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Citations Search

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ACS Chemical Biology 13(9), 2758–70. Quantitative live-cell kinetic degradation and mechanistic profiling of PROTAC mode of action. 2018

Riching, K.M., Mahan, S., Corona, C.R., McDougall, M., Vasta, J.D., Robers, M.B., Urh, M. and Daniels, D.L.

Notes: The authors use Promega HiBiT and NanoBRET™ technologies to monitor PROTAC-mediated degradation. (5081)

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ACS Med. Chem. Lett. 9(6), 546–51. Homogeneous assay for target engagement utilizing bioluminescent thermal shift. 2018

Dart, M. L., Machleidt, T., Jost, E., Schwinn, M. K., Robers, M. B., Shi, C., Kirkland, T. A., Killoran, M. P., Wilkinson, J. M., Hartnett, J. R. Zimmerman, K. and Wood, K. V.

Notes: Determining target engagement of potential therapeutics and their target protein is commonly assessed through Thermal Shift Assays (TSA). Cell-based TSA (CETSA) now provide a more physiologically relevant information on target engagement however, these assays require use of antibodies which selectively identify the target protein and commonly have problems with reproducibility. The NanoLuc® luciferase thermal shift assay (NaLTSA) uses NanoLuc® luciferase activity to measure the remaining soluble fraction of the target protein making it a simplified procedure with higher reproducibility. (5055)

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Sci. Rep. 7, 40674. Methylation specific targeting of a chromatin remodeling complex from sponges to humans. 2017

Cramer, J. M., Pohlmann, D., Gomez, F., Mark, L., Kornegay, B., Hall, C., Siraliev-Perez, E., Walavalkar, N. M., Sperlazza, M. J., Bilinovich, S., Prokop, J. W., Hill, A. L. and Williams, D. C. Jr.

Notes: The presence of methyl-cytosine binding domain (MBD) containing proteins and ability to remodel methylated chromatin was investigate in sponges. Specifically, the sponge MBD2 and GATAD2A protein interaction was monitored in cells using the NanoBRET™ Protein-Protein Interaction System. Multiple coiled-coil fusion constructs were tested to determine optimal signal intensity. Interestingly, while DNA methylation was observed in sponges, this interaction was much lower affinity than in vertebrate organisms. (5057)

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Nucl. Acids Res. 45(18), 10649–71.. Nucleic acid binding proteins affect the subcellular distribution of phosphorothioate antisense oligonucleotides. 2017

Bailey, J. K., Shen, W., Liang, X. H. and Crooke, S. T.

Notes: Modified antisense oligonucleotides (ASOs) show increased delivery and stability in cells, however these modifications have off-target effects. Localization of ASOs to cytoplasmic ribonucleoprotein (RNP) granules is observed to be mediated by RNA binding proteins, FUS and PSF. These interactions are further investigated using the NanoBRET™ Protein-Protein Interaction System. NanoLuc® tagged protein is produced using an in vitro transcription and translation system, purified, and bound to an acceptor oligonucleotide (AlexaFluor594-ASO). Various backbone and 2′ ASO modifications were screened for interaction with FUS truncations to determine the interaction domain. (5061)

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Cancer Sci. 109(2), 373–83. PRDM14 directly interacts with heat shock proteins HSP90α and glucose-regulated protein 78. 2017

Moriya, C., Taniguchi, H., Nagatoishi, S., Igarashi, H., Tsumoto, K. and Imai, K.

Notes: PRDM14 is dysregulated in a variety of cancers, including breast and pancreatic cancer, and overexpression leads to stem-cell-like phenotypes associated with aggressive tumors. Here, PRDM14 interacting proteins are identified using the HaloTag® Mammalian Pull-down System. The interactions of PRDM14 and glucoseregulated protein 78 (GRP78) and heat shock protein 90-a (HSP90a) were validated in vivo with the NanoBRET™ assay. (5053)

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Biochem. Pharmacol. 136, 62–75. Real-time analysis of the binding of fluorescent VEGF165a to VEGFR2 in living cells: Effect of receptor tyrosine kinase inhibitors and fate of internalized agonist-receptor complexes. 2017

Kilpatrick, L. E., Friedman-Ohana, R., Alcobia, D. C., Riching, K., Peach, C. J., Wheal, A. J., Briddon, S. J., Robers, M. B., Zimmerman, K., Machleidt, T., Wood, K. V., Woolard, J. and Hill, S. J.

