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1.
Nat Commun ; 11(1): 81, 2020 01 03.
Artigo em Inglês | MEDLINE | ID: mdl-31900403

RESUMO

Live-cell Raman imaging based on bioorthogonal Raman probes with distinct signals in the cellular Raman-silent region (1800-2800 cm-1) has attracted great interest in recent years. We report here a class of water-soluble and biocompatible polydiacetylenes with intrinsic ultrastrong alkyne Raman signals that locate in this region for organelle-targeting live-cell Raman imaging. Using a host-guest topochemical polymerization strategy, we have synthesized a water-soluble and functionalizable master polydiacetylene, namely poly(deca-4,6-diynedioic acid) (PDDA), which possesses significantly enhanced (up to ~104 fold) alkyne vibration compared to conventional alkyne Raman probes. In addition, PDDA can be used as a general platform for multi-functional ultrastrong Raman probes. We achieve high quality live-cell stimulated Raman scattering imaging on the basis of modified PDDA. The polydiacetylene-based Raman probes represent ultrastrong intrinsic Raman imaging agents in the Raman-silent region (without any Raman enhancer), and the flexible functionalization of this material holds great promise for its potential diverse applications.


Assuntos
Células/citologia , Imagem Molecular/métodos , Sondas Moleculares/química , Polímero Poliacetilênico/química , Análise Espectral Raman/métodos , Alquinos/química , Células/química , Células HeLa , Humanos , Imagem Molecular/instrumentação , Sondas Moleculares/síntese química , Polímero Poliacetilênico/síntese química , Análise Espectral Raman/instrumentação
2.
Org Biomol Chem ; 17(37): 8601-8610, 2019 09 25.
Artigo em Inglês | MEDLINE | ID: mdl-31528884

RESUMO

Cholesterol is an essential and ubiquitous component in mammalian cell membranes. However, its distributions and interactions with phospholipids are often elusive, partly because chemical modifications for preparing cholesterol probes often cause significant perturbations in its membrane behavior. To overcome these problems, a 2H-labeled probe (24-d-cholesterol), which perfectly retained the original membrane properties, was synthesized by a stereoselective introduction of 2H into the side chain of cholesterol. A deuterium label at the side-chain more sensitively reflects membrane fluidity than the conventional labeling at the 3 position of a sterol core (3-d-cholesterol), thus providing 24-d-cholesterol with desirable properties to report membrane ordering. Solid state 2H NMR of 24-d-cholesterol with sphingomyelins (SM) and unsaturated phosphatidylcholine in the bilayer membranes clearly revealed the partitioning ratio of cholesterol in the raft-like liquid ordered (Lo) phase and the liquid disordered phase based on cholesterol interactions with surrounding lipids in each phase. This probe turned out to be superior to the widely used 3-d-cholesterol; e.g., 24-d-cholesterol clearly revealed a 10 mol% difference in the Lo distribution ratios of cholesterol between palmitoyl-SM and stearoyl-SM. The comprehensive use of 24-d-cholesterol in solid state 2H NMR will disclose the cholesterol-lipid interactions, distribution ratio of cholesterol, and membrane ordering in model bilayers as well as more complicated biological membranes.


Assuntos
Membrana Celular/química , Colesterol/química , Sondas Moleculares/química , Fosfolipídeos/química , Colesterol/síntese química , Conformação Molecular , Sondas Moleculares/síntese química
3.
Molecules ; 24(16)2019 Aug 16.
Artigo em Inglês | MEDLINE | ID: mdl-31426440

RESUMO

Matrix metalloproteinases (MMPs) are a family of zinc- and calcium-dependent endopeptidases which are secreted or anchored in the cell membrane and are capable of degrading the multiple components of the extracellular matrix (ECM). MMPs are frequently overexpressed or highly activated in numerous human diseases. Owing to the important role of MMPs in human diseases, many MMP inhibitors (MMPIs) have been developed as novel therapeutics, and some of them have entered clinical trials. However, so far, only one MMPI (doxycycline) has been approved by the FDA. Therefore, the evaluation of the activity of a specific subset of MMPs in human diseases using clinically relevant imaging techniques would be a powerful tool for the early diagnosis and assessment of the efficacy of therapy. In recent years, numerous MMPIs labeled imaging agents have emerged. This article begins by providing an overview of the MMP subfamily and its structure and function. The latest advances in the design of subtype selective MMPIs and their biological evaluation are then summarized. Subsequently, the potential use of MMPI-labeled diagnostic agents in clinical imaging techniques are discussed, including positron emission tomography (PET), single-photon emission computed tomography (SPECT) and optical imaging (OI). Finally, this article concludes with future perspectives and clinical utility.


