells. This versatile strategy provides new opportunities in chemical biology and pharmacology.


Asunto(s)
Ácidos Borónicos/química , Portadores de Fármacos/química , Proteínas Fluorescentes Verdes/administración & dosificación , Ribonucleasas/administración & dosificación , Animales , Ácidos Borónicos/metabolismo , Células CHO , Línea Celular Tumoral , Permeabilidad de la Membrana Celular , Cricetulus , Portadores de Fármacos/metabolismo , Proteínas Fluorescentes Verdes/química , Proteínas Fluorescentes Verdes/farmacocinética , Humanos , Modelos Moleculares , Ribonucleasas/química , Ribonucleasas/farmacocinética
5.
ACS Chem Biol ; 11(1): 193-9, 2016 Jan 15.
Artículo en Inglés | MEDLINE | ID: mdl-26535807

RESUMEN

Collagen is the most abundant protein in animals. Its overproduction is associated with fibrosis and cancer metastasis. The stability of collagen relies on post-translational modifications, the most prevalent being the hydroxylation of collagen strands by collagen prolyl 4-hydroxylases (CP4Hs). Catalysis by CP4Hs enlists an iron cofactor to convert proline residues to 4-hydroxyproline residues, which are essential for the conformational stability of mature collagen. Ethyl 3,4-dihydroxybenzoate (EDHB) is commonly used as a "P4H" inhibitor in cells, but suffers from low potency, poor selectivity, and off-target effects that cause iron deficiency. Dicarboxylates of 2,2'-bipyridine are among the most potent known CP4H inhibitors but suffer from a high affinity for free iron. A screen of biheteroaryl compounds revealed that replacing one pyridyl group with a thiazole moiety retains potency and enhances selectivity. A diester of 2-(5-carboxythiazol-2-yl)pyridine-5-carboxylic acid is bioavailable to human cells and inhibits collagen biosynthesis at concentrations that neither cause general toxicity nor disrupt iron homeostasis. These data anoint a potent and selective probe for CP4H and a potential lead for the development of a new class of antifibrotic and antimetastatic agents.


Asunto(s)
Ácidos Carboxílicos/farmacología , Procolágeno-Prolina Dioxigenasa/antagonistas & inhibidores , Antineoplásicos/química , Antineoplásicos/farmacología , Antineoplásicos/toxicidad , Ácidos Carboxílicos/química , Ácidos Carboxílicos/toxicidad , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Activación Enzimática/efectos de los fármacos , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Humanos , Procolágeno-Prolina Dioxigenasa/química , Tiazoles/química , Tiazoles/farmacología
6.
Methods Mol Biol ; 1248: 55-65, 2015.
Artículo en Inglés | MEDLINE | ID: mdl-25616325

RESUMEN

Site-specific isopeptide linkages between the ε-amino group of a lysine residue in one protein and a carboxyl group in another are central to ubiquitin-mediated protein degradation and other cellular processes. These linkages are inaccessible with common recombinant DNA techniques. Here, we describe a method to link two proteins by an authentic isopeptide bond. The method unites three techniques at the forefront of molecular biology. An azidonorleucine residue is installed at a desired site in a substrate protein by nonnatural amino acid incorporation, and a phosphinothioester is installed at the C terminus of a pendant protein by expressed protein ligation. Then, the traceless Staudinger ligation is used to link the substrate and pendant proteins via an isopeptide bond. This method facilitates the study of otherwise intractable protein structure-function relationships.


Asunto(s)
Reactivos de Enlaces Cruzados/química , Péptidos/química , Proteínas/química
7.
Protein Sci ; 24(2): 182-9, 2015 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-25401704

RESUMEN

The post-translational modification of proteins with ubiquitin can take on many forms, including the decoration of substrates with polymeric ubiquitin chains. These chains are linked through one of the seven lysine residues in ubiquitin, with the potential to form a panoply of linkage combinations as the chain length increases. The ensuing structural diversity of modifications serves a variety of signaling functions. Still, some linkages are present at a much higher level than others in cellulo. Although ubiquitination is an enzyme-catalyzed process, the large disparity of abundancies led us to the hypothesis that some linkages might be intrinsically faster to form than others, perhaps directing the course of enzyme evolution. Herein, we assess the kinetics of ubiquitin dimer formation in an enzyme-free system by measuring the rate constants for thiol-disulfide interchange between appropriate ubiquitin variants. Remarkably, we find that the kinetically expedient linkages correlate with those that are most abundant in cellulo. As the abundant linkages also appear to function more broadly in cellulo, this correlation suggests that the more accessible chains were selected for global roles.