Notes: Vascular endothelial growth factor (VEGF) receptor interactions are observed using the NanoBRET™ Protein-Protein Interaction System. A novel method of labeling VEGF at a single N-terminal cysteine (TMR) to maintain full activity is presented. NanoLuc®-VEGFR2 and VEGF-TMR are used in conjunction to monitor an interaction and internalization into intracellular endosomes. The effect of receptor tyrosine kinase inhibitors such as Cediranib and vandetanib on internalization in living cells is assessed. (5060)

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Nat. Commun. 8, 14259. The tumour suppressor APC promotes HIV-1 assembly via interaction with Gag precursor protein. 2017

Miyakawa, K., Nishi, M., Matsunaga, S., Okayama, A., Anraku, M., Kudoh, A., Hirano, H., Kimura, H., Morikawa, Y., Yamamoto, N., Ono, A. and Ryo, A.

Notes: Adenomatous polyposis coli protein (APC) is shown to directly interact with HIV-1 Gag protein to stimulate Gag multimerization and spread of viral particles. Direct measurements of the Gag-Gag protein interaction were measured using the NanoBRET™ system. HeLa cells were co-transfected with Gag-HaloTag® and Gag-NanoLuc® expression vectors and BRET signal was monitored after 24 hours. ADC knockdown displayed a substantial decrease in viral production and Gag-Gag interaction. Together, the authors show ADC regulates Gag localization to the PM and viral packaging. (5058)

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Sci. Rep. 7(1), 3187. Using nanoBRET and CRISPR/Cas9 to monitor proximity to a genome-edited protein in real-time. 2017

White, C. W., Vanyai, H. K., See, H. B., Johnstone, E. K. M and Pfleger, K. D. G.

Notes: G protein coupled receptor (GPCR) oligomerization and protein interaction has been commonly investigated using bioluminescence resonance energy transfer (BRET). The need for exogenous expression of fusion proteins has been a short coming of the BRET assay. Here CRISPR/Cas9 mediated homology directed repair was used to generate protein-NanoLuc® fusions under endogenous expression. The GPCR- β-arrestin2 interaction serves as a model of this system, where interaction and internalization are monitored under conditions where the donor luciferase endogenously expressed. (5059)

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Sci. Rep. 6, 29130. Determination of GLUT1 oligomerization parameters using bioluminescent Förster resonance energy transfer. 2016

Looyenga, B., VanOpstall, C., Lee. Z., Bell, J., Lodge, E., Wrobel, K., Arnoys, E. and Louters, L.

Notes: Oligomerization of the glucose transporter (GLUT1) within this plasma membrane was assessed using the NanoBRET™ system. When expressed at high levels, GLUT1 has been shown to form tetrameric complexes with higher transport efficiency. Theoretical NanoBRET™ efficiency was determined for a variety of fluorescent protein acceptors and experiments were performed with mCherry as the NanoBRET™ acceptor. GLUT1 was shown to form a range of higher order complexes in live cells. Flow cytometry and immunoblotting were used in parallel to estimate GLUT1 density in cells. (5054)

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J. Biol. Chem. 291(53), 27134–46. Fc engineering approaches to enhance the agonism and effector functions of an anti-OX40 antibody. 2016

Zhang, D., Goldberg, M. V. and Chiu, M. L.