Assuntos
Aterosclerose/diagnóstico por imagem , Doenças Cardiovasculares/diagnóstico por imagem , Pneumopatias/diagnóstico por imagem , Inibidores de Metaloproteinases de Matriz/farmacocinética , Metaloproteinases da Matriz/química , Sondas Moleculares/farmacocinética , Neoplasias/diagnóstico por imagem , Osteoartrite/diagnóstico por imagem , Animais , Aterosclerose/metabolismo , Aterosclerose/patologia , Doenças Cardiovasculares/metabolismo , Doenças Cardiovasculares/patologia , Domínio Catalítico/genética , Modelos Animais de Doenças , Encefalomielite Autoimune Experimental/diagnóstico por imagem , Encefalomielite Autoimune Experimental/metabolismo , Encefalomielite Autoimune Experimental/patologia , Matriz Extracelular/metabolismo , Matriz Extracelular/patologia , Humanos , Pneumopatias/metabolismo , Pneumopatias/patologia , Inibidores de Metaloproteinases de Matriz/síntese química , Metaloproteinases da Matriz/genética , Metaloproteinases da Matriz/metabolismo , Imagem Molecular/métodos , Sondas Moleculares/síntese química , Esclerose Múltipla/diagnóstico por imagem , Esclerose Múltipla/metabolismo , Esclerose Múltipla/patologia , Neoplasias/metabolismo , Neoplasias/patologia , Osteoartrite/metabolismo , Osteoartrite/patologia , Tomografia por Emissão de Pósitrons/métodos , Tomografia Computadorizada de Emissão de Fóton Único/métodos
4.
Chem Commun (Camb) ; 55(62): 9080-9083, 2019 Aug 11.
Artigo em Inglês | MEDLINE | ID: mdl-31287110

RESUMO

New strategies are required for the discovery of unknown bioactive molecules produced by gut microbiota in the human host. Herein, we utilize a chemoselective probe immobilized to magnetic beads for analysis of carbonyls in human fecal samples. We identified 112 metabolites due to femtomole analysis and an increased mass spectrometric sensitivity by up to six orders of magnitude.


Assuntos
Aldeídos/análise , Fezes/química , Microbioma Gastrointestinal/fisiologia , Cetonas/análise , Sondas Moleculares/análise , Sondas Moleculares/química , Aldeídos/metabolismo , Fezes/microbiologia , Feminino , Humanos , Cetonas/metabolismo , Masculino , Sondas Moleculares/síntese química , Estrutura Molecular , Tamanho da Partícula , Propriedades de Superfície
5.
Spectrochim Acta A Mol Biomol Spectrosc ; 223: 117335, 2019 Dec 05.
Artigo em Inglês | MEDLINE | ID: mdl-31288169

RESUMO

Luminogens with aggregation-induced emission (AIE) have been used to develop a new type of molecular probes based on analyte-triggered aggregation, but it still remains a challenge to design water-soluble AIE-active probe for specific detection of metal ions. Herein, we designed and synthesized a water-soluble molecular probe with AIE property for discriminative detection of aluminum ion and lead ion. Four carboxylic acid groups were incorporated into a tetraphenylethylene unit to enhance the coordination affinity and increase water-solubility in aqueous solution. The designed probe can be selectively lighted up by aluminum ion and lead ion via coordination-triggered AIE process. Discrimination of aluminum ion and lead ions based on the probe can be achieved in quantitative manner with the assistance of suitable masking reagents. This probe was further used to image aluminum ions in living cells of seedling roots of Arabidopsis, and the results showed that this probe is capable of imaging aluminum ions in living cells avoiding the interference of lead ions, and is suited for long-term imaging due to its excellent photostability. This work expands the application scope of AIE-active probes in discriminative detection of metal ions, and provides a design direction for water-soluble AIE probes to avoid the false signals from self-precipitation under physiological conditions.


