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The discovery of the bioluminescence pathway in the fungus Neonothopanus nambi enabled engineering of eukaryotes with self-sustained luminescence. However, the brightness of luminescence in heterologous hosts was limited by performance of the native fungal enzymes. Here we report optimized versions of the pathway that enhance bioluminescence by one to two orders of magnitude in plant, fungal and mammalian hosts, and enable longitudinal video-rate imaging.
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Eucariotos , Luminescência , Animais , MamíferosRESUMO
The objective was to determine the feasibility of an Open Dialogue-inspired approach in a metropolitan, public hospital setting with predominately African American participants. Participants were ages 18-35, experienced psychosis within the past month, and involved at least one support person in their care. We evaluated domains of feasibility including implementation, adaptation, practicality, acceptability, and limited-efficacy. An organizational change model (Addressing Problems Through Organizational Change) facilitated implementation. Clinicians received three trainings and ongoing supervision. Network meetings were successfully implemented with good self-reported fidelity to principles of dialogic practice. Some adaptations (less frequent meetings and no home visits) were necessary. A subset of individuals completed research assessments over 12 months. Qualitative interviews with participants suggested the intervention was acceptable. Symptom and functional outcomes were preliminary but trended toward improvement. Implementation was feasible with relatively brief training, organizational change processes, and context-specific adaptations. Lessons learned can assist in planning a larger research study.
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Transtornos Psicóticos , Humanos , Adulto Jovem , Estudos de Viabilidade , Transtornos Psicóticos/terapia , AutorrelatoRESUMO
Enzyme-linked immunosorbent assays (ELISAs) are used extensively for the detection and quantification of biomolecules in clinical diagnostics as well as in basic research. Although broadly used, the inherent complexities of ELISAs preclude their utility for straightforward point-of-need testing, where speed and simplicity are essential. With this in mind, we developed a bioluminescence-based immunoassay format that provides a sensitive and simple method for detecting biomolecules in clinical samples. We utilized a ternary, split-NanoLuc luciferase complementation reporter consisting of two small peptides (11mer, 13mer) and a 17 kDa polypeptide combined with a luminogenic substrate to create a complete, shelf-stable add-and-read assay detection reagent. Directed evolution was used to optimize reporter constituent sequences to impart chemical and thermal stability, as well as solubility, while formulation optimization was applied to stabilize an all-in-one reagent that can be reconstituted in aqueous buffers or sample matrices. The result of these efforts is a robust, first-generation bioluminescence-based homogenous immunoassay reporter platform where all assay components can be configured into a stable lyophilized cake, supporting homogeneous, rapid, and sensitive one-step biomolecule quantification in complex human samples. This technology represents a promising alternative immunoassay format with significant potential to bring critical diagnostic molecular detection testing closer to the point-of-need.
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Testes Imunológicos , Ensaio de Imunoadsorção Enzimática , Humanos , Imunoensaio , Indicadores e Reagentes , Luciferases/genéticaRESUMO
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.
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Marcadores de Afinidade/química , Azidas/química , Reagentes de Ligações Cruzadas/química , Diazometano/análogos & derivados , Hidrocarbonetos Clorados/química , Proteômica/métodos , Marcadores de Afinidade/efeitos da radiação , Azidas/efeitos da radiação , Cromatografia Líquida , Reagentes de Ligações Cruzadas/efeitos da radiação , Dasatinibe/análogos & derivados , Dasatinibe/farmacologia , Dasatinibe/efeitos da radiação , Diazometano/efeitos da radiação , Histona Desacetilases/análise , Histona Desacetilases/química , Humanos , Hidrocarbonetos Clorados/efeitos da radiação , Hidrolases/química , Células K562 , Espectrometria de Massas , Propranolol/análogos & derivados , Propranolol/farmacologia , Propranolol/efeitos da radiação , Proteínas Quinases/análise , Proteínas Quinases/química , Receptores Adrenérgicos alfa 2/análise , Receptores Adrenérgicos alfa 2/química , Raios Ultravioleta , Vorinostat/análogos & derivados , Vorinostat/farmacologia , Vorinostat/efeitos da radiaçãoRESUMO
