Abstract
RAS mutations are the most common oncogenic drivers across human cancers, but there remains a paucity of clinically-validated pharmacological inhibitors of RAS, as druggable pockets have proven difficult to identify. Here, we identify two RAS-binding Affimer proteins, K3 and K6, that inhibit nucleotide exchange and downstream signaling pathways with distinct isoform and mutant profiles. Affimer K6 binds in the SI/SII pocket, whilst Affimer K3 is a non-covalent inhibitor of the SII region that reveals a conformer of wild-type RAS with a large, druggable SII/α3 pocket. Competitive NanoBRET between the RAS-binding Affimers and known RAS binding small-molecules demonstrates the potential to use Affimers as tools to identify pharmacophores. This work highlights the potential of using biologics with small interface surfaces to select unseen, druggable conformations in conjunction with pharmacophore identification for hard-to-drug proteins.
Highlights
RAS mutations are the most common oncogenic drivers across human cancers, but there remains a paucity of clinically-validated pharmacological inhibitors of RAS, as druggable pockets have proven difficult to identify
All three Affimer proteins caused a significant reduction in the amount of KRAS immunoprecipitated, with K3 being the most potent with a 79% reduction compared to a control Affimer in which the variable regions are AAAA and AAE, respectively, while K6 and K37 showed 40% reductions (p < 0.0001, p = 0.0307 and p = 0.0439, respectively; One-way ANOVA with Dunnett’s post hoc test)
The biochemical and cellular profiles of the Affimer proteins used in this study are comparable with scaffold-based biologics that have previously been identified that inhibit RAS, again with nanomolar affinities and IC50 values[16,17,19,20,21,22,38]
Summary
RAS mutations are the most common oncogenic drivers across human cancers, but there remains a paucity of clinically-validated pharmacological inhibitors of RAS, as druggable pockets have proven difficult to identify. KRAS being the most frequently affected member; especially in pancreatic, lung, and colon cancer[1] This makes RAS a strong therapeutic target, but despite having been identified as a drug target for over 30 years, only recently have compounds been developed that show promise in pre-clinical trials[2]. Targeting of RAS has been explored using scaffold-based biologics, besides the cyclic peptide KRpep-2d Antibodies and their alternatives have been developed that bind RAS with nanomolar affinities, inhibiting nucleotide exchange, interactions with RAF, and activation of downstream pathways concurrent with negative impacts on RAS-induced cell growth and transformation[16,17,18,19,20,21]. We explore the possibility of using Affimer proteins, an established biologic with a small probe surface formed by two variable regions[26] known to bind at protein interaction ‘hotspots’[25,27,28], to probe RAS for druggable pockets and conformers that might be amenable to small-molecule inhibition
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