Assuntos
Alumínio/análise , Arabidopsis/química , Chumbo/análise , Imagem Molecular , Sondas Moleculares/química , Raízes de Plantas/química , Plântula/química , Água/química , Sobrevivência Celular , Íons , Sondas Moleculares/síntese química , Solubilidade , Espectrometria de Fluorescência , Estilbenos/química
6.
Molecules ; 24(13)2019 Jul 04.
Artigo em Inglês | MEDLINE | ID: mdl-31277409

RESUMO

We report the development of a new colorimetric probe (L-ol) for investigations of the redox process regulated by hypochlorous acid (HOCl) and glutathione (GSH). The HOCl/GSH redox-switching cycle process was investigated in detail by UV-vis absorption spectroscopy, colorimetric analysis assay and high-resolution mass spectrometry (HRMS). The switchable absorbance responses were attributed to the HOCl-induced oxidation of the p-methoxyphenol unit to the benzoquinone derivative (L-one) and sequential reduction of L-one to hydroquinone (L-ol') by GSH. In phosphate-buffered saline (PBS) buffer, the absorbance of L-ol at 619 nm underwent a remarkable bathochromic-shift, accompanied by a color change from pale yellow to blue in the presence of HOCl. With further addition of GSH, the absorbance of L-one exclusively recovered to the original level. Meanwhile, the blue-colored solution returned to the naive pale yellow color in the presence of GSH. The detection limits for HOCl and GSH were calculated to be 6.3 and 96 nM according to the IUPAC criteria. Furthermore, L-ol-loaded chromatography plates have been prepared and successfully applied to visualize and quantitatively analyze HOCl in several natural waters.


Assuntos
Colorimetria/métodos , Glutationa/análise , Ácido Hipocloroso/análise , Benzoquinonas/química , Cor , Hidroquinonas/química , Sondas Moleculares/síntese química , Sondas Moleculares/química , Oxirredução , Espectrofotometria Ultravioleta , Fatores de Tempo , Água/química
7.
Nat Chem ; 11(7): 629-637, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31209299

RESUMO

In DNA, the loss of a nucleobase by hydrolysis generates an abasic site. Formed as a result of DNA damage, as well as a key intermediate during the base excision repair pathway, abasic sites are frequent DNA lesions that can lead to mutations and strand breaks. Here we present snAP-seq, a chemical approach that selectively exploits the reactive aldehyde moiety at abasic sites to reveal their location within DNA at single-nucleotide resolution. Importantly, the approach resolves abasic sites from other aldehyde functionalities known to exist in genomic DNA. snAP-seq was validated on synthetic DNA and then applied to two separate genomes. We studied the distribution of thymine modifications in the Leishmania major genome by enzymatically converting these modifications into abasic sites followed by abasic site mapping. We also applied snAP-seq directly to HeLa DNA to provide a map of endogenous abasic sites in the human genome.


Assuntos
DNA/genética , Genoma/genética , Análise de Sequência de DNA/métodos , Aldeídos/química , Sequência de Bases , DNA/química , Dano ao DNA/genética , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/genética , Técnicas de Silenciamento de Genes , Células HeLa , Humanos , Leishmania major/genética , Sondas Moleculares/síntese química , Sondas Moleculares/química , Timina/química , Uracila-DNA Glicosidase/química
8.
Molecules ; 24(9)2019 May 08.
Artigo em Inglês | MEDLINE | ID: mdl-31072045

RESUMO

A novel sensing system has been designed for the detection of cupric ions. It is based on the quenched fluorescence signal of carbon dots (CDs), which were carbonized from poly(vinylpyrrolidone) (PVP) and L-Cysteine (CYS). Cupric ions interact with the nitrogen and sulfur atoms on surface of the CDs to form an absorbed complex; this results in strong quenching of the fluorescence of the CDs via a fast metal-to-ligand binding affinity. The synthesized water-soluble CDs also exhibited a quantum yield of 7.6%, with favorable photoluminescent properties and good photostability. The fluorescence intensity of the CDs was very stable in high ionic strength (up to 1.0 M NaCl) and over a wide range of pH levels (2.0-12.0). This facile method can therefore develop a sensor that offers reliable, fast, and selective detection of cupric ions with a detection limit down to 0.15 µM and a linear range from 0.5 to 7.0 µM (R2 = 0.980). The CDs were used for cell imaging, observed that they were low toxicity to Tramp C1 cells and exhibited blue and green and red fluorescence under a fluorescence microscope. In summary, the CDs exhibited excellent fluorescence properties, and could be applied to the selective and sensitive detection of cupric ion and multicolor cell imaging.


