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

Version of Record: August 3, 2018
Version of Record: July 10, 2018

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Nicolas Bery

Abimael Cruz-Migoni

Carole JR Bataille

Camilo E Quevedo

Hanna Tulmin

Ami Miller

Angela Russell

Simon EV Phillips

Stephen B Carr

Terence H Rabbitts

(2018)
BRET-based RAS biosensors that show a novel small molecule is an inhibitor of RAS-effector protein-protein interactions

eLife 7:e37122.

https://doi.org/10.7554/eLife.37122
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Figures
Tables
Additional files

9 figures, 2 tables and 2 additional files

Figures

Figure 1 with 3 supplements

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RAS-effector BRET biosensors and interference of KRAS-effector interactions by a RAS-binding compound.

An outline of the BRET2-based RAS biosensor system is shown in A. RAS bound to the plasma membrane (PM) is fused at its amino terminal end to the RLuc8 moiety (donor). When a protein fused to the …
https://doi.org/10.7554/eLife.37122.003

Figure 1—figure supplement 1

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Optimization of the RAS biosensors.

(A) Optimization of the donor and acceptor linker length. Top panel shows KRAS^G12D/iDAb RAS optimization and the bottom panel shows KRAS^G12D/CRAF RBD optimization. The red stars indicate the linker …
https://doi.org/10.7554/eLife.37122.004

Figure 1—figure supplement 2

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Validation of the RAS biosensors with the anti-iDAb RAS.

(A) Total GFP² and RLuc8 levels from the BRET titration curves in Figure 1B. (B) Representative BRET titration curves of KRAS^S17N and RAS binders (RBDs and iDAb RAS) with total GFP² and RLuc8 …
https://doi.org/10.7554/eLife.37122.005

Figure 1—figure supplement 3

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3344 inhibits RAS-RBD interactions.

(A) Chemical structure of 3344. (B) ¹H NMR and (C) ¹³C NMR spectra of 3344 were recorded on a Bruker Avance spectrometer (600 MHz) at room temperature in a solution of the deuterated solvent (CDCl₃).…
https://doi.org/10.7554/eLife.37122.006

Figure 2 with 1 supplement

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BRET biosensors of KRAS^G12 mutants and full-length CRAF are inhibited by compound 3344.

A biosensor for the full-length CRAF^S257L (CRAF^FL) protein was made and tested for interaction with mutants of KRAS glycine 12. For A and B, the plasmids expressing BRET pair KRAS^G12D/CRAF^FL was …
https://doi.org/10.7554/eLife.37122.007

Figure 2—figure supplement 1

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Interactions of KRAS^G12X mutants and full-length CRAF are inhibited by 3344.

(A) BRET titration curves of KRAS mutants with full-length CRAF^S257L (CRAF^FL). KRAS^G12D interacts with GFP²-CRAF^FL while it gives a low BRET ratio with CRAF^FL-GFP². The dominant negative KRAS^S17N …
https://doi.org/10.7554/eLife.37122.008

Figure 3 with 1 supplement

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Wild-type KRAS and CRAF biosensor interaction-induced signaling is impaired by 3344.

The BRET KRAS^WT/CRAF^FL pair was tested for interaction after EGF stimulation of HEK293T cells in presence of competitors. In A, cells were transfected with plasmids to express the KRAS^WT biosensor …
https://doi.org/10.7554/eLife.37122.009

Figure 3—figure supplement 1

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3344 inhibits KRAS^WT/CRAF^FL interaction induced by EGF treatment.

(A) BRET titration curves of KRAS^WT with CRAF^FL. After EGF stimulation (50 ng/mL), KRAS^WT contacts CRAF^FL as indicated by an increase of the BRET_max value. (B) Controls from Figure 3B. The …
https://doi.org/10.7554/eLife.37122.010

Figure 4 with 1 supplement

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Interaction between mutant KRAS and full-length PI3Kα BRET pair interaction is impeded by 3344.

The BRET signal produced from the interaction of the KRAS^G12D and full-length PI3Kα (PI3Kα^FL) was obtained by transfecting HEK239T cells with plasmids encoding this BRET pair. In A, cells were …
https://doi.org/10.7554/eLife.37122.011

Figure 4—figure supplement 1

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Interaction of KRAS^G12D with PI3Kα^FL is inhibited by 3344.