Asunto(s)
Ubiquitina/química , Disulfuros/química , Humanos , Modelos Moleculares , Multimerización de Proteína , Ubiquitinación
8.
Chem Sci ; 6(1): 752-755, 2015 Jan 01.
Artículo en Inglés | MEDLINE | ID: mdl-25544883

RESUMEN

A diazo compound is shown to convert carboxylic acids to esters efficiently in an aqueous environment. The basicity of the diazo compound is critical: low basicity does not lead to a reaction but high basicity leads to hydrolysis. This reactivity extends to carboxylic acid groups in a protein. The ensuing esters are hydrolyzed by human cellular esterases to regenerate protein carboxyl groups. This new mode of chemical modification could enable the key advantages of prodrugs to be translated from small-molecules to proteins.

9.
Sci Transl Med ; 6(229): 229ra43, 2014 Mar 26.
Artículo en Inglés | MEDLINE | ID: mdl-24670687

RESUMEN

The blockbuster chemotherapy drug paclitaxel is widely presumed to cause cell death in tumors as a consequence of mitotic arrest, as it does at concentrations routinely used in cell culture. However, we determine here that paclitaxel levels in primary breast tumors are well below those required to elicit sustained mitotic arrest. Instead, cells in these lower concentrations of drug proceed through mitosis without substantial delay and divide their chromosomes on multipolar spindles, resulting in chromosome missegregation and cell death. Consistent with these cell culture data, most mitotic cells in primary human breast cancers contain multipolar spindles after paclitaxel treatment. Contrary to the previous hypothesis, we find that mitotic arrest is dispensable for tumor regression in patients. These results demonstrate that mitotic arrest is not responsible for the efficacy of paclitaxel, which occurs because of chromosome missegregation on highly abnormal, multipolar spindles. This mechanistic insight may be used to improve selection of future antimitotic drugs and to identify a biomarker with which to select patients likely to benefit from paclitaxel.


Asunto(s)
Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/patología , Segregación Cromosómica/efectos de los fármacos , Paclitaxel/uso terapéutico , Huso Acromático/patología , Adulto , Anciano , Puntos de Control del Ciclo Celular/efectos de los fármacos , Muerte Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Femenino , Humanos , Interfase/efectos de los fármacos , Persona de Mediana Edad , Mitosis/efectos de los fármacos , Paclitaxel/toxicidad , Huso Acromático/efectos de los fármacos , Carga Tumoral/efectos de los fármacos , Células Tumorales Cultivadas
10.
J Physiol ; 546(Pt 3): 717-31, 2003 Feb 01.
Artículo en Inglés | MEDLINE | ID: mdl-12562999

RESUMEN

Transport of the amino acid GABA into neurons and glia plays a key role in regulating the effects of GABA in the vertebrate retina. We have examined the modulation of GABA-elicited transport currents of retinal horizontal cells by glutamate, the likely neurotransmitter of vertebrate photoreceptors. Enzymatically isolated external horizontal cells of skate were examined using whole-cell voltage-clamp techniques. GABA (1 mM ) elicited an inward current that was completely suppressed by the GABA transport inhibitors tiagabine (10 microM) and SKF89976-A (100 microM), but was unaffected by 100 microM picrotoxin. Prior application of 100 microM glutamate significantly reduced the GABA-elicited current. Glutamate depressed the GABA dose-response curve without shifting the curve laterally or altering the voltage dependence of the current. The ionotropic glutamate receptor agonists kainate and AMPA also reduced the GABA-elicited current, and the effects of glutamate and kainate were abolished by the ionotropic glutamate receptor antagonist 6-cyano-7-nitroquinoxaline. NMDA neither elicited a current nor modified the GABA-induced current, and metabotropic glutamate analogues were also without effect. Inhibition of the GABA-elicited current by glutamate and kainate was reduced when extracellular calcium was removed and when recording pipettes contained high concentrations of the calcium chelator BAPTA. Caffeine (5 mM) and thapsigargin (2 nM), agents known to alter intracellular calcium levels, also reduced the GABA-elicited current, but increases in calcium induced by depolarization alone did not. Our data suggest that glutamate regulates GABA transport in retinal horizontal cells through a calcium-dependent process, and imply a close physical relationship between calcium-permeable glutamate receptors and GABA transporters in these cells.


Asunto(s)
Proteínas Portadoras/metabolismo , Ácido Glutámico/fisiología , Proteínas de la Membrana/metabolismo , Proteínas de Transporte de Membrana , Transportadores de Anión Orgánico , Retina/metabolismo , Rajidae/fisiología , Animales , Calcio/metabolismo , Proteínas Portadoras/fisiología , Relación Dosis-Respuesta a Droga , Antagonistas de Aminoácidos Excitadores/farmacología , Antagonistas del GABA/farmacología , Proteínas Transportadoras de GABA en la Membrana Plasmática , Ácido Glutámico/administración & dosificación , Ácido Glutámico/farmacología , Membranas Intracelulares/metabolismo , Ácido Kaínico/farmacología , Proteínas de la Membrana/fisiología , Técnicas de Placa-Clamp , Retina/citología , Retina/efectos de los fármacos , Retina/fisiología , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol Propiónico/farmacología
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