Abstract C038: High throughput application of the NanoBiT Biochemical Assay for the discovery of selective p110α isoform binders that block its interaction with KRAS

作者：Mohamed Ismail, Julian Downward, Jonathan Tart, Gareth M. Davies, Tiziana Monteverde, David C. Hancock, Santosh Adhikari, Christopher J. Stubbs, Carolyn Blackett, Geoffrey A. Holdgate, Jason G. Kettle · 发表于：Molecular Cancer Therapeutics · 年份：2023 · DOI：10.1158/1535-7163.targ-23-c038 · 研究领域：Cancer-related Molecular Pathways、Ubiquitin and proteasome pathways、Cancer, Hypoxia, and Metabolism

Abstract RAS proteins contribute to the activation of p110α by directly interacting with its RAS binding domain (RBD), resulting in the promotion of cellular functions such as cell growth, proliferation and survival. Previously, we have shown that blocking the interaction of p110α with oncogenic RAS, by introducing specific mutations in the RBD, had a significant impact on tumor initiation and growth in mouse models, whilst having little effect on the health of normal adult mice. These studies highlight the significance of the p110α/KRAS protein-protein interaction (PPI) in tumor progression and maintenance and strongly suggest its importance as a drug target. Thus, we initiated a drug discovery project aiming to identify molecules that bind to p110α and perturb its interaction with KRAS. However, the rather weak binding affinity of the p110α/KRAS interaction (Kd = 3 μM) and the poor solubility of p110α raised significant issues when considering the use of currently available biochemical assays. The NanoLuc® Binary Technology (NanoBiT®) assay was originally developed to detect PPIs in live mammalian cells. However, the assay in its current state was not ideal for high throughput screening (HTS) due to the requirement for large-scale cell based assays. Therefore, we developed the NanoBiT Biochemical Assay (NBBA) as a more suitable and cost-effective approach. The NBBA centres on expressing modified target proteins of interest in mammalian cells and then screening cell lysates ...