Assuntos
Carbono/química , Cobre/análise , Imagem Tridimensional/métodos , Sondas Moleculares/síntese química , Pontos Quânticos/química , Animais , Carbono/toxicidade , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Fluorescência , Íons , Camundongos Transgênicos , Sondas Moleculares/química , Espectroscopia Fotoeletrônica , Pontos Quânticos/toxicidade , Espectrometria de Fluorescência , Espectrofotometria Ultravioleta , Espectroscopia de Infravermelho com Transformada de Fourier , Poluentes Químicos da Água/análise
9.
Org Biomol Chem ; 17(17): 4326-4334, 2019 04 24.
Artigo em Inglês | MEDLINE | ID: mdl-30976765

RESUMO

An unnatural monosaccharide with a C6-azide, Ac36AzGalNAc, has been developed as a potent and selective probe for O-GlcNAc-modified proteins. Combined with click chemistry, we demonstrate that Ac36AzGalNAc can robustly label O-GlcNAc glycosylation in a wide range of cell lines. Meanwhile, cell imaging and LC-MS/MS proteomics verify its selective activity on O-GlcNAc. More importantly, the protocol presented here provides a general methodology for tracking, capturing and identifying unnatural monosaccharide modified proteins in cells or cell lysates.


Assuntos
Galactosamina/química , Sondas Moleculares/química , N-Acetilglucosaminiltransferases/análise , beta-N-Acetil-Hexosaminidases/análise , Animais , Células Cultivadas , Galactosamina/análogos & derivados , Galactosamina/síntese química , Humanos , Camundongos , Sondas Moleculares/síntese química , Estrutura Molecular , N-Acetilglucosaminiltransferases/metabolismo , beta-N-Acetil-Hexosaminidases/metabolismo
10.
Anal Chim Acta ; 1061: 110-121, 2019 Jul 11.
Artigo em Inglês | MEDLINE | ID: mdl-30926029

RESUMO

Highly selective enrichment of N-linked glycopeptides and phosphopeptides from complex biological samples is extremely important prior to mass spectrometry analysis due to their low abundance as well as numerous extrinsic interferences. In this work, l-cysteine (L-Cys)-modified multifunctional metal-organic frameworks denoted as Fe3O4@PDA@MIL-125@Au@L-Cys (mMIL-125@Au@L-Cys) were prepared by modifications step by step. By combining hydrophilic interaction chromatography (HILIC) with metal oxide affinity chromatography (MOAC), the as-prepared material was firstly utilized to identify N-linked glycopeptides and phosphopeptides from tryptic digests of horseradish peroxidase (HRP) and beta-casein (ß-casein), respectively, with the help of matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) and exhibited outstanding sensitivity (0.1 fmol µL-1), great reusability (5 circles) and high selectivity (1: 100). Based on this, it was further applied into the enrichment of glycopeptides and phosphopeptides from tryptic digests of 100 µg human crystalline lens proteins. In the end, 81 N-linked glycopeptides corresponding to 35 glycoproteins and 175 phosphopeptides ascribed to 55 phosphorylated proteins were identified, respectively. The remarkable results were benefitted from the merits of improved hydrophilicity from L-Cys, strong affinity of TiO centers, numerous reaction sites on the large surface of MOFs and superparamagnetism from Fe3O4 cores. The design of mMIL-125@Au@L-Cys not only served as a multifunctional probe for efficient identification of N-linked glycopeptides and phosphopeptides in human crystalline lens, but also set a precedent for fabricating more MOFs with post-modifications for further proteomics research.