Telebehavioral health emerged as an important practice during the coronavirus disease 2019 (COVID-19) pandemic as an opportunity for continued evidence-based mental health intervention, while minimizing exposure to coronavirus contagion. Though preliminary research suggests feasibility and positive outcomes of telebehavioral health practice for people with schizophrenia spectrum and other psychotic disorders, there is limited research about implementation and effectiveness of this practice (Kasckow et al., 2014). This case series highlights the transition from in-person to telebehavioral health practice of a Cognitive Behavioral Social Skills Training for Schizophrenia group due to the COVID-19 pandemic. This article summarizes: (a) the staff procedures needed to transition the group from in-person to telebehavioral health, (b) participant outcome data, (c) session attendance data, and (d) survey results from facilitators and participants about barriers and facilitators of the transition to telebehavioral health, and about how the virtual platform altered the therapeutic relationship and engagement. Participant outcome and engagement data suggest that, not only were two participants able to transition to telehealth and complete the program, but both participants also showed notable improvement in treatment engagement, goal progress, and skill acquisition. Surveys of six facilitators and one participant highlight how the transition to telebehavioral health had treatment advantages (e.g., therapeutic relationship, treatment engagement, group dynamics). Though survey results highlighted several implementation challenges in using the new virtual platform (e.g., technological connectivity, confidential space for engagement), no survey respondents reported that participation in this program resulted in harm to facilitators or participants. All facilitators and one participant agreed that the transition from in-person to virtual services was easy and reduced transportation barriers. Given the limited treatment engagement for this population (Lora et al., 2012) and the importance of early intervention to maximize clinical outcomes (Black et al., 2001; Bottlender et al., 2003), unanimous facilitator and participant report about improved patient attendance and participation in treatment after the transition to telebehavioral health was critically important. Though results of this case study are promising in suggesting telebehavioral health could be a viable modality for providing psychosocial treatment to people with schizophrenia spectrum and other psychotic disorders, more rigorous study is needed.
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Firefly luciferase-based ATP detection assays are frequently used as a sensitive, cost-efficient method for monitoring hygiene in many industrial settings. Solutions of detection reagent, containing a mixture of a substrate and luciferase enzyme that produces photons in the presence of ATP, are relatively unstable and maintain only a limited shelf life even under refrigerated conditions. It is therefore common for the individual performing a hygiene test to manually prepare fresh reagent at the time of monitoring. To simplify sample processing, a liquid detection reagent with improved thermal stability is needed. The engineered firefly luciferase, Ultra-Glo™, fulfills one aspect of this need and has been valuable for hygiene monitoring because of its high resistance to chemical and thermal inactivation. However, solutions containing both Ultra-Glo™ luciferase and its substrate luciferin gradually lose the ability to effectively detect ATP over time. We demonstrate here that dehydroluciferin, a prevalent oxidative breakdown product of luciferin, is a potent inhibitor of Ultra-Glo™ luciferase and that its formation in the detection reagent is responsible for the decreased ability to detect ATP. We subsequently found that dialkylation at the 5-position of luciferin (e.g., 5,5-dimethylluciferin) prevents degradation to dehydroluciferin and improves substrate thermostability in solution. However, since 5,5-dialkylluciferins are poorly utilized by Ultra-Glo™ luciferase as substrates, we used structural optimization of the luciferin dialkyl modification and protein engineering of Ultra-Glo™ to develop a luciferase/luciferin pair that shows improved total reagent stability in solution at ambient temperature. The results of our studies outline a novel luciferase/luciferin system that could serve as foundations for the next generation of bioluminescence ATP detection assays with desirable reagent stability.
