The Francis Crick Institute
ceb17896-086b-49db-a362-024f0eeda88f_15675_-_julian_downward_v2.pdf (2.53 MB)

Development of a cell-free split-luciferase biochemical assay as a tool for screening for inhibitors of challenging protein-protein interaction targets

Download (2.53 MB)
journal contribution
posted on 2020-07-13, 11:52 authored by Rachel Cooley, Neesha Kara, Ning Sze Hui, Jonathan Tart, Chloë Roustan, Roger George, David C Hancock, Brock F Binkowski, Keith V Wood, Mohamed Ismail, Julian Downward
© 2020 Cooley R et al. Targeting the interaction of proteins with weak binding affinities or low solubility represents a particular challenge for drug screening. The NanoLuc Binary Technology (NanoBiT) 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.


Downward FC001070

ERC 834692 - RASImmune

WT 103799/Z/14/Z


Usage metrics

    The Francis Crick Institute



    Ref. manager