Assuntos
Cisteína/química , Glicopeptídeos/análise , Cristalino/química , Estruturas Metalorgânicas/química , Sondas Moleculares/química , Fosfopeptídeos/análise , Humanos , Interações Hidrofóbicas e Hidrofílicas , Estruturas Metalorgânicas/síntese química , Sondas Moleculares/síntese química , Tamanho da Partícula , Propriedades de Superfície
11.
Bioorg Chem ; 85: 505-514, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30802807

RESUMO

Human cathepsin L is a ubiquitously expressed endopeptidase and is known to play critical roles in a wide variety of cellular signaling events. Its overexpression has been implicated in numerous human diseases, including highly invasive forms of cancer. Inhibition of cathepsin L is therefore considered a viable therapeutic strategy. Unfortunately, several redundant and even opposing roles of cathepsin L have recently emerged. Selective cathepsin L probes are therefore needed to dissect its function in context-specific manner before significant resources are directed into drug discovery efforts. Herein, the development of a clickable and tagless activity-based probe of cathepsin L is reported. The probe is highly efficient, active-site directed and activity-dependent, selective, cell penetrable, and non-toxic to human cells. Using zebrafish model, we demonstrate that the probe can inhibit cathepsin L function in vivo during the hatching process. It is anticipated that the probe will be a highly effective tool in dissecting cathepsin L biology at the proteome levels in both normal physiology and human diseases, thereby facilitating drug-discovery efforts targeting cathepsin L.


Assuntos
Catepsina L/antagonistas & inibidores , Sondas Moleculares/farmacologia , Animais , Domínio Catalítico/efeitos dos fármacos , Catepsina L/química , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Química Click , Humanos , Sondas Moleculares/síntese química , Sondas Moleculares/toxicidade , Peixe-Zebra
12.
Nucleic Acids Res ; 47(6): 2727-2738, 2019 04 08.
Artigo em Inglês | MEDLINE | ID: mdl-30715502

RESUMO

Specific G-quadruplex-probing is crucial for both biological sciences and biosensing applications. Most reported probes are focused on fluorescent or colorimetric recognition of G-quadruplexes. Herein, for the first time, we reported a new specific G-quadruplex-probing technique-resonance light scattering (RLS)-based ratiometric recognition. To achieve the RLS probing of G-quadruplexes in the important physiological pH range of 7.4-6.0, four water soluble cationic porphyrin derivatives, including an unreported octa-cationic porphyrin, with large side arm substituents were synthesized and developed as RLS probes. These RLS probes were demonstrated to work well for ratiometric recognition of G-quadruplexes with high specificity against single- and double-stranded DNAs, including long double-stranded ones. The working mechanism was speculated to be based on the RLS signal changes caused by porphyrin protonation that was promoted by the end-stacking of porphyrins on G-quadruplexes. This work adds an important member in G-quadruplex probe family, thus providing a useful tool for studies on G-quadruplex-related events concerning G-quadruplex formation, destruction and changes in size, shape and aggregation. As a proof-of-concept example of applications, the RLS probes were demonstrated to work well for label-free and sequence-specific sensing of microRNA. This work also provides a simple and useful way for the preparation of cationic porphyrins with high charges.


Assuntos
Quadruplex G , Sondas Moleculares/síntese química , Ressonância Magnética Nuclear Biomolecular/métodos , Ácidos Nucleicos/análise , Porfirinas/síntese química , Sítios de Ligação , Técnicas Biossensoriais/métodos , Calorimetria/métodos , Cátions/síntese química , Cátions/química , Cátions/metabolismo , Dicroísmo Circular , Luz , Sondas Moleculares/química , Sondas Moleculares/metabolismo , Ácidos Nucleicos/isolamento & purificação , Ácidos Nucleicos/metabolismo , Imagem Óptica/métodos , Porfirinas/química , Porfirinas/metabolismo , Estrutura Secundária de Proteína , Espalhamento de Radiação
13.
Artigo em Inglês | MEDLINE | ID: mdl-30597436

