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1.
Anal Biochem ; 489: 1-8, 2015 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-26278171

RESUMO

Ligand-mediated endocytosis is a key autoregulatory mechanism governing the duration and intensity of signals emanating from cell surface receptors. Due to the mechanistic complexity of endocytosis and its emerging relevance in disease, simple methods capable of tracking this dynamic process in cells have become increasingly desirable. We have developed a bioluminescent reporter technology for real-time analysis of ligand-mediated receptor endocytosis using genetic fusions of NanoLuc luciferase with various G-protein-coupled receptors (GPCRs). This method is compatible with standard microplate formats, which should decrease work flows for high-throughput screens. This article also describes the application of this technology to endocytosis of epidermal growth factor receptor (EGFR), demonstrating potential applicability of the method beyond GPCRs.


Assuntos
Proteínas de Artrópodes/metabolismo , Endocitose , Ensaios de Triagem em Larga Escala/métodos , Luciferases/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Animais , Proteínas de Artrópodes/química , Proteínas de Artrópodes/genética , Membrana Celular/efeitos dos fármacos , Membrana Celular/enzimologia , Membrana Celular/metabolismo , Descoberta de Drogas/métodos , Endocitose/efeitos dos fármacos , Corantes Fluorescentes/química , Genes Reporter/efeitos dos fármacos , Células HEK293 , Humanos , Interleucina-6/química , Interleucina-6/genética , Interleucina-6/metabolismo , Cinética , Ligantes , Luciferases/química , Luciferases/genética , Microscopia Confocal , Microscopia de Fluorescência , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Sinais Direcionadores de Proteínas/efeitos dos fármacos , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/genética , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo
2.
Drug Metab Dispos ; 39(12): 2403-10, 2011 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-21890735

RESUMO

Cytochrome P450 (P450) assays use probe substrates to interrogate the influence of new chemical entities toward P450 enzymes. We report the synthesis and study of a family of bioluminogenic luciferin acetal substrates that are oxidized by P450 enzymes to form luciferase substrates. The luciferin acetals were screened against a panel of purified P450 enzymes. In particular, one proluciferin acetal has demonstrated sensitive and selective CYP3A4-catalyzed oxidation to a luciferin ester-K(m) and k(cat) are 2.88 µM and 5.87 pmol metabolite · min(-1) · pmol enzyme(-1), respectively. The proluciferin acetal was used as a probe substrate to measure IC(50) values of known inhibitors against recombinant CYP3A4 or human liver microsomes. IC(50) values for the known inhibitors correlate strongly with IC(50) values calculated from the traditional high-performance liquid chromatography-based probe substrate testosterone. Luciferin acetals are rapidly oxidized to unstable hemi-orthoesters by CYP3A resulting in luciferin esters and, therefore, are conducive to simple rapid CYP3A bioluminescent assays.


Assuntos
Citocromo P-450 CYP3A/metabolismo , Luciferina de Vaga-Lumes/metabolismo , Cromatografia Líquida de Alta Pressão , Inibidores do Citocromo P-450 CYP3A , Humanos , Concentração Inibidora 50 , Microssomos Hepáticos/enzimologia , Sondas Moleculares , Especificidade por Substrato
3.
Bioconjug Chem ; 20(7): 1367-74, 2009 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-19514716

RESUMO

In vivo fluorescence cancer imaging is an important tool in understanding tumor growth and therapeutic monitoring and can be performed either with endogenously produced fluorescent proteins or with exogenously introduced fluorescent probes bound to targeting molecules. However, endogenous fluorescence proteins cannot be altered after transfection, thus requiring rederivation of cell lines for each desired color, while exogenously targeted fluorescence probes are limited by the heterogeneous expression of naturally occurring cellular targets. In this study, we adapted the dehalogenase-based protein-Tag (HaloTag) system to in vivo cancer imaging, by introducing highly expressed HaloTag receptors (HaloTagR) in cancer cells coupled with a range of externally injected fluorophore-conjugated dehalogenase-reactive reactive linkers. Tumor nodules arising from a single transfected cell line were stably labeled with fluorescence varying in emission spectra from green to near-infrared. After establishing and validating a SHIN3 cell line stably transfected with HaloTagR (HaloTagR-SHIN3), in vivo spectral fluorescence imaging studies were performed in live animals using a peritoneal dissemination model. The tumor nodules arising from HaloTagR-SHIN3 could be successfully labeled by four different fluorophore-conjugated HaloTag-ligands each emitting light at different wavelengths. These fluorophores could be alternated on serial imaging sessions permitting assessment of interval growth. Fluorescence was retained in histological specimens after fixation. Thus, this tagging system proves versatile both for in vivo and in vitro imaging without requiring modification of the underlying cell line. Thus, this strategy can overcome some of the limitations associated with the use of endogenous fluorescent proteins and exogenous targeted optical agents in current use.