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Luciferina de Vaga-Lumes/química , Substâncias Luminescentes/química , Medições Luminescentes/métodos , Trifosfato de Adenosina/química , Alquilação , Indicadores e Reagentes , Luciferases de Vaga-Lume/química , Especificidade por Substrato , TemperaturaRESUMO
BACKGROUND AND OBJECTIVES: As many as 30% of individuals with a schizophrenia spectrum disorder experience obsessive-compulsive symptoms (OCS). Clozapine has demonstrated superior efficacy for the treatment of medication-resistant schizophrenia but it is also associated with an increased risk for OCS. Because pharmacologic management of clozapine-related OCS can be particularly challenging, cognitive behavioral therapy (CBT) should be considered. Nevertheless, there are few detailed accounts of CBT for OCS and schizophrenia. METHODS: The authors describe the interdisciplinary outpatient care of a client who had a 25-year history of schizoaffective disorder, bipolar type, and OCS. The case formulation was used to guide interventions to target core schemas of being dangerous and defective. The case study describes the cognitive behavioral formulation, treatment targets, treatment course, and functional and symptom response. RESULTS: The client received 21 sessions of a formulation-based CBT for psychosis protocol, which included a 6-session course of exposure with response prevention, consisting of imaginal and in vivo exposure to multiple salient harm stimuli. Reduced ratings of distress and a 50% reduction in OCS suggest that habituation and inhibitory learning occurred. The treatment of OCS resulted in the complete resolution of thought broadcasting. Subsequently, the client was more successful in his efforts to adhere to an action schedule. LIMITATIONS: The use of both the treatment approach described in this clinical case report and contemporaneous medication management preclude comment on the mechanism(s) of the therapeutic change observed in this case. CONCLUSIONS: This report presents a means of conceptualizing the interplay between thought broadcasting and harm obsessions and discusses considerations in identifying and treating individuals with similar comorbid conditions, particularly in the context of clozapine treatment for medication-resistant psychosis.
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Transtornos Bipolares e Relacionados/complicações , Clozapina/efeitos adversos , Cognição , Formação de Conceito , Transtorno Obsessivo-Compulsivo/induzido quimicamente , Transtorno Obsessivo-Compulsivo/complicações , Esquizofrenia/complicações , Esquizofrenia/tratamento farmacológico , Idoso de 80 Anos ou mais , Antipsicóticos/efeitos adversos , Transtornos Bipolares e Relacionados/tratamento farmacológico , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Comportamento Obsessivo/induzido quimicamente , Comportamento Obsessivo/complicações , Transtornos Psicóticos/complicações , Transtornos Psicóticos/tratamento farmacológico , Adulto JovemRESUMO
The ability to analyze protein function in a native context is central to understanding cellular physiology. This study explores whether tagging endogenous proteins with a reporter is a scalable strategy for generating cell models that accurately quantitate protein dynamics. Specifically, it investigates whether CRISPR-mediated integration of the HiBiT luminescent peptide tag can easily be accomplished on a large-scale and whether integrated reporter faithfully represents target biology. For this purpose, a large set of proteins representing diverse structures and functions, some of which are known or potential drug targets, were targeted for tagging with HiBiT in multiple cell lines. Successful insertion was detected for 86% of the targets, as determined by luminescence-based plate assays, blotting, and imaging. In order to determine whether endogenously tagged proteins yield more representative models, cells expressing HiBiT protein fusions either from endogenous loci or plasmids were directly compared in functional assays. In the tested cases, only the edited lines were capable of accurately reproducing the anticipated biology. This study provides evidence that cell lines expressing HiBiT fusions from endogenous loci can be rapidly generated for many different proteins and that these cellular models provide insight into protein function that may be unobtainable using overexpression-based approaches.
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Medições Luminescentes/métodos , Proteínas Luminescentes/análise , Proteínas/análise , Sistemas CRISPR-Cas , Linhagem Celular , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , PlasmídeosRESUMO
Targeting the interaction of proteins with weak binding affinities or low solubility represents a particular challenge for drug screening. The NanoLuc â ® Binary Technology (NanoBiT â ®) was originally developed to detect protein-protein interactions in live mammalian cells. Here we report the successful translation of the NanoBit cellular assay into a biochemical, cell-free format using mammalian cell lysates. We show that the assay is suitable for the detection of both strong and weak protein interactions such as those involving the binding of RAS oncoproteins to either RAF or phosphoinositide 3-kinase (PI3K) effectors respectively, and that it is also effective for the study of poorly soluble protein domains such as the RAS binding domain of PI3K. Furthermore, the RAS interaction assay is sensitive and responds to both strong and weak RAS inhibitors. Our data show that the assay is robust, reproducible, cost-effective, and can be adapted for small and large-scale screening approaches. The NanoBit Biochemical Assay offers an attractive tool for drug screening against challenging protein-protein interaction targets, including the interaction of RAS with PI3K.
