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Antiviral Res. 154, 44-50. A Guinea pig cytomegalovirus resistant to the DNA maturation inhibitor BDCRB. 2018

Ourahmane, A., Sauer, A., Nixon, D.E., Murphy, C., Mondello, M., Douglass, Chiu E., Siegmund, S., Wang, J,B, and McVoy, M.A.

Notes: BDCRB selectively disrupts DNA packing in cytomegalovirus, a process which is critical for herpesvirus life-cycle. In this paper, the authors use guinea pig cytomegalovirus as a model for human infection and derive a resistant virus to characterize the BDCRB mechanism of action. Recombinant viruses containing an expression cassette for NanoLuc luciferase in either a wild-type or L406P mutant cytomegalovirus background were constructed. Luciferase activity was used as a proxy for viral yield in BDCRB treated or untreated cells. The L406P mutation did not show significant resistance to BDCRB treatment.  (5108)

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PLos ONE 13(4), e0195879. A novel nanoluciferase-based system to monitor Trypanosoma cruzi infection in mice by bioluminescence imaging. 2018

Silberstein, E., Serna, C., Fragoso, S.P., Nagarkatti, R., and Debrabant, A.

Notes: Infection progression remains poorly characterized in Chagas disease, a life-long infection caused by T. cruzi. To understand the development of the disease, a bioluminescent imaging system for live mice infected with NanoLuc-labeled transgenic T. cruzi was developed. Initial expression of NanoLuc luciferase was determined in TcCOL cells and cell lysates using the Nano-Glo Live Cell assay and the Nano-Glo Luciferase Assay System, respectively. Luminescence could be detected as early as 14 days post-infection and was focused in the abdomen. Further, luminescence remained detectable for up to 222 days post infection, while parasite number in blood decreased after 21 days to the limit of detection.  (5110)

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115(25), E5776-E5785. ABC transporter content diversity in Streptococcus pneumoniae impacts competence regulation and bacteriocin production. 2018

Wang, C.Y., Patel, N., Wholey, W.Y., and Dawid, S.

Notes: S. pneumoniae uses the ABC transporters ComAB and BlpAB in natural competence and bacterial predation through bacteriocin secretion. NanoLuc and RFluc constructs with upstream comAB and blpAB promoters were used to look at transcriptional activation. Also, bacteriocin secretion by ComAB and BlpAB was determined using the HiBiT system. BlpI was tagged with HiBiT and luciferase activity was monitored in both wild-type and mutant transporter backgrounds. Both ComAB and BlpAB were able to respond to and secrete BlpI. (5137)

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Anal. Biochem. 555, 67–72. Antibody-free detection of cellular neddylation dynamics of Cullin1. 2018

Schwinn, M.K., Hoang, T., Yang, X., Zhao, X., Ma, J., Li, P., Wood, K.V., Mallender, W.D., Bembenek, M.E. and Yan, Z.H.

Notes: The post-transcriptional modification of neddylation helps regulate protein activity, stability and localization. NanoLuc® Binary Technology (NanoBiT) was used to monitor covalent neddylation of Cul1 in a cellular context. SmBiT-Nedd8 and Cul1-LgBiT were expressed in HEK293 cells and luminescence was monitored as a response to a Nedd8-Cul1 covalent protein modification. Further, neddylation was monitored in the presence of both inhibitors and activators, and a concentration- and time-dependent response in luminescence was observed. (5076)

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Cell 173. Co-optation of Tandem DNA Repeats for the Maintenance of Mesenchymal Identity. 2018

Balestrieri, C., Alfarano, G., Milan, M., Tosi, V., Properini, E., Nicoli P., Palamidessi, A., Scita, G., Diaferia, G.R., Natoli, G.

Notes: Tandem repeats (TMs) are thought to be unstable genetic elements, however the presence of regulator binding motifs with TMs may allow them to function as cis-regulatory elements. ZEB1, a transcriptional repressor which promotes mesenchymal maintenance, was shown to occupy TR regions near loci relevant to epithelial identity. The minimum number of repeat regions for ZEB1 binding and transcriptional repression was determined using the Nano-Glo Dual Luciferase Assay System. ZEB1 motifs within TRs were cloned upstream of the NanoLuc reporter. Relative luminescence was measured, and repression was correlated with motif number. (5124)

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Nat. Commun. 9(1), 1305. Designer exosomes produced by implanted cells intracerebrally deliver therapeutic cargo for Parkinson's disease treatment. 2018

Kojima, R., Bojar, D., Rizzi, G., Hamri, G.C., El-Baba, M.D., Saxena, P., Ausländer, S., Tan, K.R., Fussenegger, M.

