Structural and functional characterization of a DARPin which inhibits Ras nucleotide exchange

Guillard, Sandrine; Kolasinska-Zwierz, Paulina; Debreczeni, J.; Breed, J.; Zhang, Jing; Béry, Nicolas; Marwood, Rose; Tart, Jon; Overman, R.; Stocki, Paweł; Mistry, Bina; Phillips, Christopher; Rabbitts, Terence H.; Jackson, Ronald H.; Minter, Ralph

doi:10.1038/ncomms16111

Cited by 86 publications

(117 citation statements)

References 43 publications

(62 reference statements)

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“…Although the RBDvs are a unique tool for studying Ras-dependent signaling processes, several intracellular affinity reagents targeting GTP-bound Ras have recently been published 6,8,9,10 . This raises the question of how the RBDvs compare to these engineered proteins.…”

Section: Discussionmentioning

confidence: 99%

“…Whether this observation is due to a comparable avidity effect or incomplete release of the antibody from the endosomes is unclear 10 . In addition to these examples, two designed ankyrin repeat proteins (DARPins) with specificity to either the GDP-bound inactive state (K27) or the GTP-bound active state (K55) of Ras have recently been reported 6 . Both DARPins inhibit Ras signaling, however, it remains to be seen if the observed effects also translate to other cell types and organoids.…”

Section: Discussionmentioning

confidence: 99%

“…An alternative, more direct strategy for inhibiting Ras-signaling using engineered proteins is to prevent the recruitment of downstream effectors or block the activation of Ras by GEFs. To this end, several affinity reagents have been generated that compete with Ras effectors by selectively binding to the active or inactive state of Ras 6,7,8,9,10,11,12 . Interestingly, recent work showed that a subset of these affinity reagents had only effects in cells at high concentrations 9,10,13 .…”

Section: Main Textmentioning

confidence: 99%

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Competitive Inhibitors of Ras Effector Binding

Wiechmann

Maisonneuve

Grebbin

et al. 2019

Preprint

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The small GTPases H, K and NRas are molecular switches that are indispensable for the correct regulation of cellular proliferation and growth. Mutations in Ras are associated with cancer and result in unwanted activation of signaling processes caused by aberrant recruitment of downstream effector proteins. In this study, we have engineered variants of the Ras-binding domain (RBD) of CRAF kinase that bind with highly improved affinity the effector binding site of Ras. Structural characterization demonstrates how the engineered RBD variants outcompete effector binding and inhibit Ras signaling in cells leading to apoptosis, growth arrest and senescence. The optimized RBD variants provide new insights in Ras biology and enabled the functional stratification of Ras dependency in patient-derived colorectal cancer organoids. One sentence summary:Inhibition of effector kinase binding to Ras induces senescence in non-tumorigenic cells and reveals Ras dependency in patient-derived cancer organoids.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Main Textmentioning

confidence: 99%

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Competitive Inhibitors of Ras Effector Binding

Wiechmann

Maisonneuve

Grebbin

et al. 2019

Preprint

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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%

“…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%

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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Bifunctional Small‐Molecule Ligands of K‐Ras Induce Its Association with Immunophilin Proteins

Zhang

Shokat

2019

Angewandte Chemie

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Here we report the design, synthesis and characterization of bifunctional chemical ligands that induce the association of Ras with ubiquitously expressed immunophilin proteins such as FKBP12 and cyclophilin A. We show this approach is applicable to two distinct Ras ligand scaffolds, and that both the identity of the immunophilin ligand and the linker chemistry affect compound efficacy in biochemical and cellular contexts. These ligands bind to Ras in an immunophilin-dependent fashion and mediate the formation of tripartite complexes of Ras, immunophilin and the ligand. The recruitment of cyclophilin A to GTP-bound Ras blocks its interaction with B-Raf in biochemical assays. Our study demonstrates the feasibility of ligand-induced association of Ras with intracellular proteins and suggests it as a promising therapeutic strategy for Rasdriven cancers.

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Structural and functional characterization of a DARPin which inhibits Ras nucleotide exchange

Cited by 86 publications

References 43 publications

Competitive Inhibitors of Ras Effector Binding

Competitive Inhibitors of Ras Effector Binding

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

Bifunctional Small‐Molecule Ligands of K‐Ras Induce Its Association with Immunophilin Proteins

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