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G protein-coupled receptors (GPCRs) are prominent targets to new therapeutics for a range of diseases. Comprehensive assessments of their cellular interactions with bioactive compounds, particularly in a kinetic format, are imperative to the development of drugs with improved efficacy. Hence, we developed complementary cellular assays that enable equilibrium and real-time analyses of GPCR ligand engagement and consequent activation, measured as receptor internalization. These assays utilize GPCRs genetically fused to an N-terminal HiBiT peptide (1.3 kDa), which produces bright luminescence upon high-affinity complementation with LgBiT, an 18-kDa subunit derived from NanoLuc. The cell impermeability of LgBiT limits signal detection to the cell surface and enables measurements of ligand-induced internalization through changes in cell-surface receptor density. In addition, bioluminescent resonance energy transfer is used to quantify dynamic interactions between ligands and their cognate HiBiT-tagged GPCRs through competitive binding with fluorescent tracers. The sensitivity and dynamic range of these assays benefit from the specificity of bioluminescent resonance energy transfer and the high signal intensity of HiBiT/LgBiT without background luminescence from receptors present in intracellular compartments. These features allow analyses of challenging interactions having low selectivity or affinity and enable studies using endogenously tagged receptors. Using the ß-adrenergic receptor family as a model, we demonstrate the versatility of these assays by utilizing the same HiBiT construct in analyses of multiple aspects of GPCR pharmacology. We anticipate that this combination of target engagement and proximal functional readout will prove useful to the study of other GPCR families and the development of new therapeutics.
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Técnicas de Transferência de Energia por Ressonância de Bioluminescência/métodos , Membrana Celular/metabolismo , Luciferases/metabolismo , Luminescência , Fragmentos de Peptídeos/análise , Receptores Adrenérgicos beta 2/metabolismo , Regulação Alostérica , Ligação Competitiva , Transferência de Energia , Células HEK293 , Humanos , Cinética , Ligantes , Fragmentos de Peptídeos/metabolismo , Ligação Proteica , Transporte ProteicoRESUMO
In recent years, behavioral health professionals have expressed increased interest in engaging in social justice advocacy in public health care systems. In this article, we use an ecological framework to explore opportunities for social justice advocacy in such systems and challenges associated with such efforts. We propose that ecological models are well-suited to conceptualize and address the various contexts that affect behavioral health needs, and we emphasize the importance of considering the multitude of increasingly superordinate systems within which behavioral health professionals work when pursuing advocacy initiatives. We outline the central tenets of ecological models, apply them to social justice advocacy, and provide examples of advocacy within and across ecological systems. Finally, we reflect on future directions for behavioral health professionals interested in using an ecological framework to guide their own advocacy efforts, with and on behalf of patients and communities, in public health care systems and affiliated institutions.
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Private institutions of higher education in the United States were hesitant to institute programs of distance learning for fear that they could not maintain the quality of the education they had delivered in face-to-face programs. Vanderbilt University allowed their School of Nursing to embark on such an endeavor in 1996, as long as quality measures were incorporated. The result has been a comprehensive resource support team using Quality Matters and an increase in program rankings.
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Educação a Distância , Estados Unidos , UniversidadesRESUMO
Ligand binding assays routinely employ fluorescently-labeled protein ligands to quantify the extent of binding. These ligands are commonly generated through chemical modification of accessible lysine residues, which often results in heterogeneous populations exhibiting variable binding properties. This could be remedied by quantitative, site-specific labeling. Recently, we reported on a single-step method integrating recombinant protein purification with 2-cyanobenzothiazole (CBT) condensation for labeling a proteolytically exposed N-terminal cysteine. Here, using three growth factors, we show that unlike random lysine labeling, this site-specific approach yielded homogeneous populations of growth factors that were quantitatively labeled at their N-termini and retained their binding characteristics. We demonstrate the utility of this labeling method through the development of a novel assay that quantifies the capacity of antibodies to block receptor-ligand interactions (i.e. antibody blockade). The assay uses bioluminescence resonance energy transfer (BRET) to detect binding of CBT-labeled growth factors to their cognate receptors genetically fused to NanoLuc luciferase. The ability of antibodies to block these interactions is quantified through decrease in BRET. Using several antibodies, we show that the assay provides reliable quantification of antibody blockade in a cellular context. As demonstrated here, this simple method for generating uniformly-labeled proteins has potential to promote more accurate and robust ligand binding assays.
