Development of a cell-free split-luciferase biochemical assay as a tool for screening for inhibitors of challenging protein-protein interaction targets.
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Date
2020-01-01ICR Author
Author
Cooley, R
Kara, N
Hui, NS
Tart, J
Roustan, C
George, R
Hancock, DC
Binkowski, BF
Wood, KV
Ismail, M
Downward, J
Type
Journal Article
Metadata
Show full item recordAbstract
Targeting the interaction of proteins with weak binding affinities or low solubility represents a particular challenge for drug screening. The NanoLuc â ® Binary Technology (NanoBiT â ®) was originally developed to detect protein-protein interactions in live mammalian cells. Here we report the successful translation of the NanoBit cellular assay into a biochemical, cell-free format using mammalian cell lysates. We show that the assay is suitable for the detection of both strong and weak protein interactions such as those involving the binding of RAS oncoproteins to either RAF or phosphoinositide 3-kinase (PI3K) effectors respectively, and that it is also effective for the study of poorly soluble protein domains such as the RAS binding domain of PI3K. Furthermore, the RAS interaction assay is sensitive and responds to both strong and weak RAS inhibitors. Our data show that the assay is robust, reproducible, cost-effective, and can be adapted for small and large-scale screening approaches. The NanoBit Biochemical Assay offers an attractive tool for drug screening against challenging protein-protein interaction targets, including the interaction of RAS with PI3K.
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Research team
Signal Transduction & Molecular Pharmacology
Lung Cancer Group
Language
eng
Date accepted
2020-01-29
License start date
2020-01
Citation
Wellcome open research, 2020, 5 pp. 20 - ?
Publisher
F1000 Research Ltd