RESUMEN
Small-molecule binding assays to target proteins are a core component of drug discovery and development. While a number of assay formats are available, significant drawbacks still remain in cost, sensitivity, and throughput. To improve assays by capitalizing on the power of DNA sequence analysis, we have developed an assay method that combines DNA encoding with split-and-pool sample handling. The approach involves affinity labeling of DNA-linked ligands to a protein target. Critically, the labeling event assesses ligand binding and enables subsequent pooling of several samples. Application of a purifying selection on the pool for protein-labeled DNAs allows detection of ligand binding by quantification of DNA barcodes. We demonstrate the approach in both ligand displacement and direct binding formats and demonstrate its utility in determination of relative ligand affinity, profiling ligand specificity, and high-throughput small-molecule screening.
Asunto(s)
Marcadores de Afinidad/química , Análisis de Secuencia de ADN/métodos , Bibliotecas de Moléculas Pequeñas/química , ADN/química , ADN/metabolismo , Colorantes Fluorescentes/química , Ensayos Analíticos de Alto Rendimiento/métodos , Ligandos , Unión Proteica , Proteínas/química , Proteínas/metabolismo , Bibliotecas de Moléculas Pequeñas/metabolismoRESUMEN
The mammalian membrane-bound O-acyltransferase (MBOAT) superfamily is involved in biological processes including growth, development and appetite sensing. MBOATs are attractive drug targets in cancer and obesity; however, information on the binding site and molecular mechanisms underlying small-molecule inhibition is elusive. This study reports rational development of a photochemical probe to interrogate a novel small-molecule inhibitor binding site in the human MBOAT Hedgehog acyltransferase (HHAT). Structure-activity relationship investigation identified single enantiomer IMP-1575, the most potent HHAT inhibitor reported to-date, and guided design of photocrosslinking probes that maintained HHAT-inhibitory potency. Photocrosslinking and proteomic sequencing of HHAT delivered identification of the first small-molecule binding site in a mammalian MBOAT. Topology and homology data suggested a potential mechanism for HHAT inhibition which was confirmed by kinetic analysis. Our results provide an optimal HHAT tool inhibitor IMP-1575 (Ki =38â nM) and a strategy for mapping small molecule interaction sites in MBOATs.
Asunto(s)
Acetiltransferasas/antagonistas & inhibidores , Marcadores de Afinidad/química , Bibliotecas de Moléculas Pequeñas/química , Acetiltransferasas/metabolismo , Sitios de Unión , Humanos , Cinética , Luz , Palmitoil Coenzima A/antagonistas & inhibidores , Palmitoil Coenzima A/metabolismo , Piridinas/química , Piridinas/metabolismo , Bibliotecas de Moléculas Pequeñas/metabolismo , Relación Estructura-ActividadRESUMEN
Membrane proteins are important drug targets; however, ligand discovery for membrane proteins is highly challenging due to their hydrophobic nature. We show that membrane proteins may be specifically labelled with a DNA tag by DNA-programmed affinity labelling (DPAL), thereby enabling the screening of chemical compounds against membrane proteins directly on live cells.
Asunto(s)
Marcadores de Afinidad/química , ADN/análisis , Proteínas de la Membrana/antagonistas & inhibidores , Bibliotecas de Moléculas Pequeñas/farmacología , Células HeLa , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Ligandos , Estructura Molecular , Bibliotecas de Moléculas Pequeñas/químicaRESUMEN
Identification of physiologically relevant targets for lead compounds emerging from drug discovery screens is often the rate-limiting step toward understanding their mechanism of action and potential for undesired off-target effects. To this end, we developed a streamlined chemical proteomic approach utilizing a single, photoreactive cleavable chloroalkane capture tag, which upon attachment to bioactive compounds facilitates selective isolation of their respective cellular targets for subsequent identification by mass spectrometry. When properly positioned, the tag does not significantly affect compound potency and membrane permeability, allowing for binding interactions with the tethered compound (probe) to be established within intact cells under physiological conditions. Subsequent UV-induced covalent photo-cross-linking "freezes" the interactions between the probe and its cellular targets and prevents their dissociation upon cell lysis. Targets cross-linked to the capture tag are then efficiently enriched through covalent capture onto HaloTag coated beads and subsequent selective chemical release from the solid support. The tag's built-in capability for selective enrichment eliminates the need for ligation of a capture tag, thereby simplifying the workflow and reducing variability introduced through additional operational steps. At the same time, the capacity for adequate cross-linking without structural optimization permits modular assembly of photoreactive chloroalkane probes, which reduces the burden of customized chemistry. Using three model compounds, we demonstrate the capability of this approach to identify known and novel cellular targets, including those with low affinity and/or low abundance as well as membrane targets with several transmembrane domains.