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Anticorpos Bloqueadores/análise , Corantes Fluorescentes/química , Proteômica/métodos , Anticorpos Bloqueadores/metabolismo , Becaplermina/genética , Becaplermina/metabolismo , Benzopiranos/química , Benzotiazóis/química , Cetuximab/farmacologia , Fator de Crescimento Epidérmico/genética , Fator de Crescimento Epidérmico/metabolismo , Células HEK293 , Humanos , Indóis/química , Ligantes , Medições Luminescentes/métodos , Nitrilas/química , Panitumumabe/farmacologia , Ligação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Reprodutibilidade dos Testes , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Vascular endothelial growth factor (VEGF) is an important mediator of endothelial cell proliferation and angiogenesis via its receptor VEGFR2. A common tumor associated with elevated VEGFR2 signaling is infantile hemangioma that is caused by a rapid proliferation of vascular endothelial cells. The current first-line treatment for infantile hemangioma is the ß-adrenoceptor antagonist, propranolol, although its mechanism of action is not understood. Here we have used bioluminescence resonance energy transfer and VEGFR2 genetically tagged with NanoLuc luciferase to demonstrate that oligomeric complexes involving VEGFR2 and the ß2-adrenoceptor can be generated in both cell membranes and intracellular endosomes. These complexes are induced by agonist treatment and retain their ability to couple to intracellular signaling proteins. Furthermore, coupling of ß2-adrenoceptor to ß-arrestin2 is prolonged by VEGFR2 activation. These data suggest that protein-protein interactions between VEGFR2, the ß2-adrenoceptor, and ß-arrestin2 may provide insight into their roles in health and disease.
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Receptores Adrenérgicos beta 2/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Técnicas de Transferência de Energia por Ressonância de Bioluminescência , Células Cultivadas , Corantes Fluorescentes/química , Células HEK293 , Humanos , Ligantes , Luciferases/química , Luciferases/metabolismo , Ligação Proteica , Receptores Adrenérgicos beta 2/química , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/química , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genéticaRESUMO
Intracellular target affinity and residence time are fundamental aspects of pharmacological mechanism (Lu and Tonge, Curr Opin Chem Biol 14:467-474, 2010). Although various robust biochemical approaches exist to measure these binding characteristics, analysis of compound binding with isolated targets may not accurately reflect engagement in the milieu of living cells. To realize the influence of cellular context, methods are needed that are capable of quantifying affinity and residence time in the presence of the intracellular factors that may impact target engagement. Bioluminescence resonance energy transfer (BRET) offers a solution for intracellular target engagement when quantitative metrics or kinetic analyses are required.
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Descoberta de Drogas/métodos , Transferência Ressonante de Energia de Fluorescência , Medições Luminescentes , Técnicas de Cultura de Células , Linhagem Celular , Transferência Ressonante de Energia de Fluorescência/métodos , Ensaios de Triagem em Larga Escala , Humanos , Medições Luminescentes/métodos , Sondas Moleculares/química , Sondas Moleculares/metabolismo , Permeabilidade , Reprodutibilidade dos TestesRESUMO
Fluorescent VEGF-A isoforms have been evaluated for their ability to discriminate between VEGFR2 and NRP1 in real-time ligand binding studies in live cells using BRET. To enable this, we synthesized single-site (N-terminal cysteine) labeled versions of VEGF165a, VEGF165b, and VEGF121a. These were used in combination with N-terminal NanoLuc-tagged VEGFR2 or NRP1 to evaluate the selectivity of VEGF isoforms for these two membrane proteins. All fluorescent VEGF-A isoforms displayed high affinity for VEGFR2. Only VEGF165a-TMR bound to NanoLuc-NRP1 with a similar high affinity (4.4 nM). Competition NRP1 binding experiments yielded a rank order of potency of VEGF165a > VEGF189a > VEGF145a. VEGF165b, VEGF-Ax, VEGF121a, and VEGF111a were unable to bind to NRP1. There were marked differences in the kinetic binding profiles of VEGF165a-TMR for NRP1 and VEGFR2. These data emphasize the importance of the kinetic aspects of ligand binding to VEGFR2 and its co-receptors in the dynamics of VEGF signaling.
