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1.
Chem Sci ; 10(20): 5373-5381, 2019 May 28.
Artigo em Inglês | MEDLINE | ID: mdl-31191895

RESUMO

Enzymes that consume and produce nucleoside polyphosphate (NPP) anions represent major targets in drug discovery. For example, protein kinases are one of the largest classes of drug targets in the fight against cancer. The accurate determination of enzyme kinetics and mechanisms is a critical aspect of drug discovery research. To increase confidence in the selection of lead drug compounds it is crucial that pharmaceutical researchers have robust, affordable assays to measure enzyme activity accurately. We present a simple, sensitive microplate assay for real-time monitoring of a range of pharmaceutically important enzyme reactions that generate NPP anions, including kinases and glycosyltransferases. Our assay utilises a single, stable europium(iii) complex that binds reversibly to NPP anions, signalling the dynamic changes in NPP product/substrate ratio during an enzyme reaction using time-resolved luminescence. This supramolecular approach to enzyme monitoring overcomes significant limitations in existing assays, obviating the need for expensive antibodies or equipment, chemically labelled substrates or products and isolation or purification steps. Our label and antibody-free method enables rapid and quantitative analysis of enzyme activities and inhibition, offering a potentially powerful tool for use in drug discovery, suitable for high-throughput screening of inhibitors and accurate measurements of enzyme kinetic parameters.

2.
Chemistry ; 24(42): 10745-10755, 2018 Jul 25.
Artigo em Inglês | MEDLINE | ID: mdl-29761917

RESUMO

The ability to study cellular metabolism and enzymatic processes involving adenosine triphosphate (ATP) is impeded by the lack of imaging probes capable of signalling the concentration and distribution of intracellular ATP rapidly, with high sensitivity. We report here the first example of a luminescent lanthanide complex capable of visualizing changes in the concentration of ATP in the mitochondria of living cells. Four cationic europium(III) complexes [Eu.1-4]+ have been synthesized and their binding capabilities towards nucleoside polyphosphate anions examined in aqueous solution at physiological pH. Complexes [Eu.1]+ and [Eu.3]+ bearing hydrogen bond donor groups in the pendant arms showed excellent discrimination between ATP, ADP and monophosphate species. Complex [Eu.3]+ showed relatively strong binding to ATP (logKa =5.8), providing a rapid, long-lived luminescent signal that enabled its detection in a highly competitive aqueous medium containing biologically relevant concentrations of Mg2+ , ADP, GTP, UTP and human serum albumin. This EuIII complex responds linearly to ATP within the physiological concentration range (1-5 mm), and was used to continuously monitor the apyrase-catalyzed hydrolysis of ATP to ADP in vitro. We demonstrate that [Eu.3]+ can permeate mammalian (NIH-3T3) cells efficiently and localize to the mitochondria selectively, permitting real-time visualization of elevated mitochondrial ATP levels following treatment with a broad spectrum kinase inhibitor, staurosporine, as well as depleted ATP levels upon treatment with potassium cyanide under glucose starvation conditions.


Assuntos
Trifosfato de Adenosina/metabolismo , Antígenos CD/química , Apirase/química , Európio/química , Íons/química , Mitocôndrias/metabolismo , Trifosfato de Adenosina/química , Animais , Humanos , Luminescência , Mitocôndrias/química
3.
Chem Commun (Camb) ; 54(50): 6635-6647, 2018 Jun 19.
Artigo em Inglês | MEDLINE | ID: mdl-29790500

RESUMO

Enzymes play critical roles in the regulation of cellular function and are implicated in numerous disease conditions. Reliable and practicable assays are required to study enzyme activity, to facilitate the discovery of inhibitors and activators of enzymes related to disease. In recent years, a variety of enzyme assays have been devised that utilise luminescent lanthanide(iii) complexes, taking advantage of their high detection sensitivities, long luminescence lifetimes, and line-like emission spectra that permit ratiometric and time-resolved analyses. In this Feature article, we focus on recent progress in the development of enzyme activity assays based on lanthanide(iii) luminescence, covering a variety of strategies including Ln(iii)-labelled antibodies and proteins, Ln(iii) ion encapsulation within defined peptide sequences, reactivity-based Ln(iii) probes, and discrete Ln(iii) complexes. Emerging approaches for monitoring enzyme activity are discussed, including the use of anion responsive lanthanide(iii) complexes, capable of molecular recognition and luminescence signalling of polyphosphate anions.