Asunto(s)
Marcadores de Afinidad/química , Azidas/química , Reactivos de Enlaces Cruzados/química , Diazometano/análogos & derivados , Hidrocarburos Clorados/química , Proteómica/métodos , Marcadores de Afinidad/efectos de la radiación , Azidas/efectos de la radiación , Cromatografía Liquida , Reactivos de Enlaces Cruzados/efectos de la radiación , Dasatinib/análogos & derivados , Dasatinib/farmacología , Dasatinib/efectos de la radiación , Diazometano/efectos de la radiación , Histona Desacetilasas/análisis , Histona Desacetilasas/química , Humanos , Hidrocarburos Clorados/efectos de la radiación , Hidrolasas/química , Células K562 , Espectrometría de Masas , Propranolol/análogos & derivados , Propranolol/farmacología , Propranolol/efectos de la radiación , Proteínas Quinasas/análisis , Proteínas Quinasas/química , Receptores Adrenérgicos alfa 2/análisis , Receptores Adrenérgicos alfa 2/química , Rayos Ultravioleta , Vorinostat/análogos & derivados , Vorinostat/farmacología , Vorinostat/efectos de la radiaciónRESUMEN
The reversible interaction between an affinity ligand and a complementary receptor has been widely explored in purification systems for several biomolecules. The development of tailored affinity ligands highly specific toward particular target biomolecules is one of the options in affinity purification systems. However, both genetic and chemical modifications in proteins and peptides widen the application of affinity ligand-tag receptors pairs toward universal capture and purification strategies. In particular, this chapter will focus on two case studies highly relevant for biotechnology and biomedical areas, namely the affinity tags and receptors employed on the production of recombinant fusion proteins, and the chemical modification of phosphate groups on proteins and peptides and the subsequent specific capture and enrichment, a mandatory step before further proteomic analysis.
Asunto(s)
Marcadores de Afinidad/química , Cromatografía de Afinidad , Proteínas Recombinantes de Fusión , Biotecnología , Proteómica , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/aislamiento & purificaciónRESUMEN
GTP-binding proteins are among the most important enzyme families that are involved in a plethora of biological processes. However, owing to the enormous diversity of the nucleotide-binding protein family, comprehensive analyses of the expression level, structure, activity, and regulatory mechanisms of GTP-binding proteins remain challenging with the use of conventional approaches. The many advances in mass spectrometry (MS) instrumentation and data acquisition methods, together with a variety of enrichment approaches in sample preparation, render MS a powerful tool for the comprehensive characterizations of the activities and expression levels of various GTP-binding proteins. We review herein the recent developments in the application of MS-based techniques, together with general and widely used affinity enrichment approaches, for the proteome-wide and targeted capture, identification, and quantification of GTP-binding proteins. The working principles, advantages, and limitations of various strategies for profiling the expression level, activity, posttranslational modifications, and interactome of GTP-binding proteins are discussed. It can be envisaged that future applications of MS-based proteomics will lead to a better understanding about the roles of GTP-binding proteins in different biological processes and human diseases. © 2020 John Wiley & Sons Ltd. Mass Spec Rev.