Assuntos
Diagnóstico por Imagem/métodos , Corantes Fluorescentes/análise , Neoplasias Ovarianas/diagnóstico , Proteínas/análise , Proteínas/genética , Animais , Sítios de Ligação , Linhagem Celular Tumoral , Endoscopia , Feminino , Fluorescência , Expressão Gênica , Humanos , Ligantes , Camundongos , Neoplasias Ovarianas/patologia , Transfecção
4.
BMC Cell Biol ; 9: 17, 2008 Apr 02.
Artigo em Inglês | MEDLINE | ID: mdl-18384686

RESUMO

BACKGROUND: The ability to specifically label proteins within living cells can provide information about their dynamics and function. To study a membrane protein, we fused a multi-functional reporter protein, HaloTag, to the extracellular domain of a truncated integrin. RESULTS: Using the HaloTag technology, we could study the localization, trafficking and processing of an integrin-HaloTag fusion, which we showed had cellular dynamics consistent with native integrins. By labeling live cells with different fluorescent impermeable and permeable ligands, we showed spatial separation of plasma membrane and internal pools of the integrin-HaloTag fusion, and followed these protein pools over time to study bi-directional trafficking. In addition to combining the HaloTag reporter protein with different fluorophores, we also employed an affinity tag to achieve cell capture. CONCLUSION: The HaloTag technology was used successfully to study expression, trafficking, spatial separation and real-time translocation of an integrin-HaloTag fusion, thereby demonstrating that this technology can be a powerful tool to investigate membrane protein biology in live cells.


Assuntos
Bioensaio/métodos , Corantes Fluorescentes/metabolismo , Genes Reporter/genética , Proteômica/métodos , Proteínas Recombinantes de Fusão/metabolismo , Coloração e Rotulagem/métodos , Animais , Linhagem Celular , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Citoplasma/metabolismo , Humanos , Integrinas/metabolismo , Proteínas Luminescentes/metabolismo , Conformação Proteica , Estrutura Terciária de Proteína/fisiologia , Transporte Proteico/fisiologia , Proteínas Recombinantes de Fusão/genética
5.
ACS Chem Biol ; 13(9): 2758-2770, 2018 09 21.
Artigo em Inglês | MEDLINE | ID: mdl-30137962

RESUMO

A new generation of heterobifunctional small molecules, termed proteolysis targeting chimeras (PROTACs), targets proteins for degradation through recruitment to E3 ligases and holds significant therapeutic potential. Despite numerous successful examples, PROTAC small molecule development remains laborious and unpredictable, involving testing compounds for end-point degradation activity at fixed times and concentrations without resolving or optimizing for the important biological steps required for the process. Given the complexity of the ubiquitin proteasomal pathway, technologies that enable real-time characterization of PROTAC efficacy and mechanism of action are critical for accelerating compound development, profiling, and improving guidance of chemical structure-activity relationship. Here, we present an innovative, modular live-cell platform utilizing endogenous tagging technologies and apply it to monitoring PROTAC-mediated degradation of the bromodomain and extra-terminal family members. We show comprehensive real-time degradation and recovery profiles for each target, precisely quantifying degradation rates, maximal levels of degradation ( Dmax), and time frame at Dmax. These degradation metrics show specific PROTAC and family member-dependent responses that are closely associated with the key cellular protein interactions required for the process. Kinetic studies show cellular ternary complex stability influences potency and degradation efficacy. Meanwhile, the level of ubiquitination is highly correlated to degradation rate, indicating ubiquitination stemming from productive ternary complex formation is the main driver of the degradation rate. The approaches applied here highlight the steps at which the choice of E3 ligase handle can elicit different outcomes and discern individual parameters required for degradation, ultimately enabling chemical design strategies and rank ordering of potential therapeutic compounds.


