Abstract C038: High throughput application of the NanoBiT Biochemical Assay for the discovery of selective p110α isoform binders that block its interaction with KRAS
Authors: Ismail Mohamed (Soly) Soliman , Downward Julian , Tart Jonathan , Davies Gareth , Monteverde Tiziana , Hancock David , Adhikari Santosh , Stubbs Christopher , Blackett Carolyn , Holdgate Geoff , Kettle Jason 
Published: December 2023

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 rather than live cells. We have previously shown that the NBBA is suitable for detection of both strong and weak PPIs, as exemplified by KRAS/RAF and KRAS/PI3K. Here we present the first application of NBBA for HTS of the KRAS/p110α PPI, using tagged Sm-KRAS-G12D and Lg-p110α. The NBBA proved to be stable and produced good Z’ factor metrics. Our primary screen of more than 720,000 compounds on KRAS/p110α cell lysates produced around 8,000 initial hits. A concentration response screen comparing KRAS/p110α with two other p110 isoforms, p110δ and p110γ, identified selective p110α-specific compounds and enabled derivation of IC50s for these hits. We identified around 30 compounds showing greater than 20-fold selectivity towards p110α versus p110δ and p110γ with IC50 &lt;2μM. Using 1D-NMR (1 – Dimension Nuclear Magnetic Resonance) we identified 9 compounds that binds directly to purified p110α. The positive and most potent hits will be followed up by co-crystallization with p110α to aid further elucidation of the nature of the interaction and extended optimisation of these compounds.

Citation Format: Mohamed (Soly) Soliman Ismail, Julian Downward, Jonathan Tart, Gareth Davies, Tiziana Monteverde, David Hancock, Santosh Adhikari, Christopher Stubbs, Carolyn Blackett, Geoff Holdgate, Jason Kettle. High throughput application of the NanoBiT Biochemical Assay for the discovery of selective p110α isoform binders that block its interaction with KRAS [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C038.