Assuntos
Enzimas/química , Elementos da Série dos Lantanídeos/química , Substâncias Luminescentes/química , Anticorpos/química , Complexos de Coordenação/química , Complexos de Coordenação/metabolismo , Ensaios Enzimáticos/métodos , Imunoensaio/métodos , Luminescência , Substâncias Luminescentes/metabolismo , Ligação Proteica , Proteínas/metabolismo
4.
Chem Commun (Camb) ; 53(94): 12626-12629, 2017 Nov 23.
Artigo em Inglês | MEDLINE | ID: mdl-29131206

RESUMO

We report the application of a stable cationic europium complex [Eu.1]+ in a continuous-read luminescence assay for kinase activity. [Eu.1]+ binds reversibly to ATP and ADP in water, at neutral pH, in the presence of Mg2+ ions, providing distinctive luminescence responses that permits the kinase-catalysed conversion of ATP to ADP to be monitored in real-time.


Assuntos
Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Complexos de Coordenação/química , Európio/química , Medições Luminescentes/métodos , Fosfotransferases/análise , Fosfotransferases/metabolismo
5.
Chem Commun (Camb) ; 51(54): 10879-82, 2015 Jul 11.
Artigo em Inglês | MEDLINE | ID: mdl-26054741

RESUMO

Two luminescent probes [Eu.L¹â»²]⁺ are reported for the rapid detection of fluoride in water. Probes [Eu.L¹â»²]⁺ exhibit exceptional enhancements in Eu emission in the presence of fluoride, permitting its selective determination within the environmentally relevant concentration range (20-210 µM).


Assuntos
Complexos de Coordenação/química , Európio/química , Fluoretos/análise , Medições Luminescentes , Água/química , Íons/química
6.
Dalton Trans ; 44(11): 4791-803, 2015 Mar 21.
Artigo em Inglês | MEDLINE | ID: mdl-25341077

RESUMO

The development of the brightest luminescent europium(iii) complexes is traced, including analysis of the C3-symmetric core complex based on a functionalized triazacyclononane and identification of the most suitable strongly absorbing chromophore. Strategies for the synthesis of the complexes, including enantiopure analogues, are outlined and opportunities for applications in time-resolved microscopy and spectral imaging emphasised. Practicable examples are introduced, including selective organelle staining for cellular optical imaging at 65 nm resolution and the development of new bioassays using time-resolved FRET methods.


Assuntos
Bioensaio/métodos , Corantes/síntese química , Európio/química , Imagem Óptica/métodos , Compostos Organometálicos/síntese química , Animais , Técnicas de Química Sintética , Corantes/química , Corantes/metabolismo , Humanos , Compostos Organometálicos/química , Compostos Organometálicos/metabolismo
7.
Chemistry ; 20(48): 15768-74, 2014 Nov 24.
Artigo em Inglês | MEDLINE | ID: mdl-25303281

RESUMO

Two tripodal fluorescent probes Zn⋅L(1,2) have been synthesised, and their anion-binding capabilities were examined by using fluorescence spectroscopy. Probe Zn⋅L(1) allows the selective and ratiometric detection of adenosine triphosphate (ATP) at physiological pH, even in the presence of several competing anions, such as ADP, phosphate and bicarbonate. The probe was applied to the real-time monitoring of the apyrase-catalysed hydrolysis of ATP, in a medium that mimics an extracellular fluid.


Assuntos
Trifosfato de Adenosina/análise , Trifosfato de Adenosina/química , Ânions/química , Apirase/química , Complexos de Coordenação/síntese química , Corantes Fluorescentes/síntese química , Quinolinas/química , Zinco/química , Catálise , Complexos de Coordenação/química , Corantes Fluorescentes/química , Concentração de Íons de Hidrogênio , Cinética , Modelos Moleculares , Espectrometria de Fluorescência , Água
8.
Chemistry ; 20(28): 8636-46, 2014 Jul 07.
Artigo em Inglês | MEDLINE | ID: mdl-24938657

RESUMO

A series of europium and terbium complexes based on a functionalized triazacyclononane carboxylate or phosphinate macrocyclic ligand is described. The influence of the anionic group, that is, carboxylate, methylphosphinate, or phenylphosphinate, on the photophysical properties was studied and rationalized on the basis of DFT calculated structures. The nature, number, and position of electron-donating or electron-withdrawing aryl substituents were varied systematically within the same phenylethynyl scaffold in order to optimize the brightness of the corresponding europium complexes and investigate their two-photon absorption properties. Finally, the europium complexes were examined in cell-imaging applications, and selected terbium complexes were studied as potential oxygen sensors.


