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

Rachel Cooley,Chloë Roustan,David C Hancock,Ning Sze Hui,Keith V Wood,Jonathan Tart,Brock F Binkowski,Julian Downward,Roger George,Neesha Kara,Mohamed Ismail

doi:10.12688/wellcomeopenres.15675.1

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.

Highlights

While enzymatic activities have generally been the preferred starting point for the development of drugs targeting biological processes, there are not always suitable tractable enzymatic targets in signalling pathways of interest
HTRF assay is suitable for detecting the RAS/RAF interaction but not RAS/phosphoinositide 3-kinase (PI3K) We chose homogeneous time-resolved fluorescence (HTRF) as an initial biochemical screening assay since it has previously been shown to be a suitable format for the measurement of active KRAS/CRAF-RAS binding domains (RBDs) binding interactions[20]
To determine if a similar specific response could be seen with p110α we used the full-length p110α fused to GST produced in baculovirus, as the isolated PI3K and their RBDs are known to be poorly soluble[21]

Summary

Introduction

While enzymatic activities have generally been the preferred starting point for the development of drugs targeting biological processes, there are not always suitable tractable enzymatic targets in signalling pathways of interest. In such cases, the transfer of biological signals is likely to be dependent on specific protein-protein interactions (PPIs), which can make attractive alternative targets for drug discovery. For optimal biochemical assay screening, one of the protein concentrations should be close to the Kd (dissociation constant) of the complex binding affinity. Weak binding protein complexes require more screening reagents when using biochemical assays such as HTRF (Homogeneous Time Resolved Fluorescence) which may be prohibitively expensive for high throughput screening[1,2]

Methods

Results

Conclusion