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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Embargo End Date
ICR Authors
Authors
Cooley, R
Kara, N
Hui, NS
Tart, J
Roustan, C
George, R
Hancock, DC
Binkowski, BF
Wood, KV
Ismail, M
Downward, J
Kara, N
Hui, NS
Tart, J
Roustan, C
George, R
Hancock, DC
Binkowski, BF
Wood, KV
Ismail, M
Downward, J
Document Type
Journal Article
Date
2020-01-01
Date Accepted
2020-01-29
Abstract
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.
Citation
Wellcome open research, 2020, 5 pp. 20 - ?
Source Title
Publisher
F1000 Research Ltd
ISSN
2398-502X
eISSN
2398-502X
Research Team
Signal Transduction & Molecular Pharmacology
Lung Cancer Group
Lung Cancer Group