Asunto(s)
Proteínas de Unión al GTP/análisis , Proteínas de Unión al GTP/metabolismo , Espectrometría de Masas/métodos , Proteómica/métodos , Marcadores de Afinidad/química , Animales , Biotinilación , Electroforesis/métodos , Proteínas de Unión al GTP/química , Proteínas de Unión al GTP/aislamiento & purificación , Guanina/química , Humanos , Procesamiento Proteico-PostraduccionalRESUMEN
In vitro and ex vivo development of novel therapeutic agents requires reliable and accurate analyses of the cell conditions they were preclinical tested for, such as apoptosis. The detection of apoptotic cells by annexin V (AV) coupled to fluorophores has often shown limitations in the choice of the dye due to interference with other fluorescent-labeled cell markers. The SNAP-tag technology is an easy, rapid and versatile method for functionalization of proteins and was therefore used for labeling AV with various fluorophores. We generated the fusion protein AV-SNAP and analyzed its capacity for the specific display of apoptotic cells in various assays with therapeutic agents. AV-SNAP showed an efficient coupling reaction with five different fluorescent dyes. Two selected fluorophores were tested with suspension, adherent and peripheral blood cells, treated by heat-shock or apoptosis-inducing therapeutic agents. Flow cytometry analysis of apoptotic cells revealed a strong visualization using AV-SNAP coupled to these two fluorophores exemplary, which was comparable to a commercial AV-Assay-kit. The combination of the apoptosis-specific binding protein AV with the SNAP-tag provides a novel solid method to facilitate protein labeling using several, easy to change, fluorescent dyes at once. It avoids high costs and allows an ordinary exchange of dyes and easier use of other fluorescent-labeled cell markers, which is of high interest for the preclinical testing of therapeutic agents in e.g. cancer research.
Asunto(s)
Apoptosis/fisiología , Colorantes Fluorescentes/química , Coloración y Etiquetado/métodos , Marcadores de Afinidad/química , Anexina A5/química , Anexina A5/metabolismo , Células Sanguíneas/metabolismo , Línea Celular Tumoral , Citometría de Flujo/métodos , Humanos , Neoplasias/metabolismo , Proteínas/química , Proteínas/metabolismo , TecnologíaRESUMEN
The inositol pyrophosphates (PP-InsPs) are a ubiquitous group of highly phosphorylated eukaryotic messengers. They have been linked to a panoply of central cellular processes, but a detailed understanding of the discrete signaling events is lacking in most cases. To create a more mechanistic picture of PP-InsP signaling, we sought to annotate the mammalian interactome of the most abundant inositol pyrophosphate 5PP-InsP5. To do so, triplexed affinity reagents were developed, in which a metabolically stable PP-InsP analog was immobilized in three different ways. Application of these triplexed reagents to mammalian lysates identified between 300 and 400 putative interacting proteins. These interactomes revealed connections between 5PP-InsP5 and central cellular regulators, such as lipid phosphatases, protein kinases, and GTPases, and identified protein domains commonly targeted by 5PP-InsP5. Both the triplexed affinity reagents, and the proteomic datasets, constitute powerful resources for the community, to launch future investigations into the multiple signaling modalities of inositol pyrophosphates.
Asunto(s)
Marcadores de Afinidad/química , GTP Fosfohidrolasas/metabolismo , Fosfatos de Inositol/metabolismo , Monoéster Fosfórico Hidrolasas/metabolismo , Proteínas Quinasas/metabolismo , Cromatografía Líquida de Alta Presión , GTP Fosfohidrolasas/química , Células HCT116 , Células HEK293 , Humanos , Fosfatos de Inositol/química , Monoéster Fosfórico Hidrolasas/química , Unión Proteica , Dominios Proteicos , Proteínas Quinasas/química , Proteoma/análisis , Transducción de Señal , Espectrometría de Masas en TándemRESUMEN
Histone deacetylase (HDAC) is a major class of deacetylation enzymes. Many HDACs exist in large protein complexes in cells and their functions strongly depend on the complex composition. The identification of HDAC-associated proteins is highly important in understanding their molecular mechanisms. Although affinity probes have been developed to study HDACs, they were mostly targeting the direct binder HDAC, while other proteins in the complex remain underexplored. We report a DNA-based affinity labeling method capable of presenting different probe configurations without the need for preparing multiple probes. Using one binding probe, 9 probe configurations were created to profile HDAC complexes. Notably, this method identified indirect HDAC binders that may be inaccessible to traditional affinity probes, and it also revealed new biological implications for HDAC-associated proteins. This study provided a simple and broadly applicable method for characterizing protein-protein interactions.