Notes: Obstacles in message transfer and designer exosome production have hindered the use of exosomes as therapeutic agents. These authors describe a genetically encoded device, EXOsomal transfect into cells (EXOtic), which addresses these obstacles. A screen for genes that enhance exosome production was conducted by fusing an exosome marker to NanoLuc luciferase. Luminescence was measured using the Nano-Glo Luciferase Assay. Identified genes yielded a 15- to 40-fold increase in signal.  (5111)

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FEBS J. 285, 46–71. Dynamic control of RSK complexes by phosphoswitch-based regulation. 2018

Gógl, G., Biri-Kovács, B., Póti, Á.L., Vadászi, H., Szeder, B., Bodor, A., Schlosser, G., Ács, A., Turiák, L., Buday, L., Alexa, A., Nyitray, L. and Reményi, A.

Notes: The NanoBiT® PPI MCS Starter System was used to build assays to study the interaction dynamics of RSK1 with ERK2 and MAGl-1 in live cells upon EGF treatment. Various structure-based mutations were studied by transiently transfecting cells with constructions using FuGENE® HD Reagent and assayed with Nano-Glo® Live Cell Reagent. The authors were able to determine which mutations in RSK1 affected MAGl-1 dissociation and determined that it was dependent on the presence of the PDZ-binding motif. (4969)

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Nat. Commun. 9(1), 1988. Factor XIIIA-expressing inflammatory monocytes promote lung squamous cancer through fibrin cross-linking. 2018

Porrello, A. et al.

Notes: A novel mechanism of tumor metastasis utilizing inflammatory monocytes (IMs) and Factor XIIIA is described. Specifically, Factor XIIIA serves as a scaffold stimulating cancer cell invasion through fibrin cross-linking. IM are recruited to tumor cells through the inflammatory chemokine, CCL2. Mice were injected with NanoLuc expressing lung squamous cancer cells and tumor progression was measured using the Nano-Glo Luciferase Assay. Treatment with a CCL2 inhibitor, preventing IM recruitment to tumor cells, displayed a significant decrease in tumor progression. (5109)

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Sci. Rep. 8(1), 16624. High-throughput neutralization assay for multiple flaviviruses based on single-round infectious particles using dengue virus type 1 reporter replicon. 2018

Matsuda, M., Yamanaka, A., Yato, K. Yoshii, K., Watashi, K., Aizaki, H., Konishi, E., Takasaki, T., Kato, T., Muramatsu, M., Wakita, T., and Suzuki, R.

Notes: This paper details one example of using NanoLuc luciferase to create reporter viruses. NanoLuc-containing infectious particles for various types of flavivirus (e.g., Dengue, Zika, West Nile) were used in neutralization assays with serum samples following either infection or immunization. (5129)

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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. 8, 12206. Human tissue-engineered skeletal muscle: A novel 3D in vitro model for drug disposition and toxicity after intramuscular injection. 2018

Gholobova, D., Gerard, M., Decroix, L., Desender, L., Callewaert, N., Annaert, P. and Thorrez, L.

Notes: The human bio-artificial muscle (BAM) model was used to monitor cellular response to intramuscular drug injection. The RealTime-Glo™ MT Cell Viability Assay was used to observe pro-NanoLuc® substrate dispersal within the tissue after compound injection. Luminescence was monitored on the GloMax®-Multi Microplate Multimode Reader. (5181)

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ChemMedChem 13(1), 48-66. Identification and Optimization of 4-Anilinoquinolines as Inhibitors of Cyclin G Associated Kinase. 2018

Asquith, C.R.M. et al.

Notes: Cyclin G associated kinase (GAK) is a central regulator of viral and bacterial host cell entry. The synthesis and characterization of selective and potent GAK inhibitors is described. Inhibitors were assessed for ability to compete for GAK binding using the NanoBRET Target Engagement system. Briefly, a BRET signal was observed with the NanoLuc-GAK fusion in the presence of a red-shifted dye specific to the GAK ATP binding site. Upon treatment with GAK inhibitors, the dye was competed away, and a loss of BRET signal was observed. (5130)

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Gene 640, 14-20. Isolation and characterization of a stress-responsive gene encoding a CHRD domain-containing protein from a halotolerant green alga. 2018

Ishinishi, R., Matsuura, H., Tanaka, S., Nozawa, S., Tanada, K., Kawashita, N., Fujiyama, K., Miyasaka, H., and Hirata, K.