Assuntos
Proteólise/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação/efeitos dos fármacos , Descoberta de Drogas/métodos , Avaliação Pré-Clínica de Medicamentos/métodos , Células HEK293 , Humanos , Cinética
6.
J Immunol Methods ; 431: 11-21, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-26851520

RESUMO

Receptor-mediated antibody internalization is a key mechanism underlying several anti-cancer antibody therapeutics. Delivering highly toxic drugs to cancer cells, as in the case of antibody drug conjugates (ADCs), efficient removal of surface receptors from cancer cells and changing the pharmacokinetics profile of the antibody drugs are some of key ways that internalization impacts the therapeutic efficacy of the antibodies. Over the years, several techniques have been used to study antibody internalization including radiolabels, fluorescent microscopy, flow cytometry and cellular toxicity assays. While these methods allow analysis of internalization, they have limitations including a multistep process and limited throughput and are generally endpoint assays. Here, we present a new homogeneous method that enables time and concentration dependent measurements of antibody internalization. The method uses a new hydrophilic and bright pH sensor dye (pHAb dye), which is not fluorescent at neutral pH but becomes highly fluorescent at acidic pH. For receptor mediated antibody internalization studies, antibodies against receptors are conjugated with the pHAb dye and incubated with the cells expressing the receptors. Upon binding to the receptor, the dyes conjugated to the antibody are not fluorescent because of the neutral pH of the media, but upon internalization and trafficking into endosomal and lysosomal vesicles the pH drops and dyes become fluorescent. The enabling attributes of the pHAb dyes are the hydrophilic nature to minimize antibody aggregation and bright fluorescence at acidic pH which allows development of simple plate based assays using a fluorescent reader. Using two different therapeutic antibodies--Trastuzumab (anti-HER2) and Cetuximab (anti-EGFR)--we show labeling with pHAb dye using amine and thiol chemistries and impact of chemistry and dye to antibody ration on internalization. We finally present two new approaches using the pHAb dye, which will be beneficial for screening a large number of antibody samples during early monoclonal development phase.


Assuntos
Anticorpos/análise , Corantes Fluorescentes/química , Piperazinas/química , Rodaminas/química , Anticorpos/imunologia , Linhagem Celular Tumoral , Cetuximab/química , Cetuximab/imunologia , Ensaio de Imunoadsorção Enzimática , Humanos , Concentração de Íons de Hidrogênio , Estrutura Molecular , Trastuzumab/química , Trastuzumab/imunologia
7.
Curr Chem Genomics ; 6: 48-54, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-23115613

RESUMO

Many fluorescent sensors are currently available for in vitro bio-physiological microscopic imaging. The ability to label cells in living animals with these fluorescent sensors would help translate some of these assays into in vivo applications. To achieve this goal, the first step is to establish a method for selectively labeling target cells with exogenous fluorophores. Here we tested whether the HaloTag® protein tagging system provides specific labeling of xenograft tumors in living animals. After systemic delivery of fluorophore-conjugated ligands, we performed whole animal planar fluorescent imaging to determine uptake in tag-expressing HCT116 xenografts. Our results demonstrate that HaloTag ligands containing red or near-infrared fluorophores have enhanced tumor uptake and are suitable for non-invasive in vivo imaging. Our proof-of-concept results establish feasibility for using HaloTag technology for bio-physiological imaging in living animals.

8.
Curr Chem Genomics ; 6: 55-71, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-23248739

RESUMO

Our fundamental understanding of proteins and their biological significance has been enhanced by genetic fusion tags, as they provide a convenient method for introducing unique properties to proteins so that they can be examinedin isolation. Commonly used tags satisfy many of the requirements for applications relating to the detection and isolation of proteins from complex samples. However, their utility at low concentration becomes compromised if the binding affinity for a detection or capture reagent is not adequate to produce a stable interaction. Here, we describe HaloTag® (HT7), a genetic fusion tag based on a modified haloalkane dehalogenase designed and engineered to overcome the limitation of affinity tags by forming a high affinity, covalent attachment to a binding ligand. HT7 and its ligand have additional desirable features. The tag is relatively small, monomeric, and structurally compatible with fusion partners, while the ligand is specific, chemically simple, and amenable to modular synthetic design. Taken together, the design features and molecular evolution of HT7 have resulted in a superior alternative to common tags for the overexpression, detection, and isolation of target proteins.

