scholarly journals Discovery of Influenza Polymerase PA–PB1 Interaction Inhibitors Using an In Vitro Split-Luciferase Complementation-Based Assay

2019 ◽  
Vol 15 (1) ◽  
pp. 74-82 ◽  
Author(s):  
Jiantao Zhang ◽  
Yanmei Hu ◽  
Nan Wu ◽  
Jun Wang
Keyword(s):  
Split Luciferase Complementation ◽  
Split Luciferase ◽  
Influenza Polymerase

Relationship of anti-proliferative activity of TAS-108, a steroid anti-estrogen, and high expression of ERα in MCF7 cells.

2013 ◽  
Vol 31 (15_suppl) ◽  
pp. e11566-e11566
Author(s):  
Ye-Whang Cheong ◽  
Young Choi Kim ◽  
Tae-Hoon Kim
Keyword(s):  
Proliferative Activity ◽  
Firefly Luciferase ◽  
Hek293 Cells ◽  
Mcf7 Cells ◽  
Phase Ii Clinical Study ◽  
The Media ◽  
Split Luciferase Complementation ◽  
Split Luciferase

e11566 Background: TAS-108 is an anti-estrogen that binds to both ERα and ERβ and has potent anti-proliferative activity in vitro and in vivo assay. Recently, the phase II clinical study of TAS-108 showed that it has competitive clinical benefic with lower level of adverse effects. However, the effect of TAS-108 on molecular mechanism, including ER dimerization, remains largely uninvestigated. Methods: The plasmids containing ERα or ERβ fused with N- and C-terminal fragments of firefly luciferase were constructed for split luciferase complementation assays. The resulting plasmids were transfected in HEK293 cells for 24 hr and TAS-108 was added to the media for 6 hr. For RT-qPCR of AREG and TFF1 and proliferation assay, ERβ was transfected in MCF7 cells for 24hr. The proliferation level was determined by measuring the fluorescent level induced by Prestoblue reagent. Results: Using split luciferase complementation assay, we determined that TAS-108 induced ERα/β heterodimerization about 100-fold more potently than ERα/α homodimerization. Increasing the ERβ level by transfection in MCF7 cells (MCF7-ERβ), potency of TAS-108 was increased significantly; 30nM TAS-108 was suitable to block the E2 mediated increase of AREG and TFF1 expression and significantly decrease proliferation of MCF7-ERβ. Conclusions: Taken together, these data suggest that TAS-108 has a higher affinity for ERα/β heterodimerization than ERα/α homodimerization and TAS-108 shows more effective anti-proliferative activity by blocking the expression of E2-induced proliferative genes when coupled with increased levels of ERβ. These findings also underscore the molecular role of ERβ in the biology and suggest the possibility of the compound inducing ERα/β heterodimerization as anti-breast cancer drugs.

Assay methods for small ubiquitin-like modifier (SUMO)–SUMO-interacting motif (SIM) interactions in vivo and in vitro using a split-luciferase complementation system

2014 ◽  
Vol 448 ◽  
pp. 92-94 ◽  
Author(s):  
Mikako Hirohama ◽  
Arnout R.D. Voet ◽  
Takeaki Ozawa ◽  
Hisato Saitoh ◽  
Yoichi Nakao ◽  
...  
Keyword(s):  
Split Luciferase Complementation ◽  
Assay Methods ◽  
Split Luciferase

scholarly journals Identification of Compounds Targeting Hepatitis B Virus Core Protein Dimerization through a Split Luciferase Complementation Assay

2018 ◽  
Vol 62 (12) ◽  
Author(s):  
Xia-Fei Wei ◽  
Chun-Yang Gan ◽  
Jing Cui ◽  
Ying-Ying Luo ◽  
Xue-Fei Cai ◽  
...  
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Protein Interactions ◽  
Core Protein ◽  
Cell Model ◽  
Protein Protein Interactions ◽  
B Virus ◽  
Split Luciferase Complementation ◽  
Split Luciferase

ABSTRACTThe capsid of the hepatitis B virus is an attractive antiviral target for developing therapies against chronic hepatitis B infection. Currently available core protein allosteric modulators (CpAMs) mainly affect one of the two major types of protein-protein interactions involved in the process of capsid assembly, namely, the interaction between the core dimers. Compounds targeting the interaction between two core monomers have not been rigorously screened due to the lack of screening models. We report here a cell-based assay in which the formation of core dimers is indicated by split luciferase complementation (SLC). Making use of this model, 2 compounds, Arbidol (umifenovir) and 20-deoxyingenol, were identified from a library containing 672 compounds as core dimerization regulators. Arbidol and 20-deoxyingenol inhibit the hepatitis B virus (HBV) DNA replicationin vitroby decreasing and increasing the formation of core dimer and capsid, respectively. Our results provided a proof of concept for the cell model to be used to screen new agents targeting the step of core dimer and capsid formation.

scholarly journals A Detection Method for GLUT4 Exocytosis Based on Spontaneous Split Luciferase Complementation

2019 ◽  
Vol 35 (8) ◽  
pp. 835-838 ◽  
Author(s):  
Mizuki ENDO ◽  
Masashi MIYASAKI ◽  
Qiaojing LI ◽  
Genki KAWAMURA ◽  
Takeaki OZAWA
Keyword(s):  
Detection Method ◽  
Split Luciferase Complementation ◽  
Split Luciferase

scholarly journals A Split Luciferase Complementation Assay for the Quantification of β-Arrestin2 Recruitment to Dopamine D2-Like Receptors

2020 ◽  
Vol 21 (17) ◽  
pp. 6103
Author(s):  
Lisa Forster ◽  
Lukas Grätz ◽  
Denise Mönnich ◽  
Günther Bernhardt ◽  
Steffen Pockes
Keyword(s):  
Kinase Inhibitors ◽  
Radioligand Binding ◽  
Binding Studies ◽  
Time Resolved ◽  
Protein Activation ◽  
Kinetic Information ◽  
Split Luciferase Complementation ◽  
Homogeneous Assay ◽  
Radioligand Binding Studies ◽  
Split Luciferase

Investigations on functional selectivity of GPCR ligands have become increasingly important to identify compounds with a potentially more beneficial side effect profile. In order to discriminate between individual signaling pathways, the determination of β-arrestin2 recruitment, in addition to G-protein activation, is of great value. In this study, we established a sensitive split luciferase-based assay with the ability to quantify β-arrestin2 recruitment to D2long and D3 receptors and measure time-resolved β-arrestin2 recruitment to the D2long receptor after agonist stimulation. We were able to characterize several standard (inverse) agonists as well as antagonists at the D2longR and D3R subtypes, whereas for the D4.4R, no β-arrestin2 recruitment was detected, confirming previous reports. Extensive radioligand binding studies and comparisons with the respective wild-type receptors confirm that the attachment of the Emerald luciferase fragment to the receptors does not affect the integrity of the receptor proteins. Studies on the involvement of GRK2/3 and PKC on the β-arrestin recruitment to the D2longR and D3R, as well as at the D1R using different kinase inhibitors, showed that the assay could also contribute to the elucidation of signaling mechanisms. Its broad applicability, which provides concentration-dependent and kinetic information on receptor/β-arrestin2 interactions, renders this homogeneous assay a valuable method for the identification of biased agonists.

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