Small molecule inhibitors of RAS-effector protein interactions derived using an intracellular antibody fragment

Abstract

Targeting specific protein–protein interactions (PPIs) is an attractive concept for drug development, but hard to implement since intracellular antibodies do not penetrate cells and most small-molecule drugs are considered unsuitable for PPI inhibition. A potential solution to these problems is to select intracellular antibody fragments to block PPIs, use these antibody fragments for target validation in disease models and finally derive small molecules overlapping the antibody-binding site. Here, we explore this strategy using an anti-mutant RAS antibody fragment as a competitor in a small-molecule library screen for identifying RAS-binding compounds. The initial hits are optimized by structure-based design, resulting in potent RAS-binding compounds that interact with RAS inside the cells, prevent RAS-effector interactions and inhibit endogenous RAS-dependent signalling. Our results may aid RAS-dependent cancer drug development and demonstrate a general concept for developing small compounds to replace intracellular antibody fragments, enabling rational drug development to target validated PPIs.

Bioluminescence Resonance Energy Transfer 2 (BRET2)‐Based RAS Biosensors to Characterize RAS Inhibitors

“…If the RLuc8 donor fusion molecule catalyzed the coelenterazine 400a and the GFP 2 acceptor fusion molecule is located less than 10 nm from the donor, a transfer of energy will occur from the donor to the acceptor. This method has been widely used to study PPI (Bery et al., ; Felce et al., ; Mercier et al., ) and also PPI inhibition by small molecules (Beautrait et al., ; Corbel et al., ; Corbel et al., ; Lavoie et al., ; Mazars & Fahraeus, ; Quevedo et al., ) or macromolecules (Bery et al., ; Guillard et al., ; Spencer‐Smith et al., ). It offers several advantages: it is a very sensitive technique as it is based on luminescence, there is no background signal from cellular autofluorescence.…”

“…Therefore, using this BRET‐based RAS biosensors toolbox, we characterized the cellular properties of various RAS inhibitors comprising anti‐RAS Design Ankyrin Repeat Proteins (DARPins) (Guillard et al., ) and anti‐RAS intracellular domain antibody (iDAb RAS) (Bery et al., ) macromolecules and antibody derived anti‐RAS compounds (Bery et al., ; Quevedo et al., ).…”

“…We also used as acceptor the full-length CRAF and PI3Kα effectors to study their respective downstream signaling pathways. Therefore, using this BRET-based RAS biosensors toolbox, we characterized the cellular properties of various RAS inhibitors comprising anti-RAS Design Ankyrin Repeat Proteins (DARPins) (Guillard et al, 2017) and anti-RAS intracellular domain antibody (iDAb RAS) (Bery et al, 2018) macromolecules and antibody derived anti-RAS compounds Quevedo et al, 2018).Here we describe a set of protocols that include step-by-step instructions to monitor the interaction between RAS and a partner (Basic Protocol 1), to identify inhibitors of RAS PPIs including macromolecules (Basic Protocol 2) and small molecules (Basic Protocol 3). We also describe a protocol to detect the effect of RAS inhibitors on the RAS downstream pathways by immunoblot (Basic Protocol 4) and finally a protocol explaining how to calculate the BRET ratio obtained in the previous protocols (Basic Protocol 5).…”

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

“…This paucity of agents has been driven by the lack of clearly druggable pockets on the surface of RAS. However, recent work has identified two pockets that may be amenable for drug binding [3][4][5][6][7][8][9][10][11] . The first of these, the SI/II-pocket, exists between the Switch I and Switch II regions of RAS in an area involved in the binding of the nucleotide exchange factor, Son of Sevenless (SOS).…”

“…The first of these, the SI/II-pocket, exists between the Switch I and Switch II regions of RAS in an area involved in the binding of the nucleotide exchange factor, Son of Sevenless (SOS). Several groups have independently developed compounds that bind this pocket with varying affinities and efficacies, predominately in the micromolar range [5][6][7][8] , except for BI-2852 which has nanomolar binding affinity and efficacy 11 . The second, the SII-pocket, is located under the Switch II loop and was identified using a tethered warhead approach relying on the reactive nature of the cysteine in the G12C mutant 9,10 .…”