BRET-based RAS biosensors that show a novel small molecule is an inhibitor of RAS-effector protein-protein interactions

Béry, Nicolas; Cruz-Migoni, A.; Bataille, Carole J. R.; Quevedo, Camilo; Tulmin, Hanna; Miller, Arthur I.; Russell, Angela J.; Phillips, Simon E. V.; Carr, S.B.; Rabbitts, Terence H.

doi:10.7554/elife.37122

Cited by 47 publications

(52 citation statements)

References 74 publications

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“…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.…”

Section: Commentarymentioning

confidence: 99%

“…Accordingly, we have engineered genetically encoded Bioluminescence Resonance Energy Transfer 2 (BRET2)‐based RAS biosensors (Bery et al., ). These biosensors are optimized and comprised a full‐length RAS donor molecule (wild‐type and mutant K, N, and HRAS), to respect its intracellular localization, and several different known acceptor partners like PI3Kα, PI3Kγ and CRAF RAS binding domains (RBD) and the RAS associating (RA) domain of RALGDS.…”

Section: Introductionmentioning

confidence: 99%

“…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., ).…”

Section: Introductionmentioning

confidence: 99%

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

Béry

Rabbitts

2019

CP Cell Biology

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Protein-protein interactions (PPIs) are principle biological processes that control normal cell growth, differentiation, and homeostasis but are also crucial in diseases such as malignancy, neuropathy, and infection. Despite the importance of PPIs in biology, this target class has been very challenging to convert to therapeutics. In the last decade, much progress has been made in the inhibition of PPIs involved in diseases, but many remain difficult such as RAS-effector interactions in cancers. We describe here a protocol for using Bioluminescence Resonance Energy Transfer 2 (BRET2)-based RAS biosensors to detect and characterize RAS PPI inhibition by macromolecules and small molecules. This method could be extended to any other small GTPases or any other PPIs of interest. C 2019 by John Wiley & Sons, Inc.Keywords: biosensors r BRET r protein-protein interaction inhibition r RAS family GTPases of 22RAS associating (RA) domain of RALGDS. 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). Figure 1Use of the BRET-based RAS biosensors to assess a PPI. (A) A schematic representation of the BRET-based biosensors. A protein (P1) is fused to the donor moiety RLuc8 and a protein (P2 or P3) is tagged with the acceptor moiety GFP 2 . If P1 does not bind to P2, it only produces a background BRET signal. However, when P1 interacts with P2, it induces a BRET signal, if the RLuc8 and GFP 2 domains are within 10 nm. This BRET signal can be decreased by addition of a competitor (either by a macromolecule or a small molecule inhibitor). (B) A representative donor saturation assay between KRAS G12D , KRAS WT and KRAS S17N (donors) and CRAF RBD (acceptor) with total GFP 2 and RLuc8 controls. Where error bars are presented, these correspond to mean values ± SD of quadruplicates technical repeats. Day 2: Cell transfection2. Mix together a fixed amount of RLuc8 construct (typically 50 ng) and a varying amount of GFP 2 construct (see quantity in Table 2).The pEF-myc-cyto empty plasmid is used to equalize total amount of transfected DNA between wells. The total DNA amount transfected per well is 1.6 μg. ...

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Section: Commentarymentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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

Béry

Rabbitts

2019

CP Cell Biology

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“…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.…”

Section: Introductionmentioning

confidence: 99%

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

Haza

Martin

Rao

et al. 2020

Preprint

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Telephone +44 (0)113 34 37099 † These authors contributed equally.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. We have identified two RASbinding Affimer proteins, K3 and K6, that inhibit nucleotide exchange and downstream signalling pathways with distinct isoform and mutant profiles. Affimer K6 is the first biologic to bind in the SI/SII pocket, whilst Affimer K3 is the first noncovalent inhibitor of the SII region, revealing a novel RAS conformer with a large, druggable SII/α3 pocket. This work demonstrates the potential of using biologics with small interface surfaces to select novel druggable conformations in conjunction with pharmacophore identification for hard-to-drug proteins.

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“…Indeed, recent studies have demonstrated that several molecules that bind to either RAS or effector proteins can inhibit RAS signaling by disrupting the interaction between RAS and effector proteins. These include peptides and proteins [3][4][5][6], antibody and antibody fragment [7,8], and small molecule compounds such as sulindac sulfide and its derivatives [9][10][11][12], MCP compounds [13,14], Kobe0065 and Kobe2602 [15], rigosertib [16], Abd series compounds [17], and compound 3344 [18]. Moreover, several independent groups developed small molecules that selectively inhibit oncogenic KRAS (G12C) mutant by covalently binding to Cys-12 to disrupt its effector interaction; these include acrylamide analogue 12 [19], GDP analogues (SML-8-73-1, SML-10-70-1) [20], ARS-853 [21,22] and ARS-1620 [23].…”

Section: Introductionmentioning

confidence: 99%

Development of split luciferase complementation probes sensing KRAS/effector interaction

Miyamoto¹,

Ishihara²,

Sawa³

2019

Translational and Regulatory Sciences

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KRAS is the most frequently mutated oncogene in human cancers. Clinically effective therapy targeting KRAS, however, has not been developed to date despite the decades of intensive efforts. Among various therapeutic approaches taken, blocking the interaction between KRAS and the effector proteins is one of the effective strategies to inhibit KRAS signaling in diseases. Here we have developed the highly-sensitive split ELuc (Emerald Luc, an enhanced luciferase from click beetle) probes which detect the interaction between KRAS (wild-type, G12C and G12D active mutants) and the effector proteins such as BRAF, CRAF, and three RalGEFs (RalGDS, RGL2, and RGL3). The probe pairs transiently co-transfected into 293T cells yielded significantly strong luminescence signals in all combination of KRAS and effector probes but not with negative control probes, indicating the specificity and the sensitivity of the probes. As expected, KRAS G12C and G12D active mutant probes yielded higher signals than KRAS wild-type probe in any combination with effector probes. The novel probe systems described here will be useful tools for the discovery of KRAS genotype-or effector-specific inhibitors.

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BRET-based RAS biosensors that show a novel small molecule is an inhibitor of RAS-effector protein-protein interactions

Cited by 47 publications

References 74 publications

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

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

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

Development of split luciferase complementation probes sensing KRAS/effector interaction

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