Notes: The CL58 gene from the halotolerant green alga Chlamydomonas reinhardtii was characterized. The mRNA levels of CL58 increased in response to increased copper levels and lower temperature. Further, a NanoLuc fusion showed the expressed protein is secreted. While the function of CL58 remains unknown, here initial characterization shows it is involved in stress response.  (5112)

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epub ahead of print. Kinase inhibitors: the road ahead 2018

Ferguson, F.M. and Gray, N.

Notes: This review mentions, under kinome-wide techniques, the NanoBRET™ assay for measuring the cellular target engagement across 178 members of a kinome using a technique involving competition between a compound of interest and a reversible cell-permeable energy transfer probe for binding to NanoLuc-tagged kinase in live cells. The authors note that, with the assay's fluorescent readout, it is amenable to higher throughput than mass spec-based techniques. (4964)

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Biochem. Pharmacol. 148, 298–307. Molecular dissection of the human A3 adenosine receptor coupling with β-arrestin2. 2018

Storme, J., Cannaert, A., Van Craenenbroeck, K. and Stove, C.P.

Notes: The NanoBiT Protein-Protein Interaction System was utilized to investigate the interaction of human A3 adenosine receptor, a G protein-coupled receptor, and β-arrestin2. Specifically, the importance of specific C-terminal phosphorylation sites for A3AR and β-arrestin2 coupling was determined using protein truncations. To determine the optimal fusion tags for this assay, multiple constructs were tested in the presence of an A3AR selective agonist. Unlike previous studies in rat models, C-terminal human A3AR truncations displayed no major impact on β-arrestin2 coupling. (5077)

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J. Biol. Chem. 293(23), 8750–8760. Quantitative interaction analysis permits molecular insights into functional NOX4 NADPH oxidase heterodimer assembly. 2018

O'Neill, S., Mathis, M., Kovačič, L., Zhang, S., Reinhardt, J., Scholz, D., Schopfer, U., Bouhelal, R. and Knaus, U.G.

Notes: The assembly of the NOX4- p22phox integral membrane complex, involved in electron transfer and reactive oxygen species generation, was investigated using the NanoBiT Protein-Protein Interaction System. A total of 31 Small BiT/Large BiT NOX4 or p22phox fusions were tested, and the most active pairs were further analyzed for catalytic activity. Residues involved in complex formation and catalysis were determined using mutational analysis and tested for luciferase activity. Together, the authors identified an approach for determining membrane protein assembly that can be used in the future as a drug discovery tool. (5074)

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Anal. Chem. 90(3), 2230-2237. Recombinant Peptidomimetic-Nano Luciferase Tracers for Sensitive Single-Step Immunodetection of Small Molecules. 2018

Ding, Y., Hua, X., Chen, H., Liu, F., González-Sapien, G., and Wang, M.

Notes: Phage display uses protein fusions to surface exposed membrane proteins as screening technique for protein-protein and protein-peptide interactions. Here this technique is applied for detection of imidaclothiz, a small molecule. A NanoLuc Luciferase fusion to cyclic 8-amino-acid peptidomimetic is used as a reporter for antibody detection of a imidaclothiz. Further, the authors develop two novel techniques, bioluminescent enzyme immunoassay (BLEIA) and a bioluminescence lateral flow immunoassay (BLLFIA), for use with the NanoLuc Luciferase. (5105)

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Sci. Rep. 8(1), 8812. Role of Porphyromonas gingivalis outer membrane vesicles in oral mucosal transmission of HIV. 2018

Dong. X.H., Ho, M.H., Liu. B., Hildreth, J., Dash, C., Goodwin, J.S., Balasubramaniam, M., Chen, C.H., and Xie, H.

Notes: Mucosal microbiota has been shown to influence HIV-1 infection. Here, P. gingivalis outer membrane vesicles (OMVs) show stimulation of receptor-independent HIV-1 entry into epithelial cells. A secretory NanoLuc luciferase is fused to the nef gene of the HIV genome, cell supernatant is collected, and luciferase activity is measured as a proxy for viral replication. The presence of P. gingivalis vesicles increased viral infectivity approximately 10-fold. (5107)

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Anal. Chem. 89, 9527–36. Activity-based detection of consumption of synthetic cannabinoids in authentic urine samples using a stable cannabinoid reporter system. 2017

Cannaert, A., Franz, F., Auwärter, V. and Stove, C.P.