RESUMO

Hydrogen sulfide (H2S) has been regarded as an important gas transmitter playing vital role in cytoprotective processes and redox signaling. It is very meaningful to monitor and analyze it in biosystem for obtaining important physiological and pathological information. Despite numerous fluorescent probes for cellular H2S have been reported in past decades, only a few have capability to detect mitochondrial H2S with near-infrared (NIR) emission. Therefore, a new mitochondria-targeting NIR fluorescent probe (Mito-NSH) for detection of cellular H2S was developed by introducing 2,4-dinitrophenyl ether into a novel dye (Mito-NOH). A large "turn-on" NIR fluorescence response was obtained due to thiolysis of ether to hydroxyl group when Mito-NSH was treated with NaHS. Moreover, Mito-NSH could quantitatively detect H2S at concentration ranging from 0 to 30 µM with a detection limit of 68.2 nM, and it exerts some superior optical properties, such as large stokes shift (107 nm), highly selectively mitochondria location, fast response and high selectivity to H2S. More impressively, it was successfully applied to imaging exogenous and endogenously generated H2S in living HeLa cells via confocal fluorescence microscopy.


Assuntos
Corantes Fluorescentes/química , Sulfeto de Hidrogênio/análise , Imagem Tridimensional , Mitocôndrias/metabolismo , Espectroscopia de Luz Próxima ao Infravermelho/métodos , Morte Celular , Corantes Fluorescentes/síntese química , Células HeLa , Humanos , Sondas Moleculares/síntese química , Sondas Moleculares/química , Espectrometria de Fluorescência
14.
Chembiochem ; 20(9): 1155-1160, 2019 05 02.
Artigo em Inglês | MEDLINE | ID: mdl-30600897

RESUMO

The mitochondrion is one of the most important organelles in the eukaryotic cell. Characterization of the mitochondrial proteome is a prerequisite for understanding its cellular functions at the molecular level. Here we report a proteomics method based on mitochondrion-targeting groups and click chemistry. In our strategy, three different mitochondrion-targeting moieties were each augmented with a clickable handle and a cysteine-reactive group. Fluorescence-based bioimaging and fractionation experiments clearly showed that most signals arising from the labels were localized in the mitochondria of cells, as a result of covalent attachment between probe and target proteins. The three probes had distinct profiling characteristics. Furthermore, we successfully identified more than two hundred mitochondrial proteins. The results showed that different mitochondrion-targeting groups targeted distinct proteins with partial overlap. Most of the labeled proteins were localized in the mitochondrial matrix and inner mitochondrial membrane. Our results provide a tool for chemoproteomic analysis of mitochondrion-related proteins.


Assuntos
Mitocôndrias/química , Proteínas Mitocondriais/análise , Sondas Moleculares/química , Proteoma/análise , Alquinos/síntese química , Alquinos/química , Cromatografia Líquida , Química Click , Corantes Fluorescentes/química , Células HeLa , Humanos , Proteínas Mitocondriais/química , Sondas Moleculares/síntese química , Oligopeptídeos/síntese química , Oligopeptídeos/química , Proteoma/química , Proteômica/métodos , Rodaminas/química , Espectrometria de Massas em Tandem
15.
Org Lett ; 21(3): 679-682, 2019 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-30645132

RESUMO

A convergent total synthesis of the siderophore coelichelin is described. The synthetic route also provided access to acetyl coelichelin and other congeners of the parent siderophore. The synthetic products were evaluated for their ability to bind ferric iron and promote growth of a siderophore-deficient strain of the Gram-negative bacterium Pseudomonas aeruginosa under iron restriction conditions. The results of these studies indicate coelichelin and several derivatives serve as ferric iron delivery vehicles for P. aeruginosa.


Assuntos
Ferro/metabolismo , Oligopeptídeos/síntese química , Oligopeptídeos/metabolismo , Pseudomonas aeruginosa/metabolismo , Sideróforos/síntese química , Sideróforos/metabolismo , Sondas Moleculares/síntese química , Sondas Moleculares/química , Sondas Moleculares/metabolismo , Sondas Moleculares/farmacologia , Oligopeptídeos/química , Oligopeptídeos/farmacologia , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/crescimento & desenvolvimento , Sideróforos/química , Sideróforos/farmacologia
16.
Bioorg Med Chem Lett ; 29(2): 148-154, 2019 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-30528696