9.
ACS Chem Biol ; 6(5): 401-6, 2011 May 20.
Artigo em Inglês | MEDLINE | ID: mdl-21226520

RESUMO

Chromophore-assisted light inactivation (CALI) is a potentially powerful tool for the acute disruption of a target protein inside living cells with high spatiotemporal resolution. This technology, however, has not been widely utilized, mainly because of the lack of an efficient chromophore as the photosensitizing agent for singlet oxygen ((1)O(2)) generation and the difficulty of covalently labeling the target protein with the chromophore. Here we choose eosin as the photosensitizing chromophore showing 11-fold more production of ((1)O(2)) than fluorescein and about 5-fold efficiency in CALI of ß-galactosidase by using an eosin-labeled anti-ß-galactosidase antibody compared with the fluorescein-labeled one. To covalently label target protein with eosin, we synthesize a membrane-permeable eosin ligand for HaloTag technology, demonstrating easy labeling and efficient inactivation of HaloTag-fused PKC-γ and aurora B in living cells. These antibody- and HaloTag-based CALI techniques using eosin promise effective biomolecule inactivation that is applicable to many cell biological assays in living cells.


Assuntos
Amarelo de Eosina-(YS)/farmacologia , Fármacos Fotossensibilizantes/farmacologia , beta-Galactosidase/antagonistas & inibidores , Aurora Quinase B , Aurora Quinases , Corantes Fluorescentes/farmacologia , Células HeLa , Humanos , Lasers , Ligantes , Luz , Proteína Quinase C/efeitos da radiação , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Serina-Treonina Quinases/efeitos da radiação , Oxigênio Singlete , beta-Galactosidase/imunologia , beta-Galactosidase/efeitos da radiação
10.
Am J Transl Res ; 3(4): 392-403, 2011 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-21904659

RESUMO

Among the many molecular imaging techniques, reporter gene imaging has been a dynamic area of research. The HaloTag protein is a modified haloalkane dehalogenase which was designed to covalently bind to synthetic ligands (i.e. the HaloTag ligands [HTL]). Covalent bond formation between the HaloTag protein and the chloroal-kane within the HTL occurs rapidly under physiological conditions, which is highly specific and essentially irreversible. Over the years, HaloTag technology has been investigated for various applications such as in vitro/in vivo imaging, protein purification/trafficking, high-throughput assays, among others. The goal of this study is to explore the use of the HaloTag protein as a novel reporter gene for positron emission tomography (PET) imaging. By attaching a HaloTag -reactive chloroalkane to 1, 4, 7-triazacyclononane-N, N', N"-triacetic acid (NOTA) through hydrophilic linkers, the resulting NOTA-conjugated HTLs were labeled with (64)Cu and tested for PET imaging in living mice bearing 4T1-HaloTag-ECS tumors, which stably express the HaloTag protein on the cell surface. Significantly higher uptake of (64)Cu-NOTA-HTL-S (which contains a short hydrophilic linker) in the 4T1-HaloTag-ECS than the non-HaloTag-expressing 4T1 tumors was observed, which demonstrated the HaloTag specificity of (64)Cu-NOTA-HTL-S and warranted future investigation of the HaloTag protein as a PET reporter gene.

