Abstract B24: Development of small-molecule RAS inhibitors using Affimer reagents

Abstract

Abstract Ras proteins are small GTPases that are mutationally activated in around 30% of all human cancers. Oncogenic mutations in Ras trigger uncontrolled cellular differentiation and division through uninhibited Ras-GTP signaling. Despite major efforts in developing inhibitors, lack of treatments directly targeting Ras in cancer led to the current assumption that Ras is undruggable. A new approach, which involves use of biologics, has shown great potential for development of Ras inhibitors, as demonstrated by recent increase in the number of antibody mimetic reagents targeting Ras, including single domain antibodies, monobodies and DARPins. We have developed modulators of Ras activity using novel artificial binding proteins, termed Affimers. Affimers are small 91-amino-acid scaffold proteins that constrain one or two randomized nine amino acid loop regions for molecular recognition. Affimers isolated against KRas, the most commonly mutated Ras family member, displayed low nanomolar binding affinities, were shown to be effective at inhibiting nucleotide exchange and blocked interaction between Ras and its effector Raf. When expressed in mammalian cells, Affimers bound with endogenous Ras and inhibited Ras-mediated signaling. Site-directed mutagenesis of Affimer variable regions revealed three residues critical for binding and inhibition. X-ray crystal structure of Affimer in complex with KRas demonstrated that these residues bind into a hydrophobic pocket on Ras, previously described with small molecules. Currently, we are determining whether this interaction gives insight into new modes of therapeutic development using molecular docking in attempt to mimic the Affimer residues with small molecules. This project provides a unique opportunity to further our understanding of Ras biology through the development of reagents that modulate Ras activity. In addition, structural insights into mode of inhibition allow the modelling and design of small-molecule compounds, providing novel therapeutic strategies to slow Ras-addicted tumor growth. Citation Format: Katarzyna Haza, Heather Martin, Christian Tiede, Kevin Tipping, Chi Trinh, Holly Foster, Rachel Trowbridge, Richard Foster, Thomas Edwards, Alexander Breeze, Michael McPherson, Darren Tomlinson. Development of small-molecule RAS inhibitors using Affimer reagents [abstract]. In: Proceedings of the AACR Special Conference on Targeting RAS-Driven Cancers; 2018 Dec 9-12; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(5_Suppl):Abstract nr B24.