Asunto(s)
Marcadores de Afinidad/química , ADN/química , Histona Desacetilasas/metabolismo , Acetilación , ADN/metabolismo , Células HeLa , Inhibidores de Histona Desacetilasas/química , Inhibidores de Histona Desacetilasas/metabolismo , Histona Desacetilasas/química , Humanos , Luz , Unión Proteica , Isoformas de Proteínas/metabolismoRESUMEN
Here, we describe a new immersion-based clearing method suitable for optical clearing of thick adult human brain samples while preserving its lipids and lipophilic labels such as 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI). This clearing procedure is simple, easy to implement, and allowed for clearing of 5 mm thick human brain tissue samples within 12 days. Furthermore, we show for the first time the advantageous effect of the Periodate-Lysine-Paraformaldehyde (PLP) fixation as compared to the more commonly used 4% paraformaldehyde (PFA) on clearing performance.
Asunto(s)
Encéfalo/citología , Fijación del Tejido/métodos , Marcadores de Afinidad/química , Animales , Encéfalo/anatomía & histología , Carbocianinas/química , Colorantes Fluorescentes/química , Formaldehído/química , Humanos , Lípidos/química , Lisina/química , Ratones , Ácido Peryódico/química , PorcinosRESUMEN
The inert chemical property of RNA modification N6-methyladenosine (m6A) makes it very challenging to detect. Most m6A sequencing methods rely on m6A-antibody immunoprecipitation and cannot distinguish m6A and N6,2'-O-dimethyladenosine modification at the cap +1 position (cap m6Am). Although the two antibody-free methods (m6A-REF-seq/MAZTER-seq and DART-seq) have been developed recently, they are dependent on m6A sequence or cellular transfection. Here, we present an antibody-free, FTO-assisted chemical labeling method termed m6A-SEAL for specific m6A detection. We applied m6A-SEAL to profile m6A landscapes in humans and plants, which displayed the known m6A distribution features in transcriptome. By doing a comparison with all available m6A sequencing methods and specific m6A sites validation by SELECT, we demonstrated that m6A-SEAL has good sensitivity, specificity and reliability for transcriptome-wide detection of m6A. Given its tagging ability and FTO's oxidation property, m6A-SEAL enables many applications such as enrichment, imaging and sequencing to drive future functional studies of m6A and other modifications.
Asunto(s)
Adenosina/análogos & derivados , Marcadores de Afinidad/química , Adenosina/análisis , Adenosina/química , Dioxigenasa FTO Dependiente de Alfa-Cetoglutarato/química , Dioxigenasa FTO Dependiente de Alfa-Cetoglutarato/metabolismo , Humanos , Inmunoprecipitación/métodos , Metilación , ARN/química , ARN Mensajero/química , ARN Mensajero/metabolismo , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , TranscriptomaRESUMEN
Biochemical, biophysical, and structural studies of membrane proteins rely on the availability of highly pure and monodisperse membrane protein samples. One of the most powerful methods for isolation of the membrane protein of interest is affinity purification. This methodology typically relies on engineering an affinity tag into the protein of interest and an affinity resin that specifically recognizes the tag, allowing one to purify the target protein in a single step. In some cases, the affinity purification procedure is combined with additional steps to increase the purity and homogeneity of the final protein sample. Here, we describe several protocols for affinity purification of TSPO, a small membrane protein. The techniques we use include immobilized metal affinity chromatography (IMAC) and strep-II tag-based streptavidin affinity chromatography.