Notes: Agonist antibodies targeting T cells and other immune cells to simulate immune activation have shown to be promising for cancer therapeutics. Here, the NanoBRET™ Protein-Protein Interaction Assay was used to measure hexamerization of anti-OX40 antibodies on the cell surface. Specific anti-OX40 antibody mutations were analyzed for increased antibody multimerization and engagement. Mutations promoting IgG hexamerization showed enhanced agonistic activity. (5056)

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Nat Chem. Biol. 12(12), 1097–1104. Potent and selective bivalent inhibitors of BET bromodomains. 2016

Waring, M.J., Chen, H., Rabow, A.A., Walker, G., Bobby, R., Boiko, S., Bradbury, R.H., Callis, R., Clark, E., Dale, I., Daniels, D.L., Dulak, A., Flavell, L., Holdgate, G., Jowitt, T.A., Kikhney, A., McAlister, M., Méndez, J., Ogg, D., Patel, J., Petteruti, P., Robb, G.R., Robers, M.B., Saif, S., Stratton, N., Svergun, D.I., Wang, W., Whittaker, D., Wilson, D.M. and Yao, Y.

Notes: The bromodomain and extraterminal (BET) family of proteins contain two bromodomains. A probe compound, biBET, capable of binding both bromodomains of BET proteins in cis is characterized. BDR4-NanoLuc and Halo-tagged histone H3 fusions are used to monitor biBET binding with the NanoBRET Target Engagement system. Interestingly, bivalent binding lead to slower displacement of inhibitor from BDR4 and increased potency. (5078)

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J. Med. Chem. 58, 2718–36. 9H-Purine Scaffold Reveals Induced-Fit Pocket Plasticity of the BRD9 Bromodomain. 2015

Picaud, S., Strocchia, M., Terracciano, S., Lauro, G., Mendez, J., Daniels, D.L., Riccio, R., Bifulco, G., Bruno, I. and Filippakopoulos, P.

Notes: The authors used bioluminescence resonance energy transfer (BRET) to test the ability of a bromodomain 9 ligand to disrupt binding to histone. HEK 293 cells were cotransfected with a histone H3.3-HaloTag® fusion vector and either NanoLuc®-BRD9 bromodomain or NanoLuc®-full-length BRD4 fusion vector. After 24 hours, the transfected cells were trypsinized, diluted in phenol red-free DMEM with or without 10nM of HaloTag® NanoBRET™ 618 Ligand and dispensed into a 96-well plate. One of two potential BRD-disrupting compounds, 7d or 11, was adding to a final concentration of 0.005–33μM, cells were incubated for 18 hours and NanoBRET™ Nano-Glo® Substrate (final concentration 10µM) was added. Fluorescence was measured and a corrected BRET ratio calculated. Cytotoxicity was assessed after the NanoBRET™ assay by incubating the cells with the CellTiter-Glo® Reagent for 30 minutes and measuring luminescence. To examine histone H3.3 localization, HEK 293 cells were transfected with the histone H3.3-HaloTag® fusion vector using FuGENE® HD Transfection Reagent. After 24 hours, cells were labeled with 5μM HaloTag® TMR ligand for 15 minutes before washing with complete medium, incubated for 30 minutes and imaged with a confocal microscope. (4568)

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ACS Chemical Biology 10, 1797–1804. NanoBRET—A Novel BRET Platform for the Analysis of Protein–Protein Interactions. 2015

Machleidt, T, Woodroofe, C.C., Schwinn, M.K., Méndez, J.,  Robers, M.B., Zimmerman, K., Otto, P., Daniels, D.L., Kirkland, T.A., and Wood, K.V.

Notes: This paper introduces NanoBRET technology, which provides an improved alternative to conventional BRET protein interaction assays. NanoBRET assays combine the extremely bright NanoLuc luciferase with a means for tagging intracellular proteins with a long-wavelength fluorophore (HaloTag). The greater light intensity and improved spectral resolution of the NanoBRET assay results in increased detection sensitivity and dynamic range over current BRET technologies. Performance of the assay is demonstrated using several model systems, and the ability to image BRET in individual cells is illustrated. The  authors also demonstrate the application of NanoBRET in a novel assay developed for analyzing the interactions of bromodomain proteins with chromatin in living cells. (4575)

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Nat. Commun. 6, 10091 doi:10.1038/ncomms10091. Target engagement and drug residence time can be observed in living cells with BRET. 2015

Robers, M.B, Dart, M.L., Woodroofe, C.C,  Zimprich, C.A., Kirkland, T.A., Machleidt, T., Kupcho, K.R., Levin, S., Hartnett, J.R., Zimmerman, K., Niles, A.L., Ohana, R.F., Daniels, D.L., Slater, M., Wood, M.G., Cong, M., Cheng Y., and Wood, K.V.