(A) BRET titration curves of KRAS^G12D and KRAS^S17N mutants with full-length PI3Kα (PI3Kα^FL). KRAS^G12D interacts with PI3Ka^FL when the full-length regulatory subunit p85a is co-expressed along with …
https://doi.org/10.7554/eLife.37122.012

Figure 5 with 1 supplement

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Compound 3344 inhibits NRAS and HRAS-effector BRET-based biosensors.

HEK293T cells were transfected 24 hr with plasmids expressing the NRAS^Q61H (A, C) and HRAS^G12V (B, D) biosensors together with the indicated RBDs of PI3K, CRAF and RALGDS (A, B) or full-length CRAF …
https://doi.org/10.7554/eLife.37122.013

Figure 5—figure supplement 1

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iDAb RAS inhibits mutant NRAS and HRAS interaction with CRAF^FL.

(A) BRET titration curves of NRAS^Q61H and HRAS^G12V with CRAF^FL with total GFP² and RLuc8 controls. (B) BRET titration curves of NRAS^Q61H and HRAS^G12V with iDAb RAS. (**C, D**) Competition assays show …
https://doi.org/10.7554/eLife.37122.014

Figure 6

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Compound 3344 interacts in a pocket close to the switch regions of KRAS.

The interaction of mutant KRAS with compound 3344 was analyzed by X-ray crystallography. (A) KRAS^Q61H crystals were soaked with 3344 compound and crystal structures obtained from X-ray diffraction. …
https://doi.org/10.7554/eLife.37122.015

Chemical structure 1

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Chemical structure 2

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Chemical structure 3

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Tables

Table 1

Data processing and refinement statistics.

https://doi.org/10.7554/eLife.37122.016

Structure	KRAS^Q61H-3344
Data collection
PDB ID	6F76
Diffraction source	ID30A-1, ESRF
Temperature (K)	100
Wavelength (Å)	0.966
Rotation range per image (°)	0.05
Exposure time per image (s)	0.092
Space group	P 2₁2₁2₁
Molecules/asymmetric unit	6
Unit cell dimensions
a, b, c (Å)	63.17, 118.19, 155.95
α, β, γ (°)	90, 90, 90
Resolution range (Å)	77.98–2.20 (2.16–2.20)*
Total no. of reflections	295785 (13854)
Unique reflections	65992 (2888)
Completeness (%)	99.2 (87.3)
Multiplicity	4.5 (4.8)
Rmeas(I)^†	0.193 (0.997)
Rmerge^‡	0.151 (0.780)
Rpim(I)^§	0.119 (0.612)
I/sigma	5 (1.8)
CC_1/2 (%)^#	0.985 (0.513)
Refinement
No. of reflections, working set	62692 (2744)
No. of reflections, test set	3300 (144)
Rwork/Rfree	22.7/25.0
No. of atoms
Protein	8400
Water	57
Average B factors (Å²)
Protein	46.8
Ligand GTP	31.9
Water	30.1
RMSD
Bond lengths (Å)	0.014
Bond angles (°)	1.67
Ramachandran plot
Favoured regions (%)	97.1
Additionally allowed (%)	2.9
Outliers	0
MolProbity statistics
Overall score	1.11
Clash score	1.22
Rotamer outliers (%)	1.4

a*Values in parentheses are for data in the highest resolution shell.

^†Rmeas = Σ_hkl{N(hkl)/[N(hkl)−1]}^1/2 Σ_i|I_i(hkl)- < I(hkl)>|/ Σ_hkl Σ_iI_i(hkl), where I_i(hkl) is the intensity of reflection hkl. Σ_i is the sum over all i measurements of reflection hkl and N(hkl) is the multiplicity of reflection hkl.
^‡Rmerge = Σ_hkl Σ_i | I_i (hkl)–<I(hkl)>| / Σ_hkl Σ_i I_i (hkl), where I_i (hkl) is the intensity of reflection hkl and Σ_i is the sum over all I measurements of reflection hkl.