Asunto(s)
Cromatografía de Afinidad/métodos , Proteínas de la Membrana/aislamiento & purificación , Proteínas Recombinantes/aislamiento & purificación , Marcadores de Afinidad/química , Secuencia de Aminoácidos , Animales , Detergentes/química , Detergentes/farmacología , Escherichia coli , Células Eucariotas , Histidina/química , Humanos , Insectos , Resinas de Intercambio Iónico/química , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Oligopéptidos/química , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Solubilidad/efectos de los fármacos , Estreptavidina/químicaRESUMEN
The Car9 affinity tag is a dodecameric silica-binding peptide that can be fused to the N- and C-termini of proteins of interest to enable their rapid and inexpensive purification on underivatized silica in a process that typically relies on l-lysine as an eluent. Here, we show that silica paper spin columns and borosilicate multi-well plates used for plasmid DNA purification are suitable for recovering Car9-tagged proteins with high purity in a workflow compatible with high-throughput experiments. Spin columns typically yield 100 µg of biologically active material that can be recovered in minutes with low concentrations of lysine. Because of their short bed length, spin columns also offer unique advantages, as evidenced by the selective recovery of functional Car9-tagged tobacco etch virus (TEV) protease from a fused and auto-cleaved maltose binding protein (MBP) folding partner that nonspecifically binds to silica in the presence of NaCl. These additional purification modalities should increase the versatility and appeal of the Car9 tag for affinity protein purification.
Asunto(s)
Cromatografía de Afinidad/métodos , Endopeptidasas/aislamiento & purificación , Proteínas de Unión a Maltosa/aislamiento & purificación , Péptidos/química , Plásmidos/metabolismo , Dióxido de Silicio/química , Marcadores de Afinidad/química , Cromatografía de Afinidad/instrumentación , Clonación Molecular , Endopeptidasas/genética , Endopeptidasas/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Proteínas de Unión a Maltosa/genética , Proteínas de Unión a Maltosa/metabolismo , Péptidos/metabolismo , Plásmidos/química , Unión Proteica , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Cloruro de Sodio/química , Coloración y Etiquetado/métodosRESUMEN
N-glycans are involved in a variety of biological processes and associated with many diseases. However, the sensitive and specific detection of glycans via mass spectrometry (MS) remains challenging due to their relatively low abundance and low ionization efficiency. In this work, a new method termed glycan reductive amino acid coded affinity tagging (GRACAT) is developed to address these problems. Through labeling the reducing ends of glycan with a biotinylated arginine, the ionization efficiency of glycan was increased nearly 50-fold and glycan isomers were discriminated by improved signals of fragments in tandem spectra. Moreover, the strong affinity interaction between streptavidin and biotin enabled highly specific enrichment of the biotin-tagged glycans from the mixture of proteins and peptides. We successfully applied GRACAT in the profiling of N-glycome in human serum and bovine milk, in which a total of 55 and 67â¯N-glycans were detected respectively.
Asunto(s)
Marcadores de Afinidad/química , Glicómica/métodos , Leche/química , Polisacáridos/sangre , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Animales , Biotina/análogos & derivados , Bovinos , Dipéptidos/química , Humanos , Extracción en Fase Sólida , Estreptavidina/químicaRESUMEN
The CDK family plays a crucial role in the control of the cell cycle. Dysregulation and mutation of the CDKs has been implicated in cancer and the CDKs have been investigated extensively as potential therapeutic targets. Selective inhibition of specific isoforms of the CDKs is crucial to achieve therapeutic effect while minimising toxicity. We present a group of photoaffinity probes designed to bind to the family of CDKs. The site of crosslinking of the optimised probe, as well as its ability to enrich members of the CDK family from cell lysates, was investigated. In a proof of concept study, we subsequently developed a photoaffinity probe-based competition assay to profile CDK inhibitors. We anticipate that this approach will be widely applicable to the study of small molecule binding to protein families of interest.