Notes: The NanoBiT® system was used to develop CB1 and CB2 receptor activation bioassays based on β-arrestin recruitment, and stable cell lines were generated for these assays. The cell lines were used to screen for synthetic cannabinoids in urine samples and assayed using the Nano-Glo® Live Cell Reagent. (4966)

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Biochem. Biophys. Rep. 12, 40–5. Application of a novel HiBiT peptide tag for monitoring ATF4 protein expression in Neuro2a cells. 2017

Oh-Hashi, K., Furuta, E., Fujimura, K. and Hirata, Y.

Notes: The authors used CRISPR/Cas9 editing to tag endogenous ATF4 with HiBiT and then studied changes in abundance following various cell treatments. (4928)

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J. Lipid Res. 58, 1722–1729. Cell-based, bioluminescent assay for monitoring the interaction between PCSK9 and the LDL receptor. 2017

Duellman, S.J., Machleidt, T., Cali, J.J. and Vidugiriene, J.

Notes: Researchers used NanoLuc® Luciferase technology to monitor the interaction between and LDLR and PCSK9. (4931)

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ACS Chemical Biology Epub before print. CRISPR-Mediated Tagging of Endogenous Proteins with a Luminescent Peptide 2017

Schwinn, M.K., Machleidt, T., Zimmerman, K., Eggers, C.T., Dixon, A.S., Hurst, R., Hall, M.P., Encell, L.P., Binkowski, B.F. and Wood, K.V.

Notes: Using CRISPR/Cas9 gene editing, HiBiT was tagged to the C terminus of HIF1α and several of its downstream transcriptional target proteins. The Nano-Glo® Lytic Detection System was used to quantify HiBiT-tagged endogenous protein levels, and the Nano-Glo® Live Cell Assay System was used for real-time detection in live cells. Bioluminescence was detected using the GloMax® Discover System. This study demonstrated the ability to efficiently tag endogenous proteins with HiBiT, allowing fast and sensitive quantification of the response dynamics in their regulated expression and covalent modifications. (4869)

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J. Med. Chem. 60, 4869–81. Design and discovery of N-(2-Methyl-5'-morpholino-6'-((tetrahydro-2H-pyran-4-yl)oxy)-[3,3'-bipyridin]-5-yl)-3-(trifluoromethyl)benzamide (RAF709): A potent, selective, and efficacious RAF inhibitor targeting RAS mutant cancers. 2017

Nishiguchi, G.A., Rico, A., Tanner, H., Aversa, R.J., Taft, B.R., Subramanian, S., Setti, L., Burger, M.T., Wan, L., Tamez, V., Smith, A., Lou, Y., Barsanti, P.A., Appleton, B.A., Mamo, M., Tandeske, L., Dix, I., Tellew, J.E., Huang, S., Mathews Griner, L.A., Cooke, V.G., Van Abbema, A., Merritt, H., Ma, S., Gampa, K., Feng, F., Yuan, J., Wang, Y., Haling, J.R., Vaziri, S., Hekmat-Nejad, M., Jansen, J.M., Polyakov, V., Zang, R., Sethuraman, V., Amiri, P., Singh, M., Lees, E., Shao, W., Stuart, D.D., Dillon, M.P. and Ramurthy, S.

Notes: The major goal of this study was to identify selective RAF inhibitors that would suppress the RAF-MEK-ERKK pathway. A NanoBiT® cRAF:bRAF interaction assay was used to study the potency of identified compounds to stabilize this dimer using dose-response assays in transfected HCT116 cells (4967)

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Anal. Biochem. 522, 10-17. Development of a bimolecular luminescence complementation assay for RGS: G protein interactions in cells. 2017

Bodle, C.R., Hayes, M.P., O'Brien, J.B. and Roman, D.L.

Notes: NanoBit™ technology was used to characterize interactions between regulators of G protein signaling proteins (RGS proteins) and target G proteins in this study. (4898)

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Virus Res. 243, 69–74. Development of a rapid and quantitative method for the analysis of viral entry and release using the NanoLuc® luciferase complementation assay. 2017

Sasaki, M., Anindita, P.D., Phongphaew, W., Carr, M., Kobayashi, S., Orba, Y. and Sawa, H.

Notes: The authors developed quantitative methods for the detection of cellular entry and release of subviral and flavivirus-like particles (SVPs, VLPs) by tagging the particles with HiBiT. The HiBiT tag was used for quantitation of the particles, and cellular entry was studied using a LgBiT-expressing stable Vero cell line. (4930)

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