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Cell Chemical Biology 25, 206–14. Quantitative, wide-spectrum kinase profiling in live cells for assessing the effect of cellular ATP on target engagement. 2018

Vasta, J.D., Corona, C.R., Wilkinson, J., Zimprich, C.A., Hartnett, J.R., Ingold, M.R., Zimmerman, K., Machleidt, T., Kirkland, T.A., Huwiler, K.G., Ohana, R.F., Slater, M., Otto, P.,Cong, M., Wells, C.I., Berger, B-T., Hanke, T., Glas, C., Ding, K., Drewry, D.H., Huber, K.V.M., Willson, T.M., Knapp, S., Müller, S., Meisenheimer, P.L., Fan, F., Wood, K.V. and Robers, M.B.

Notes: The researchers demonstrate use of the NanoBRET™ Target Engagement Intracellular Kinase Assays to report on kinase target engagement in real time for quantitative inhibitor profiling of 178 kinases, including over 40 integral membrane receptors. (4936)

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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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Sci. Signal. 8 (405), ra123. DOI: 10.1126/scisignal.aab4068. Distinct profiles of functional discrimination among G proteins determine the actions of G protein–coupled receptors. 2015

Masuho, I., Ostrovskaya, O., Kramer, G.M.,  Jones, C.D., Xie, K., and Martemyanov, K.A.

Notes: These authors studied the interactions between G-protein coupled receptors (GPCRs)  and 13 different G-proteins using a NanoBRET assay. NanoBRET assays were performed using protein partners labeled with NanoLuc luciferase or Venus yellow fluorescent protein. The improvement in signal-to-noise ratio achieved using the NanoBRET method enabled resolution of signals from previously intractable G-proteins and GPCRs. The authors demonstrated that GPCRs engage multiple G-proteins with distinct patterns of activity or “fingerprints”. This differential engagement of multiple target G-proteins was revealed by quantitative analysis of G-protein activation kinetics.  (4588)

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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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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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