Asunto(s)
Marcadores de Afinidad/química , Antineoplásicos/química , Reactivos de Enlaces Cruzados/química , Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Isoformas de Proteínas/química , Inhibidores de Proteínas Quinasas/química , Unión Competitiva , Ensayos de Selección de Medicamentos Antitumorales , Espectrometría de Masas , Estructura Molecular , Procesos Fotoquímicos , Roscovitina , Relación Estructura-ActividadRESUMEN
In the present study, EMA (ethidium monoazide) treatment was applied to a silty-sand reference soil prior to DNA extraction to enable a differentiation between dead and living cells. For this purpose, a reference soil was spiked with Listeria monocytogenes cells or cell equivalents, respectively. With the purpose of evaluating optimum treatment conditions, different EMA concentrations have been tested. However, the results remained largely inconclusive. Furthermore, varied dark incubation periods allowing EMA to penetrate dead cells did not allow the selective removal of DNA from membrane-compromised cells in downstream analyses. In contrast to undiluted soil, an effect of EMA treatment during DNA extraction could be observed when using a 1:10 dilution of the reference soil; however, the effect has not been sufficiently selective to act on heat-treated cells only. Although the application of EMA to soil requires further evaluation, the procedure harbors future potential for improving DNA-based approaches in microbial ecology studies.
Asunto(s)
Marcadores de Afinidad/química , Azidas/química , Técnicas Bacteriológicas/métodos , ADN Bacteriano/química , Listeria monocytogenes/fisiología , Viabilidad Microbiana , Recuento de Colonia Microbiana , ADN Bacteriano/genética , ADN Bacteriano/aislamiento & purificación , Listeria monocytogenes/genética , Listeria monocytogenes/aislamiento & purificación , Reacción en Cadena de la Polimerasa , Microbiología del SueloRESUMEN
Reported herein is a relebactam-derived fluorogenic reagent for covalent labeling of serine ß-lactamases (SBLs), which are the major causes of bacterial resistance to ß-lactam antibiotics. This highly selective imaging reagent generates over 300-fold stronger near-infrared fluorescence signals upon covalently bonding to SBLs, allowing wash-free visualization of live antimicrobial-resistant bacteria.
Asunto(s)
Marcadores de Afinidad/farmacología , Compuestos de Azabiciclo/farmacología , Enterobacter cloacae/aislamiento & purificación , Colorantes Fluorescentes/farmacología , Inhibidores de beta-Lactamasas/farmacología , beta-Lactamasas/química , Marcadores de Afinidad/síntesis química , Marcadores de Afinidad/química , Compuestos de Azabiciclo/síntesis química , Compuestos de Azabiciclo/química , Enterobacter cloacae/enzimología , Fluoresceínas/síntesis química , Fluoresceínas/química , Fluoresceínas/farmacología , Colorantes Fluorescentes/síntesis química , Colorantes Fluorescentes/química , Indoles/síntesis química , Indoles/química , Indoles/farmacología , Inhibidores de beta-Lactamasas/síntesis química , Inhibidores de beta-Lactamasas/químicaRESUMEN
To identify a potent and selective σ2 receptor ligand appropriate for development as a positron emission tomography (PET) tracer, several fluorinated analogues of the spirocyclic lead compounds trans- and cis-6 (N-(2,4-dimethylbenzyl)-3-methoxy-3,4-dihydrospiro[[2]benzopyran-1,1'-cyclohexan]-4'-amine) were designed. In multistep syntheses, a fluorine atom was introduced directly or as a 2-fluoroethoxy moiety on the 2-benzopyran scaffold, on the dimethylbenzylamino moiety, or on the central amino moiety. The σ1 and σ2 receptor affinity was determined in receptor binding studies with radioligands. With respect to σ2 affinity and σ2 /σ1 selectivity, cis-N-(2,4-dimethylbenzyl)-5-fluoro-3-methoxy-3,4-dihydrospiro[[2]benzopyran-1,1'-cyclohexan]-4'-amine (cis-15 c, Ki (σ2 )=51â nm) and cis-N-[4-(fluoromethyl)-2-methylbenzyl]-3-methoxy-3,4-dihydrospiro[[2]benzopyran-1,1'-cyclohexan]-4'-amine (cis-28 e, Ki (σ2 )=57â nm) are the most promising ligands. The combination of both structural elements in one molecule, cis-N-[4-(fluoromethyl)-2-methylbenzyl]-5-fluoro-3-methoxy-3,4-dihydrospiro[[2]benzopyran-1,1'-cyclohexan]-4'-amine (cis-28 c: Ki (σ2 )=874â nm), resulted in decreased σ2 and σ1 affinity. Methylation of secondary amines led to three tertiary methylamines with moderate affinity for both σ receptor subtypes.