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Neuropilina-1/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Transferência de Energia , Corantes Fluorescentes/metabolismo , Células HEK293 , Células Endoteliais da Veia Umbilical Humana , Humanos , Ligantes , Medições Luminescentes , Ligação Proteica , Isoformas de Proteínas/metabolismo , Rodaminas/metabolismoRESUMO
Protein thermal shift assays (TSAs) provide a means for characterizing target engagement through ligand-induced thermal stabilization. Although these assays are widely utilized for screening libraries and validating hits in drug discovery programs, they can impose encumbering operational requirements, such as the availability of purified proteins or selective antibodies. Appending the target protein with a small luciferase (NanoLuc) allows coupling of thermal denaturation with luminescent output, providing a rapid and sensitive means for assessing target engagement in compositionally complex environments such as permeabilized cells. The intrinsic thermal stability of NanoLuc is greater than mammalian proteins, and our results indicate that the appended luciferase does not alter thermal denaturation of the target protein. We have successfully applied the NanoLuc luciferase thermal shift assay (NaLTSA) to several clinically relevant protein families, including kinases, bromodomains, and histone deacetylases. We have also demonstrated the suitability of this assay method for library screening and compound profiling.
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Neddylation is a posttranslational modification that regulates protein stability, activity, and subcellular localization. Here, we describe a new tool for exploring the neddylation cycle of cullin1 (Cul1) directly in a cellular context. This assay utilizes the NanoLuc® Binary Technology (NanoBiT) to monitor the covalent neddylation status of Cul1. A stable clonal cell line derived from HEK293 was developed that expressed a C-terminus LgBiT tagged-Cul1 and N-terminus SmBiT tagged-Nedd8. Using this cell line, we screened inhibitors that are known to disrupt Nedd8 biology and demonstrated that both inhibitors of Nedd8-activating enzyme (NAE) and Constitutive photomorphogenesis 9 signalosome (CSN) complex produce concentration and time dependent signal decreases and increases, respectively. The kinetics of both responses could be monitored in real time and demonstrated that modulation of the Nedd8 pathway occurs rapidly. Further characterization of the cellular components of this cell line was performed in order to quantify the various levels of Cul1, Nedd8 and NAE and determined to be near endogenous levels. There was no difference between control and stably transfected cell lines in viability studies of NAE and CSN inhibitors. Taken together, these results suggest that the NanoBiT assay can be used to monitor Cul1 neddylation specifically and in real time.
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Bioensaio/métodos , Proteínas Culina/metabolismo , Proteína NEDD8/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas Culina/genética , Células HCT116 , Células HEK293 , Humanos , Proteína NEDD8/genéticaRESUMO
The sensitivity of bioluminescence imaging in animals is primarily dependent on the amount of photons emitted by the luciferase enzyme at wavelengths greater than 620 nm where tissue penetration is high. This area of work has been dominated by firefly luciferase and its substrate, D-luciferin, due to the system's peak emission (~ 600 nm), high signal to noise ratio, and generally favorable biodistribution of D-luciferin in mice. Here we report on the development of a codon optimized mutant of click beetle red luciferase that produces substantially more light output than firefly luciferase when the two enzymes are compared in transplanted cells within the skin of black fur mice or in deep brain. The mutant enzyme utilizes two new naphthyl-luciferin substrates to produce near infrared emission (730 nm and 743 nm). The stable luminescence signal and near infrared emission enable unprecedented sensitivity and accuracy for performing deep tissue multispectral tomography in mice.
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Benzotiazóis/metabolismo , Besouros/enzimologia , Proteínas de Insetos/metabolismo , Luciferases/metabolismo , Animais , Benzotiazóis/química , Células HEK293 , Humanos , Proteínas de Insetos/genética , Luciferases/genética , Luminescência , Medições Luminescentes/métodos , Células MCF-7 , Camundongos Endogâmicos C57BL , Camundongos Nus , Microscopia de Fluorescência , Mutação , Espectroscopia de Luz Próxima ao InfravermelhoRESUMO
For kinase inhibitors, intracellular target selectivity is fundamental to pharmacological mechanism. Although a number of acellular techniques have been developed to measure kinase binding or enzymatic inhibition, such approaches can fail to accurately predict engagement in cells. Here we report the application of an energy transfer technique that enabled the first broad-spectrum, equilibrium-based approach to quantitatively profile target occupancy and compound affinity in live cells. Using this method, we performed a selectivity profiling for clinically relevant kinase inhibitors against 178 full-length kinases, and a mechanistic interrogation of the potency offsets observed between cellular and biochemical analysis. For the multikinase inhibitor crizotinib, our approach accurately predicted cellular potency and revealed improved target selectivity compared with biochemical measurements. Due to cellular ATP, a number of putative crizotinib targets are unexpectedly disengaged in live cells at a clinically relevant drug dose.