Assuntos
Alquinos/química , Compostos Aza/química , Európio/química , Compostos Organometálicos/química , Piperidinas/química , Térbio/química , Ligantes , Estrutura Molecular
9.
Dalton Trans ; 43(15): 5721-30, 2014 Apr 21.
Artigo em Inglês | MEDLINE | ID: mdl-24557335

RESUMO

The synthetic utility of europium complexes with three coordinated 4-bromopyridyl groups for chromophore elaboration has been assessed in palladium-catalysed Sonogashira coupling reactions, and in copper(i) mediated click reactions of the triazide derivative, generated in situ. The crystal structure of the Eu complex of a p-OMe-phenyl substituted triazole at 100 K is reported in which the pendant triazole sensitising moieties interdigitate in the solid-state lattice. The triazole complex can be separated into Δ and Λ enantiomers by chiral HPLC but is weakly emissive in methanol (ε 5.5 mM(-1) cm(-1); λexc 320 nm; ϕ 0.2%), contrasting with a set of four alkynyl-aryl derivatives which are one thousand times brighter and absorb strongly with broad absorption maxima in the range 332 to 360 nm. An enantiopure europium complex gives an intense CPL signal in solution that is the strongest yet reported.

10.
Chem Commun (Camb) ; 49(80): 9104-6, 2013 Oct 14.
Artigo em Inglês | MEDLINE | ID: mdl-23989726

RESUMO

A cysteine-selective tagging method in water is reported, based on rapid displacement of a pyridyl nitro-substituent in simple pyridines and lanthanide complexes. The conjugation reaction creates a short link between the tag and peptide, holding the peptide closer to the Ln(3+) ion and with reduced flexibility compared to existing methods.


Assuntos
Complexos de Coordenação/química , Cisteína/química , Elementos da Série dos Lantanídeos/química , Peptídeos/química , Proteínas/química , Piridinas/química , Íons/química , Ressonância Magnética Nuclear Biomolecular , Peptídeos/metabolismo , Proteínas/metabolismo
11.
ACS Chem Biol ; 8(9): 1955-63, 2013 Sep 20.
Artigo em Inglês | MEDLINE | ID: mdl-23802178

RESUMO

Tyrosine sulfation is a post-translational modification that enhances protein-protein interactions and may identify druggable sites in the extracellular space. The G protein-coupled receptor CXCR4 is a prototypical example with three potential sulfation sites at positions 7, 12, and 21. Each receptor sulfotyrosine participates in specific contacts with its chemokine ligand in the structure of a soluble, dimeric CXCL12:CXCR4(1-38) complex, but their relative importance for CXCR4 binding and activation by the monomeric chemokine remains undefined. NMR titrations with short sulfopeptides showed that the tyrosine motifs of CXCR4 varied widely in their contributions to CXCL12 binding affinity and site specificity. Whereas the Tyr21 sulfopeptide bound the same site as in previously solved structures, the Tyr7 and Tyr12 sulfopeptides interacted nonspecifically. Surprisingly, the unsulfated Tyr7 peptide occupied a hydrophobic site on the CXCL12 monomer that is inaccessible in the CXCL12 dimer. Functional analysis of CXCR4 mutants validated the relative importance of individual CXCR4 sulfotyrosine modifications (Tyr21 > Tyr12 > Tyr7) for CXCL12 binding and receptor activation. Biophysical measurements also revealed a cooperative relationship between sulfopeptide binding at the Tyr21 site and CXCL12 dimerization, the first example of allosteric behavior in a chemokine. Future ligands that occupy the sTyr21 recognition site may act as both competitive inhibitors of receptor binding and allosteric modulators of chemokine function. Together, our data suggests that sulfation does not ubiquitously enhance complex affinity and that distinct patterns of tyrosine sulfation could encode oligomer selectivity, implying another layer of regulation for chemokine signaling.


Assuntos
Quimiocina CXCL12/metabolismo , Peptídeos/metabolismo , Receptores CXCR4/metabolismo , Sequência de Aminoácidos , Animais , Células CHO , Quimiocina CXCL12/química , Cricetulus , Humanos , Modelos Moleculares , Peptídeos/química , Ligação Proteica , Multimerização Proteica , Receptores CXCR4/química , Tirosina/química , Tirosina/metabolismo
12.
Chemistry ; 19(29): 9511-7, 2013 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-23744790

RESUMO

A series of five europium(III) complexes has been prepared from heptadentate N5O2 ligands that possess a brightness of more than 10 mM(-1) cm(-1) in water, following excitation over the range λ=330-355 nm. Binding of several oxy anions has been assessed by emission spectral titrimetric analysis, with the binding of simple carboxylates, lactate and citrate involving a common ligation mode following displacement of the coordinated water. Selectivity for bicarbonate allows the rapid determination of this anion in human serum, with K(d)=37 mM (295 K). The complexes are internalised quickly into mammalian cells and exhibit a mitochondrial localisation at early time points, migrating after a few hours to reveal a predominant lysosomal distribution. Herein, we report the synthesis and complexation behaviour of strongly emissive europium (III) complexes that bind oxy-anions in aqueous media with an affinity and selectivity profile that is distinctively different from previously studied systems.