^§Rpim= Σ_hkl{1/[N(hkl)−1]} ^1/2 Σ_i|I_i(hkl)- < I (hkl)> |/ Σ_hkl Σ_iI_i(hkl), where I_i(hkl) is the intensity of reflection hkl, Σ_i is the sum over all i measurements of reflection hkl and N(hkl) is the multiplicity of reflection hkl.
^#CC_1/2 is Pearson’s correlation coefficient between random half data sets.

Key resources table

Reagent type (species) or resource	Designation	Source or reference	Identifiers	Additional information
Cell line (human)	HEK293T	ATCC	Cat#CRL-3216 RRID:CVCL_0063
Cell line (human)	DLD-1	ATCC	Cat#CCL-221 RRID:CVCL_0248
Cell line (human)	H358	ATCC	Cat#CRL-5807 RRID:CVCL_1559
Transfected construct (human)	pEF-RLuc8-(GGGS)₃- KRAS^G12D-CAAX plasmid	This paper	N/A	DNA/protein sequences provided in the Supplementary file 1
Transfected construct (human)	pEF-RLuc8-(GGGS)₃- KRAS^G12A-CAAX plasmid	This paper	N/A
Transfected construct (human)	pEF-RLuc8-(GGGS)₃- KRAS^G12C-CAAX plasmid	This paper	N/A
Transfected construct (human)	pEF-RLuc8-(GGGS)₃- KRAS^G12V-CAAX plasmid	This paper	N/A
Transfected construct (human)	pEF-RLuc8-(GGGS)₃- KRAS^G12R-CAAX plasmid	This paper	N/A
Transfected construct (human)	pEF-RLuc8-(GGGS)₃- NRAS^Q61H-CAAX plasmid	This paper	N/A
Transfected construct (human)	pEF-RLuc8-(GGGS)₃- HRAS^G12V-CAAX plasmid	This paper	N/A
Transfected construct (human)	pEF-RLuc8-(GGGS)₃- KRAS^S17N-CAAX plasmid	This paper	N/A
Transfected construct (human)	pEF-RLuc8-(GGGS)₃- KRAS^WT-CAAX plasmid	This paper	N/A
Transfected construct (human)	pEF-GFP²-(GGGS)₃- CRAF^S257LFL plasmid	This paper	N/A
Transfected construct (human)	pEF-PI3Kα^FL-(GGGS)₃- GFP² plasmid	This paper	N/A
Transfected construct (human)	pEF-CRAF RBD (aa 1–149)- (GGGS)₃-GFP² plasmid	This paper	N/A
Transfected construct (human)	pEF-PI3Kα RBD (aa 161–315)- (GGGS)₃-GFP² plasmid	This paper	N/A
Transfected construct (human)	pEF-PI3Kγ RBD (aa 190–315)- (GGGS)₃-GFP² plasmid	This paper	N/A	DNA/protein sequences provided in the Supplementary file 1
Transfected construct (human)	pEF-iDAb RAS-(GGGS)₂- GFP² plasmid	This paper	N/A
Transfected construct (human)	pEF-iDAb_dm RAS-(GGGS)₂- GFP² plasmid	This paper	N/A
Transfected construct (human)	pEF-iDAb control-(GGGS)₂- GFP² plasmid	This paper	N/A
Transfected construct (human)	pEF-LMO2-(GGGS)₂- RLuc8 plasmid	This paper	N/A
Transfected construct (human)	pEF-GFP²-(GGGS)₃- iDAb_dm LMO2 plasmid	This paper	N/A
Transfected construct (human)	pEF-memb-FLAG-iDAb RAS plasmid	This paper	N/A
Transfected construct (human)	pEF-memb-FLAG-iDAb control plasmid	This paper	N/A
Transfected construct (human)	pEF-iDAb RAS-myc plasmid	This paper	N/A
Transfected construct (human)	pEF-iDAb control-myc plasmid	This paper	N/A
Transfected construct (human)	pcDNA3.1-myc-p85α^FL plasmid	A gift from R. Williams and O. Perisic	N/A
Transfected construct (mouse)	pEF-RALGDS RA (aa 788–884)- (GGGS)₃-GFP² plasmid	This paper	N/A	The RALGDS RA domain corresponds to the mouse sequence
Antibody	Phospho-ERK 1/2 Rabbit antibody	Cell Signaling Technology	Cat#9101S RRID:AB_331646
