KRAS-specific inhibition using a DARPin binding to a site in the allosteric lobe

Abstract

Inhibiting the RAS oncogenic protein has largely been through targeting the switch regions that interact with signalling effector proteins. Here, we report designed ankyrin repeat proteins (DARPins) macromolecules that specifically inhibit the KRAS isoform by binding to an allosteric site encompassing the region around KRAS-specific residue histidine 95 at the helix α3/loop 7/helix α4 interface. We show that these DARPins specifically inhibit KRAS/effector interactions and the dependent downstream signalling pathways in cancer cells. Binding by the DARPins at that region influences KRAS/effector interactions in different ways, including KRAS nucleotide exchange and inhibiting KRAS dimerization at the plasma membrane. These results highlight the importance of targeting the α3/loop 7/α4 interface, a previously untargeted site in RAS, for specifically inhibiting KRAS function.

RAS-inhibiting biologics identify and probe druggable pockets including an SII-α3 allosteric site

“…The biochemical and cellular profiles of the Affimer proteins used in this study are comparable with biologics that have previously been identified that also inhibit RAS, again with nanomolar affinities and IC 50 values 14,15,[17][18][19][20]36 . However, the majority of these do not distinguish between RAS variants, and/or mutants, and structural analyses reveal that these pan-RAS inhibitors are binding in the Switch I/II region, except for the NS1 monobody that binds the α4-β6-α5 dimerization domain 18 , and the DARPins K13 and K19 14 ( Supplementary Fig.…”

“…Importantly, the K3 Affimer shows inhibition of RAS, but also demonstrates a preference for KRAS over the HRAS and NRAS variants. To our knowledge, the only other biologics to express such RAS variant specificity are DARPins K13 and K19 14 . This preferential behaviour is underpinned by the involvement of the H95 residue unique to KRAS; mutation of this residue abolished binding of both Affimer K3 and the DARPins K13 and K19 14 .…”

“…To our knowledge, the only other biologics to express such RAS variant specificity are DARPins K13 and K19 14 . This preferential behaviour is underpinned by the involvement of the H95 residue unique to KRAS; mutation of this residue abolished binding of both Affimer K3 and the DARPins K13 and K19 14 . This ablation was more complete with mutation to glutamine for the DARPins, but was still significant with Affimer K3.…”

“…Targeting of RAS has also been explored using biologics. 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 [14][15][16][17][18][19] . Amongst others these include scFvs, DARPins and monobodies 14,15,17,19,20 .…”

“…Although to date some of these have been used to assist the identification of small molecules 5,7,21 , none has directly probed druggable pockets on RAS, as the majority of these biologics tend to bind over large protein interfaces 14,15,17,20 that are difficult to mimic with small molecules 22 . As some biologics form smaller interfaces 19,23,24 there emerges the tantalising prospect that biologics could be used as tools to identify druggable pockets and novel conformers, and could also have the potential to act as pharmacophore templates for in silico-informed drug discovery.…”