Assuntos
Ânions/química , Compostos Aza/química , Bicarbonatos/análise , Bicarbonatos/química , Ácido Cítrico/química , Európio/análise , Európio/química , Piperidinas/química , Soro/química , Humanos , Ligantes , Luminescência , Soro/metabolismo
13.
J Biol Chem ; 288(14): 10024-34, 2013 Apr 05.
Artigo em Inglês | MEDLINE | ID: mdl-23408426

RESUMO

Chemokine receptors are commonly post-translationally sulfated on tyrosine residues in their N-terminal regions, the initial site of binding to chemokine ligands. We have investigated the effect of tyrosine sulfation of the chemokine receptor CCR2 on its interactions with the chemokine monocyte chemoattractant protein-1 (MCP-1/CCL2). Inhibition of CCR2 sulfation, by growth of expressing cells in the presence of sodium chlorate, significantly reduced the potency for MCP-1 activation of CCR2. MCP-1 exists in equilibrium between monomeric and dimeric forms. The obligate monomeric mutant MCP-1(P8A) was similar to wild type MCP-1 in its ability to induce leukocyte recruitment in vivo, whereas the obligate dimeric mutant MCP-1(T10C) was less effective at inducing leukocyte recruitment in vivo. In two-dimensional NMR experiments, sulfated peptides derived from the N-terminal region of CCR2 bound to both the monomeric and dimeric forms of wild type MCP-1 and shifted the equilibrium to favor the monomeric form. Similarly, MCP-1(P8A) bound more tightly than MCP-1(T10C) to the CCR2-derived sulfopeptides. NMR chemical shift mapping using the MCP-1 mutants showed that the sulfated N-terminal region of CCR2 binds to the same region (N-loop and ß3-strand) of both monomeric and dimeric MCP-1 but that binding to the dimeric form also influences the environment of chemokine N-terminal residues, which are involved in dimer formation. We conclude that interaction with the sulfated N terminus of CCR2 destabilizes the dimerization interface of inactive dimeric MCP-1, thus inducing dissociation to the active monomeric state.


Assuntos
Quimiocina CCL2/metabolismo , Regulação da Expressão Gênica , Receptores CCR2/química , Tirosina/química , Sítios de Ligação , Cálcio/metabolismo , Dimerização , Células HEK293 , Humanos , Cinética , Espectroscopia de Ressonância Magnética/métodos , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Modelos Químicos , Peptídeos/química , Fosforilação , Ligação Proteica , Processamento de Proteína Pós-Traducional , Receptores CCR2/fisiologia , Enxofre/química
14.
Chem Commun (Camb) ; 49(16): 1600-2, 2013 Feb 25.
Artigo em Inglês | MEDLINE | ID: mdl-23336102

RESUMO

The synthesis, structure and photophysical properties of a series of highly emissive europium complexes is reported. Certain complexes enter mammalian cells by macropinocytosis and stain the mitochondria selectively, allowing observation of the Eu emission in cellulo by time-gated spectral imaging.


Assuntos
Európio/análise , Európio/química , Mitocôndrias/química , Amilorida/farmacologia , Androstadienos/farmacologia , Animais , Camundongos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Modelos Moleculares , Estrutura Molecular , Células NIH 3T3 , Compostos Organometálicos/análise , Compostos Organometálicos/antagonistas & inibidores , Compostos Organometálicos/química , Temperatura , Wortmanina
15.
Chem Soc Rev ; 42(4): 1652-66, 2013 Feb 21.
Artigo em Inglês | MEDLINE | ID: mdl-22760156

RESUMO

Reversible anion binding at lanthanide centres in aqueous media has emerged as an effective means of signalling and sensing the presence of selected anions. The constitution and configuration of a wide range of anion adducts has been defined by X-ray analyses and NMR methods, and both chelating and monodentate binding modes characterised. Variation of the lanthanide ion modulates charge density, and ligand modification allows alteration of both the peripheral electrostatic gradient and the local steric demand at the metal centre. Thus, selectivity for a target anion can be engineered, and the affinity constant modulated to target the desired concentration range. Changes in anion concentration can be monitored rapidly, accurately and with high spatial resolution using optical emission spectroscopy and microscopy, facilitating the measurement of anions such as bicarbonate, lactate, citrate and urate in a variety of bio-fluids.

