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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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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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Proc. Natl. Acad. Sci. USA 111(38), 13990–5. Tyrosine phosphorylation of GluK2 up-regulates kainate receptor-mediated responses and downstream signaling after brain ischemia 2014

Zhu, Q.J., Kong, F.S., Xu, H., Wang, Y., Du, C.P., Sun, C.C., Liu, Y., Li, T. and Hou, X.Y.

Notes: In this study the authors looked for molecular mechanisms underlying the role of kainite receptors in ischemic stroke. In their studies, the researchers examined binding of Src kinase to GluK2, and the site of this interaction. A GST pulldown assay confirmed a direct interaction between GluK2 and Src in vitro. Then a bioluminescence resonance energy transfer (BRET) assay was used to examine this GluK2-Src interaction in live HEK293 cells, using NanoLuc® Luciferase as the energy donor, and HaloTag-labeled GluK2 as the energy acceptor. As reported, the co-expression of NLuc and HaloTag® fusions resulted in a significant NanoBRET ratio. The authors added untagged GluK2 as a competitor of the GluK2-HaloTag and Src-NLuc interaction, which resulted in reduction of the NanoBRET ratio. These results demonstrated that GluK2 interacts directly with Src in living cells. The GloMax® Discover Detection System was used to measure the output of these assays.

Their source of NanoLuc® Luciferase and HaloTag® tags was the NanoBRET™ PPI Starter System (Cat.# N1811, N1821). (4696)

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