Asunto(s)
Bencilaminas/química , Plomo/química , Receptores sigma/química , Compuestos de Espiro/química , Marcadores de Afinidad/química , Benzopiranos/química , Halogenación , Isomerismo , Ligandos , Estructura Molecular , Tomografía de Emisión de Positrones , Unión Proteica , Coloración y Etiquetado , Relación Estructura-ActividadRESUMEN
Advances in biocompatible organic chemistry applicable for endogenous protein modification under live-cell conditions have been longed as these can produce an important tool for the elucidation of a variety of biological phenomena. However, there are still various obstacles to be overcome, such as the limited repertories of the reaction modes, the slow reaction kinetics, and the insufficient specificity for endogenous protein modification. We have recently reported a new type of affinity-based labeling technique termed ligand-directed (LD) chemistry that does not need any genetic manipulation, which shows a sharp contrast with other strategies including peptide/enzyme-tag methods or bioorthogonal chemistry-based methods. Here we describe the general principles of LD chemistry using N-sulfonyl pyridone (SP) as a new reactive group (LDSP chemistry) that allows for endogenous protein sulfonylation with the higher labeling rate and specificity, relative to our previously reported LD chemistry on the surface of and the inside of live cells. The detailed protocols of LDSP chemistry for carbonic anhydrase labeling and imaging in vitro and in living cells are explained.
Asunto(s)
Marcadores de Afinidad/química , Piridonas/química , Coloración y Etiquetado/métodos , Humanos , Células MCF-7 , Microscopía FluorescenteRESUMEN
The divergent sequences, protein structures, and catalytic mechanisms of serine- and metallo-ß-lactamases hamper the development of wide-spectrum ß-lactamase inhibitors that can block both types of enzymes. The O-aryloxycarbonyl hydroxamate inactivators of Enterobacter cloacae P99 class C serine-ß-lactamase are unusual covalent inhibitors in that they target both active-site Ser and Lys residues, resulting in a cross-link consisting of only two atoms. Many clinically relevant metallo-ß-lactamases have an analogous active-site Lys residue used to bind ß-lactam substrates, suggesting a common site to target with covalent inhibitors. Here, we demonstrate that an O-aryloxycarbonyl hydroxamate inactivator of serine-ß-lactamases can also serve as a classical affinity label for New Delhi metallo-ß-lactamase-1 (NDM-1). Rapid dilution assays, site-directed mutagenesis, and global kinetic fitting are used to map covalent modification at Lys211 and determine KI (140 µM) and kinact (0.045 min-1) values. Mass spectrometry of the intact protein and the use of ultraviolet photodissociation for extensive fragmentation confirm stoichiometric covalent labeling that occurs specifically at Lys211. A 2.0 Å resolution X-ray crystal structure of inactivated NDM-1 reveals that the covalent adduct is bound at the substrate-binding site but is not directly coordinated to the active-site zinc cluster. These results indicate that Lys-targeted affinity labels might be a successful strategy for developing compounds that can inactivate both serine- and metallo-ß-lactamases.