Notes: This paper describes a method for using bioluminescence resonance energy transfer (BRET) to reveal the binding characteristics of a drug with selected targets in live cells. The authors used cell-permeable fluorescent tracers in a competitive binding assay to quantify drug engagement with target proteins fused to Nanoluc luciferase. Using this approach, they were able to profile isozyme-specific engagement and binding kinetics for a panel of histone deacetylase (HDAC) inhibitors. (4587)

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Angewandte Chemie International Edition 54, 6217–21. LP99: Discovery and Synthesis of the First Selective BRD7/9 Bromodomain Inhibitor 2015

Clark, P.G.K., Vieira, L.C.C., Tallant, C., Fedorov, O., Singleton, D.C., Rogers, C.M., Monteiro, O.P., Bennett, J.M., Baronio, R., Müller, S., Daniels, D.L., Méndez, J., Knapp, S., Brennan, P.E. and Dixon, D.J.

Notes: To characterize the effectiveness of LP99, a potential bromodomain inhibitor, BRD7 and BRD9 were fused with NanoLuc® luciferase and histones H3.3 and H4 were fused with HaloTag® protein for use in BRET. The two proteins were expressed in HEK 293 cells, and the histone-HaloTag® fusions were fluorescently labeled with the HaloTag® NanoBRET™ 618 Ligand. Once the NanoBRET™ Nano-Glo® Substrate was added, NanoBRET™ ratios were assessed in the presence of varying concentrations of LP99. (4567)

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ACS Med. Chem. Lett. 5, 1190–1195. 1,3-Dimethyl Benzimidazolones Are Potent, Selective Inhibitors of the BRPF1 Bromodomain. 2014

Demont, E.H., Bamborough,P., Chung,C., Craggs, P.D., Fallon, D., Gordon, L.J., Grandi, P., Hobbs, C.I., Hussain, J., Jones, E.J., Le  A., Michon, A., Mitchell, D.J., Prinjha, R.K., Roberts, A.D., Sheppard, R.J, and Watson, R.J.

Notes: In this paper the authors report on the discovery, binding mode, and structure:activity relationship of the first potent, selective series of inhibitors of the BRPF1 (bromodomain and PHD finger-containing)  bromodomain.  Bromodomains are specific protein modules present in a group of chromatin-regulator proteins responsible for “reading” acetylated lysine residues. Although some bromodomain-containing proteins (BCPs), such as those in the BET subfamily, are well characterized and have been identified as potential therapeutic targets, other BCPs, including those in the BPRF subfamily, are less well understood.  These authors set out to generate selective inhibitors of the BRPF1 domain in order to better understand the functional role of this specific bromodomain region. Using an inhibitor discovery strategy based on other known compound-bromodomain interactions, a potent, selective inhibitor of the BRPF1 bromodomain was identified, synthesized, and characterized using in vitro methods.  To demonstrate the function of this compound in live cells, the NanoBRET™ assay for protein:protein interactions (PPI) was used. The NanoBRET™ PPI assay enabled the authors to demonstrate both the cell permeability of the newly identified compound and also the ability of the compound to disrupt chromatin binding of the BRPF1 domain. NanoLuc® Luciferase-tagged BRPF1 bromodomain and HaloTag®-labeled Histone H3.3 were used for the NanoBRET™ assay in HEK293 cells. Dose-response curves performed with the NanoBRET™ assay enabled calculation of the cellular IC50 of the newly identified compound. A less active control analog compound was unable to inhibit the BRPF1 bromodomain:Histone H3.3 interaction, demonstrating assay specificity. Finally, the newly identified compound was inactive in NanoBRET™ assays using a second BRPF1 isoform containing a natural insertion, a result that was consistent with the proposed compound mode of action. Confirmation that the new identified compound can enter cells and disrupt the BRPF1 bromodomain:chromatin interaction in a cellular environment suggests that it may be a useful compound for studying the physiological role and therapeutic potential of BCPs containing the BRPF1 bromodomain. (4514)