11.
Biotechniques ; 47(3): 769-74, 2009 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-19852762

RESUMO

As a means of reliably measuring intracellular pH, we have precisely targeted 5(and 6-) carboxyseminaphthorhodafluor to cellular subcompartments. This was accomplished by combining the well-established pH-sensitive dye with a protein-based reporter system. When expressed in cells, the reporter protein is designed to covalently bind ligands composed of a functional group and a reactive linker. In order to make a pH-sensitive ligand, we chemically coupled the pH sensor to a reactive linker. Several ligands of differing linker lengths were made and tested for their pH responsiveness in vitro. The most responsive of these ligands was then evaluated for its efficacy in live cell labeling and its use as an intracellular pH sensor for ratiometric confocal microscopy. Here we show that we could target this pH sensor within mammalian cells exclusively to either the nucleus or cytoplasm. Exhibiting the versatility of this reporter technology, we were also able to specifically limit pH sensor labeling to within the trafficking pathway of integrins and directly measure pH of this environment. Results correspond well with previously published reports. Both the simplicity and flexibility of the technology used in this study make possible the development of diverse targeted microenvironmental sensors or other moieties of interest.


Assuntos
Benzopiranos/análise , Técnicas Biossensoriais/métodos , Naftóis/análise , Rodaminas/análise , Frações Subcelulares/química , Benzopiranos/química , Transporte Biológico , Linhagem Celular Tumoral , Humanos , Concentração de Íons de Hidrogênio , Ligantes , Estrutura Molecular , Naftóis/química , Rodaminas/química
12.
ACS Chem Biol ; 3(6): 373-82, 2008 Jun 20.
Artigo em Inglês | MEDLINE | ID: mdl-18533659

RESUMO

We have designed a modular protein tagging system that allows different functionalities to be linked onto a single genetic fusion, either in solution, in living cells, or in chemically fixed cells. The protein tag (HaloTag) is a modified haloalkane dehalogenase designed to covalently bind to synthetic ligands (HaloTag ligands). The synthetic ligands comprise a chloroalkane linker attached to a variety of useful molecules, such as fluorescent dyes, affinity handles, or solid surfaces. Covalent bond formation between the protein tag and the chloroalkane linker is highly specific, occurs rapidly under physiological conditions, and is essentially irreversible. We demonstrate the utility of this system for cellular imaging and protein immobilization by analyzing multiple molecular processes associated with NF-kappaB-mediated cellular physiology, including imaging of subcellular protein translocation and capture of protein--protein and protein--DNA complexes.


Assuntos
Técnicas Biossensoriais/métodos , Células/citologia , Corantes Fluorescentes/química , Medições Luminescentes/métodos , Proteínas Luminescentes/química , Coloração e Rotulagem , Animais , Sítios de Ligação , Células/metabolismo , DNA/análise , DNA/química , DNA/metabolismo , Enzimas Imobilizadas , Humanos , Hidrocarbonetos Clorados/química , NF-kappa B/análise , NF-kappa B/metabolismo , Proteínas/análise , Proteínas/química , Proteínas/metabolismo , Sensibilidade e Especificidade
13.
Bioconjug Chem ; 13(3): 468-73, 2002.
Artigo em Inglês | MEDLINE | ID: mdl-12009935

RESUMO

Dipivaloyl-5-carboxyfluorescein N-hydroxysuccinimidyl ester 1 and 5-propargylamino-2',3'-dideoxyuridine triphosphate 5 were modified with maleimide, haloacetamide, and sulfhydryl reactive functional groups to participate in cross-conjugation reactions via sulfide bonds to generate fluorescently labeled, thioether cross-conjugated terminators 10 and 11. Their DNA sequencing potential was compared with an amide cross-conjugated terminator 13, synthesized by directly coupling 5-carboxyfluorescein NHS ester with 18-ddUTP 9. These terminators (10, 11, and 13) in combination with the Thermo Sequenase II DNA polymerase, in thermal cycle sequencing experiments, revealed that the thioether cross-conjugated terminator 10 and amide cross-conjugated terminator 13 served as good terminating substrates, generating satisfactory single-color gel images and electropherograms, while the other thioether cross-conjugated and maleimide derived one 11 underwent unexpected pH and temperature induced decomposition without showing fluorescent signatures for incorporation.


Assuntos
DNA Polimerase Dirigida por DNA/química , DNA/isolamento & purificação , Corantes Fluorescentes , Análise de Sequência de DNA/métodos , Compostos de Sulfidrila/química , Compostos de Sulfidrila/metabolismo , Regiões Terminadoras Genéticas/genética , Reagentes de Ligações Cruzadas , DNA/química , DNA/metabolismo , Primers do DNA
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