RESUMO

The mitotic spindle is a microtubule-based machine that segregates a replicated set of chromosomes during cell division. Many cancer drugs alter or disrupt the microtubules that form the mitotic spindle. Microtubule-dependent molecular motors that function during mitosis are logical alternative mitotic targets for drug development. Eg5 (Kinesin-5) and Kif15 (Kinesin-12), in particular, are an attractive pair of motor proteins, as they work in concert to drive centrosome separation and promote spindle bipolarity. Furthermore, we hypothesize that the clinical failure of Eg5 inhibitors may be (in part) due to compensation by Kif15. In order to test this idea, we screened a small library of kinase inhibitors and identified GW108X, an oxindole that inhibits Kif15 in vitro. We show that GW108X has a distinct mechanism of action compared with a commercially available Kif15 inhibitor, Kif15-IN-1 and may serve as a lead with which to further develop Kif15 inhibitors as clinically relevant agents.


Assuntos
Inibidores Enzimáticos/farmacologia , Cinesina/antagonistas & inibidores , Sondas Moleculares/farmacologia , Oxindois/farmacologia , Quinazolinonas/farmacologia , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Humanos , Cinesina/metabolismo , Sondas Moleculares/síntese química , Sondas Moleculares/química , Estrutura Molecular , Oxindois/síntese química , Oxindois/química , Quinazolinonas/síntese química , Quinazolinonas/química , Relação Estrutura-Atividade
17.
Bioorg Med Chem ; 27(1): 224-229, 2019 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-30529151

RESUMO

Protein arginine methyltransferases (PRMTs) are a family of mammalian enzymes catalyzing the symmetric dimethylation (Type I), asymmetric dimethylation (Type II), or monomethylation (Type III) of arginine residues within proteins. This family is composed of 11 isozymes, however the vast majority of asymmetric and symmetric dimethylation in mammals is completed by either PRMT1 or PRMT5, respectively. In recent years, a number of chemical probes targeting this family of enzymes have been developed, but the majority of these probes lack isozyme specificity. Herein, we report the development of a chemical probe, based on a non-natural peptide sequence, which specifically labels PRMT1 over PRMT5 with high selectivity and sensitivity.


Assuntos
Isoenzimas/química , Sondas Moleculares/química , Peptídeos/química , Proteína-Arginina N-Metiltransferases/química , Sequência de Aminoácidos , Ensaios Enzimáticos , Isoenzimas/análise , Cinética , Limite de Detecção , Metilação , Sondas Moleculares/síntese química , Peptídeos/síntese química , Proteína-Arginina N-Metiltransferases/análise , Especificidade por Substrato
18.
Bioorg Med Chem Lett ; 29(3): 413-419, 2019 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-30587448

RESUMO

The voltage-gated sodium (Nav) channel is the molecular determinant of action potential in neurons. Protein-protein interactions (PPI) between the intracellular Nav1.6 C-tail and its regulatory protein fibroblast growth factor 14 (FGF14) provide an ideal and largely untapped opportunity for development of neurochemical probes. Based on a previously identified peptide FLPK, mapped to the FGF14:FGF14 PPI interface, we have designed and synthesized a series of peptidomimetics with the intent of increasing clogP values and improving cell permeability relative to the parental lead peptide. In-cell screening using the split-luciferase complementation (LCA) assay identified ZL0177 (13) as the most potent inhibitor of the FGF14:Nav1.6 channel complex assembly with an apparent IC50 of 11 µM. Whole-cell patch-clamp recordings demonstrated that ZL0177 significantly reduced Nav1.6-mediated transient current density and induced a depolarizing shift of the channel voltage-dependence of activation. Docking studies revealed strong interactions between ZL0177 and Nav1.6, mediated by hydrogen bonds, cation-π interactions and hydrophobic contacts. All together these results suggest that ZL0177 retains some key features of FGF14-dependent modulation of Nav1.6 currents. Overall, ZL0177 provides a chemical scaffold for developing Nav channel modulators as pharmacological probes with therapeutic potential of interest for a broad range of CNS and PNS disorders.