16.
Chem Asian J ; 7(11): 2621-8, 2012 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-22965665

RESUMO

The anion binding ability of a family of bis(Zn(II)-Dpa) functionalized cyclic peptides has been investigated using displacement assays with a fluorescent coumarin indicator in water, saline solution, and Krebs buffer. Non-binding side-chain steric bulk, the relative position of binding sites, and the scaffold size were all found to affect the ability of these receptors to discriminate between polyphosphate ions. Most receptors showed some selectivity for pyrophosphate over ATP and ADP in water and saline, and this selectivity was significantly enhanced in the biologically relevant Krebs buffer giving chemosensing ensembles capable of selective recognition of pyrophosphate in the presence of excess ATP.


Assuntos
Difosfatos/química , Peptídeos Cíclicos/química , Ânions/química , Difosfatos/metabolismo , Peptídeos Cíclicos/metabolismo , Ligação Proteica , Cloreto de Sódio/química , Zinco/química
17.
J Biol Chem ; 287(18): 14692-702, 2012 Apr 27.
Artigo em Inglês | MEDLINE | ID: mdl-22396538

RESUMO

Chemokine-receptor interactions regulate leukocyte trafficking during inflammation. CC chemokines exist in equilibrium between monomeric and dimeric forms. Although the monomers can activate chemokine receptors, dimerization is required for leukocyte recruitment in vivo, and it remains controversial whether dimeric CC chemokines can bind and activate their receptors. We have developed an obligate dimeric mutant of the chemokine monocyte chemoattractant protein-1 (MCP-1) by substituting Thr(10) at the dimer interface with Cys. Biophysical analysis showed that MCP-1(T10C) forms a covalent dimer with similar structure to the wild type MCP-1 dimer. Initial cell-based assays indicated that MCP-1(T10C) could activate chemokine receptor CCR2 with potency reduced 1 to 2 orders of magnitude relative to wild type MCP-1. However, analysis of size exclusion chromatography fractions demonstrated that the observed activity was due to a small proportion of MCP-1(T10C) being monomeric and highly potent, whereas the majority dimeric form could neither bind nor activate CCR2 at concentrations up to 1 µM. These observations help to reconcile previous conflicting results and indicate that dimeric CC chemokines do not bind to their receptors with affinities approaching those of the corresponding monomeric chemokines.


Assuntos
Quimiocina CCL2/metabolismo , Mutação de Sentido Incorreto , Multimerização Proteica , Substituição de Aminoácidos , Linhagem Celular , Quimiocina CCL2/química , Quimiocina CCL2/genética , Humanos , Receptores CCR2/química , Receptores CCR2/genética , Receptores CCR2/metabolismo
19.
Org Biomol Chem ; 9(9): 3471-83, 2011 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-21431170

RESUMO

We report here the design and synthesis of a family of novel backbone modified cyclic peptides, bearing dipicolylamine side chains for metal complexation and subsequent anion binding studies. Two approaches to the cyclic peptides were investigated. Initially, a stepwise approach was employed, involving solid-phase assembly of oxazole-based building blocks, followed by solution-phase macrolactamisation of the resulting linear precursor. The alternative strategy involved the formation of linear bisoxazole fragments in solution-phase, followed by a cyclodimerisation reaction. The zinc(II) complexes of these receptors bind selectively to di- and tri-phosphate ions over hydrogenphosphate.


Assuntos
Peptídeos Cíclicos/síntese química , Aminas/química , Aminoácidos/síntese química , Ânions/química , Ciclização , Dimerização , Lactamas Macrocíclicas/química , Estrutura Molecular , Oxazóis/química , Ácidos Picolínicos/química
20.
J Org Chem ; 74(8): 2992-6, 2009 Apr 17.
Artigo em Inglês | MEDLINE | ID: mdl-19292430

RESUMO

The design and synthesis of a diketopiperazine based anion receptor bearing two dipicolylamino arms complexed to zinc(II) ions is described. This receptor is readily prepared from the dipeptide precursor by a microwave-assisted intramolecular cyclization reaction. Upon addition of zinc(II), the receptor binds di- and triphosphate ions with high affinity and selectivity in aqueous solution, as determined by using a fluorescent indicator displacement assay.

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