High throughput application of the NanoBiT Biochemical Assay for the discovery of selective inhibitors of the interaction of PI3K-p110α with KRAS.

The NanoBiT Biochemical Assay (NBBA) was designed as a biochemical format of the NanoBiT cellular assay, aiming to screen weak protein-protein interactions (PPIs) in mammalian cell lysates. Here we present a High Throughput Screening (HTS) application of the NBBA to screen small molecule and fragment libraries to identify compounds that block the interaction of KRAS-G12D with phosphatidylinositol 3-kinase (PI3K) p110α. This interaction promotes PI3K activity, resulting in the promotion of cell growth, proliferation and survival, and is required for tumour initiation and growth in mouse lung cancer models, whilst having little effect on the health of normal adult mice, establishing the significance of the p110α/KRAS interaction as an oncology drug target.

Fragment-Based Discovery of Novel MUS81 Inhibitors.

MUS81 is a structure-selective endonuclease that cleaves various branched DNA structures arising from natural physiological processes such as homologous recombination and mitosis. Due to this, MUS81 is able to relieve replication stress, and its function has been reported to be critical to the survival of many cancers, particularly those with dysfunctional DNA-repair machinery. There is therefore interest in MUS81 as a cancer drug target, yet there are currently few small molecule inhibitors of this enzyme reported, and no liganded crystal structures are available to guide hit optimization.

Targeting a Novel KRAS Binding Site: Application of One-Component Stapling of Small (5-6-mer) Peptides.

RAS proteins are central in the proliferation of many types of cancer, but a general approach toward the identification of pan-mutant RAS inhibitors has remained unresolved. In this work, we describe the application of a binding pharmacophore identified from analysis of known RAS binding peptides to the design of novel peptides. Using a chemically divergent approach, we generated a library of small stapled peptides from which we identified compounds with weak binding activity.

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

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.