Assuntos
Fatores de Crescimento de Fibroblastos/antagonistas & inibidores , Sondas Moleculares/farmacologia , Canal de Sódio Disparado por Voltagem NAV1.6/metabolismo , Oligopeptídeos/farmacologia , Peptidomiméticos/farmacologia , Relação Dose-Resposta a Droga , Fatores de Crescimento de Fibroblastos/química , Fatores de Crescimento de Fibroblastos/metabolismo , Humanos , Sondas Moleculares/síntese química , Sondas Moleculares/química , Estrutura Molecular , Canal de Sódio Disparado por Voltagem NAV1.6/química , Oligopeptídeos/síntese química , Oligopeptídeos/química , Peptidomiméticos/síntese química , Peptidomiméticos/química , Ligação Proteica/efeitos dos fármacos , Relação Estrutura-Atividade
19.
J Am Chem Soc ; 140(49): 17060-17070, 2018 12 12.
Artigo em Inglês | MEDLINE | ID: mdl-30433779

RESUMO

The endoplasmic reticulum (ER) is an organelle that performs a variety of essential cellular functions via interactions with other organelles. Despite its important role, chemical tools for profiling the composition and dynamics of ER proteins remain very limited because of the labile nature of these proteins. Here, we developed ER-localizable reactive molecules (called ERMs) as tools for ER-focused chemical proteomics. ERMs can spontaneously localize in the ER of living cells and selectively label ER-associated proteins with a combined affinity and imaging tag, enabling tag-mediated ER protein enrichment and identification with liquid chromatography tandem mass spectrometry (LC-MS/MS). Using this method, we performed proteomic analysis of the ER of HeLa cells and newly assigned three proteins, namely, PAICS, TXNL1, and PPIA, as ER-associated proteins. The ERM probes could be used simultaneously with the nucleus- and mitochondria-localizable reactive molecules previously developed by our group, which enabled orthogonal organellar chemoproteomics in a single biological sample. Moreover, quantitative analysis of the dynamic changes in ER-associated proteins in response to tunicamycin-induced ER stress was performed by combining ER-specific labeling with SILAC (stable isotope labeling by amino acids in cell culture)-based quantitative MS technology. Our results demonstrated that ERM-based chemical proteomics provides a powerful tool for labeling and profiling ER-related proteins in living cells.


Assuntos
Retículo Endoplasmático/química , Sondas Moleculares/química , Proteoma/análise , Xantenos/química , Carboxiliases/análise , Carboxiliases/química , Cromatografia Líquida , Ciclofilina A/análise , Ciclofilina A/química , Retículo Endoplasmático/metabolismo , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Células HeLa , Humanos , Sondas Moleculares/síntese química , Enzimas Multifuncionais/análise , Enzimas Multifuncionais/química , Peptídeo Sintases/análise , Peptídeo Sintases/química , Proteoma/química , Proteômica/métodos , Espectrometria de Massas em Tandem , Tiorredoxinas/análise , Tiorredoxinas/química , Tunicamicina/farmacologia , Resposta a Proteínas não Dobradas/efeitos dos fármacos , Xantenos/síntese química
20.
J Am Chem Soc ; 140(49): 17120-17126, 2018 12 12.
Artigo em Inglês | MEDLINE | ID: mdl-30422648

RESUMO

The development of new bio-orthogonal ligation methods for the conjugation of native proteins is of particular importance in the field of chemical biology and biotherapies. In this work, we developed a traceless electrochemical method for protein bioconjugation. The electrochemically promoted tyrosine-click (e-Y-CLICK) allowed the chemoselective Y-modification of peptides and proteins with labeled urazoles. A low potential is applied in an electrochemical cell to activate urazole anchors in situ and on demand, without affecting the electroactive amino acids from the protein. The versatility of the electrosynthetic approach was shown on biologically relevant peptides and proteins such as oxytocin, angiotensin 2, serum bovine albumin, and epratuzumab. The fully conserved enzymatic activity of a glucose oxidase observed after e-Y-CLICK further highlights the softness of the method. The e-Y-CLICK protocols were successfully performed in pure aqueous buffers, without the need for co-solvents, scavenger or oxidizing chemicals, and should therefore significantly broaden the scope of bioconjugation.


Assuntos
Sondas Moleculares/química , Proteínas/química , Triazinas/química , Tirosina/química , Sequência de Aminoácidos , Animais , Aspergillus niger/enzimologia , Bovinos , Química Click/métodos , Técnicas Eletroquímicas/métodos , Glucose Oxidase/química , Humanos , Sondas Moleculares/síntese química , Triazinas/síntese química
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