Antibody	Total ERK 1/2 Rabbit antibody	Cell Signaling Technology	Cat#9102S RRID:AB_330744
Antibody	Phospho-MEK 1/2 Rabbit antibody	Cell Signaling Technology	Cat#9154S RRID:AB_2138017
Antibody	Total MEK 1/2 Mouse antibody	Cell Signaling Technology	Cat#4694S RRID:AB_10695868
Antibody	Phospho-AKT S473 Rabbit antibody	Cell Signaling Technology	Cat#4058S RRID:AB_331168
Antibody	Total AKT Rabbit antibody	Cell Signaling Technology	Cat#9272S RRID:AB_329827
Antibody	Pan-RAS Mouse antibody	Millipore	Cat#OP40 RRID:AB_213400
Antibody	GFP Mouse antibody	Santa Cruz Biotechnology	Cat#sc-9996 RRID:AB_627695
Antibody	β-Actin Mouse antibody	Sigma-Aldrich	Cat#A1978 RRID:AB_476692
Antibody	CMYC HRP-linked Goat antibody	Novus Biologicals	Cat#NB600-341 RRID:AB_10000717
Antibody	Anti-Mouse IgG HRP-linked antibody	Cell Signaling Technology	Cat#7076S RRID:AB_330924
Antibody	Anti-Rabbit IgG HRP-linked antibody	Cell Signaling Technology	Cat#7074S RRID:AB_2099233
Recombinant DNA reagent	pEF-myc-cyto vector	Invitrogen	Cat#V89120
Recombinant DNA reagent	pRLuc8-N3 vector	A gift from J. Felce	Felce et al., 2017
Recombinant DNA reagent	pGFP²-N3 vector	A gift from J. Felce	Felce et al., 2017
Recombinant DNA reagent	pBABEpuro-CRAF^S257L FL plasmid	Addgene	Addgene#51125
Peptide, recombinant protein	KRAS^Q61H	This paper	N/A
Peptide, recombinant protein	KRAS^G12V	This paper	N/A
Peptide, recombinant protein	Anti-RAS scFv	This paper	N/A
Peptide, recombinant protein	Recombinant Human Epidermal Growth Factor (EGF)	Life Technologies	Cat#PHG0311
Chemical compound, drug	Coelenterazine 400a	Cayman Chemical	Cat#16157
Chemical compound, drug	2-bromo-6-methoxyphenol	This paper	N/A
Chemical compound, drug	3-bromobenzene-1,2-diol	This paper	N/A
Chemical compound, drug	5-bromo-2,3- dihydrobenzo[b][1,4]dioxine	This paper	N/A
Chemical compound, drug	5-(4-chloro-3- methoxyphenyl)−2,3- dihydrobenzo[b][1,4]dioxine	This paper	N/A
Chemical compound, drug	4-(2,3-dihydrobenzo[b] [1,4]dioxin-5-yl)-N-(4- (dimethylamino) methyl)phenyl)-2- methoxyaniline	This paper	N/A
Software, algorithm	Image J	National Institutes of Health	https://imagej.nih.gov/ij/download.html RRID:SCR_003070
Software, algorithm	Prism 7.0 c	GraphPad	https://www.graphpad.com/scientific-software/prism/ RRID:SCR_002798
Software, algorithm	PROCHECK	Laskowski et al. (1993a)	http://www.ccp4.ac.uk/html/procheck_man/index.html
Software, algorithm	REFMAC	Murshudov et al. (1997)	http://www.ccp4.ac.uk/html/refmac5.html RRID:SCR_014225
Software, algorithm	MolProbity	Chen et al. (2010)	http://molprobity.biochem.duke.edu/ RRID:SCR_014226
Software, algorithm	Phenix	Adams et al. (2010)	https://www.phenix-online.org/ RRID:SCR_014224
Software, algorithm	PyMOL	Schrodinger	https://pymol.org/2/ RRID:SCR_000305
Other	Opti-MEM I Reduced Serum Medium, no phenol red	Thermo-Fisher	Cat#11058021
Other	ViewPlate, White 96-well plate, clear bottom for tissue culture	PerkinElmer	Cat#6005181
Other	BRET2 Dual Emission optical module	PerkinElmer	Cat#2100–8140
Other	Envision instrument, Multilabel Reader	PerkinElmer	Cat#2103