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Proc. Natl. Acad. Sci. USA 112, 148–153. Activation of Rab8 guanine nucleotide exchange factor Rabin8 by ERK1/2 in response to EGF signaling. 2014

Wang, J., Ren, J., Wu, B., Feng, S., Cai, G., Tuluc, F., Peränen, J. and Guo, W.

Notes: To investigate whether protein conformation of Rabin8 plays a role in autoinhibition, the authors created a Rabin8 fusion construct with NanoLuc® luciferase at the N terminus and HaloTag® protein at the C terminus so that they can use BRET as an indication of protein conformation. A t-SRARE protein, syntaxin-4 (STX4), which is known to have a closed conformation, was constructed with the same NanoLuc® luciferase-STX4-HaloTag® protein configuration for use as a positive control. Both the control STX4 protein and Rabin8 were expressed in E. coli, the NanoBRET™ Nano-Glo® Substrate added and fluorescence measured. As a negative control, TEV protease was used to cleave the HaloTag® sequence from the protein fusions, eliminating the NanoBRET™ signal. NanoBRET™ signals were determined from experiments comparing Rabin8 with a gain-of-function Rabin8 mutant, exposing Rabin8 to constitutively active ERK2 or a kinase-dead ERK2 and assessing wildtype Rabin8 versus Rabin8-4D, where the aspartates acted as phosphorylation mimics. (4566)

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Chem. Biol. 21, 1463–75. EZH2 inhibitor efficacy in non-Hodgkin's lymphoma does not require suppression of H3K27 monomethylation. 2014

Bradley WD, Arora S, Busby J, Balasubramanian S, Gehling VS, Nasveschuk CG, Vaswani RG, Yuan CC, Hatton C, Zhao F, Williamson KE, Iyer P, Méndez J, Campbell R, Cantone N, Garapaty-Rao S, Audia JE, Cook AS, Dakin LA, Albrecht BK, Harmange JC, Daniels DL, Cummings RT, Bryant BM, Normant E, Trojer P.

Notes: In this paper the authors report on the identification and characterization of small-molecule inhibitors of the histone lysine methyltransferase Enhancer of Zeste Homolog 2 (EZH2). EZH2 has been identified as a potential oncology target because of its role in histone lysine methylation and associated involvement in the manipulation of chromatin structure. EZH2 is the catalytic component of the polycomb repressive complex 2 (PRC2), and recurrent mutations in EZH2 have been discovered in multiple cancer cell types resulting in aberrantly high H3 lysine 27 (H3K27) trimethylation levels. The authors identified compound CPI-360 as a potent inhibitor of EZH2 methyl transferase activity that functions on the basis of S-adenosyl-L-methionine (SAM)-competition and demonstrated that treatment of cells with CP1-360 resulted in reduced di and tri, but not mono, methylation of H3K27 in a dose-dependent manner. Using NanoBRET™ protein:protein interaction assays, the authors were able to demonstrate in cells that the inhibition of EZH2 activity was not due to disruption of the PRC2 complex and also did not affect binding of EZH2 to chromatin, presenting a novel mechanism for the inhibition of EZH2 activity. The authors further demonstrate how inhibiting EZH2 methyl transferase activity affects H3K27 methylation and transcriptional patterns in lymphoma cell lines and demonstrate the effect of EZH2 inhibitors on growth and viability of lymphoma cells in vitro using Cell Titer-Glo® Luminescent Cell Viability Assay and in in vivo xenograft models. (4562)

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