Journal: eLife

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

doi: 10.7554/eLife.37122

Figure Lengend Snippet: 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 GFP 2 moiety (acceptor) does not bind to RAS, it only produces a background BRET signal. However, when an acceptor binds to RAS, it induces a BRET signal, if the luciferase and GFP domains are within 100 Å. The BRET signal can be decreased by addition of a competitor (either by a macrodrug or a small molecule inhibitor). The interaction titration of full-length KRAS G12D -CAAX (for simplicity, the CAAX motif is omitted in all the RAS constructs described hereafter) with the four effector acceptor proteins and the effect on intracellular protein levels are shown in B and C . Competition assays show the specificity of the RAS biosensors in D (iDAb) and E (RAS-binding compounds). In D , the non-relevant anti-LMO2 iDAb (called hereafter iDAb control, Ctl) serves as a negative control and anti-RAS iDAb (herein named iDAb RAS) serves as a positive control. In E , 3344 (black bars) decreases KRAS G12D /effector domain interactions in a dose-dependent manner showing its broad range of inhibition. Cells were treated with 5, 10 and 20 μM of 3344 (black bars), Abd-2 (grey bars) or DMSO alone (white bars) as the negative control. Statistical analysis was performed with a one-way ANOVA followed by Dunnett’s post-hoc tests (*p<0.05, ***p<0.001, ****p<0.0001). Each experiment was repeated three ( B, D ) or four times ( E ). Where error bars are presented, these correspond to mean values ± SD of biological repeats ( B, D–E ). See also , , and .

Article Snippet: The sample preparation is exemplified as follows: for a 5 μM GST-KRAS G12V sample: 55 μM of the 3344 compound (1.1 μL of a 10 mM solution in DMSO- d 6 ) was added to an Eppendorf before sequential addition of the D 2 O PBS buffer (194.0 μL) and GST-KRAS G12V (4.9 μL of a 205 μM solution, the protein is in an H 2 O buffer for stability reason).

Techniques: Clinical Proteomics, Membrane, Luciferase, Titration, Construct, Binding Assay, Control, Negative Control, Positive Control, Inhibition

Journal: eLife

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

doi: 10.7554/eLife.37122

Figure Lengend Snippet: ( A ) Chemical structure of 3344. ( B ) 1 H NMR and ( C ) 13 C NMR spectra of 3344 were recorded on a Bruker Avance spectrometer (600 MHz) at room temperature in a solution of the deuterated solvent (CDCl 3 ). The field was locked by external referencing to the relevant deuteron resonance. Chemical shifts are reported in parts per million (ppm). ( D ) NMR Carr-Purcell-Meiboom-Gill (CPMG) evaluation of 3344 Kd. Dose-dependent CPMG spectra of 3344 (at a fixed concentration of 55 μM) were recorded on a Bruker Avance spectrometer (700 MHz) at room temperature against an array of concentration of GST-KRAS G12V (0 to 20 μM, left hand panel). The amount of protein was increased from 0 μM until the signals of the compound completely disappear in the proton NMR (here 20 μM). The integrations of the proton acquired were all compared to the compound alone (0 μM of protein) in order to obtain a percentage of decrease for each concentration of GST-KRAS G12V . Concentration and percentage of decrease were plotted and Kd fitting was run on the generated binding curve using Origin ® software (right hand panel, see Materials and methods for details). ( E ) WaterLOGSY spectra of 3344 interacting with GST-KRAS G12V -GppNHp. The proton NMR of 3344 is the lower spectrum (blue), the spectrum of 3344 with KRAS is shown in the top (green) and the inhibitory effect of added anti-RAS scFv on 3344 binding to KRAS is shown in the middle spectrum (red). ( F ) Chemical structure of Abd-2. ( G–I ) 3344 decreases KRAS G12D -iDAb dm RAS interaction in a dose-dependent manner and not with iDAb RAS or with a negative BRET-biosensor LMO2-iDAb dm LMO2. Statistical analyses were performed using a one-way ANOVA followed by Dunnett’s post-tests (*p<0.05, ****p<0.0001). ( J ) Total GFP 2 and RLuc8 levels from the BRET competition assay shown in G-I and . Each experiment was repeated four times ( G–I ). Where error bars are presented, they correspond to mean values ± SD of biological repeats.

Article Snippet: The sample preparation is exemplified as follows: for a 5 μM GST-KRAS G12V sample: 55 μM of the 3344 compound (1.1 μL of a 10 mM solution in DMSO- d 6 ) was added to an Eppendorf before sequential addition of the D 2 O PBS buffer (194.0 μL) and GST-KRAS G12V (4.9 μL of a 205 μM solution, the protein is in an H 2 O buffer for stability reason).

Techniques: Solvent, Concentration Assay, Proton NMR, Generated, Binding Assay, Software, Competitive Binding Assay

Journal: eLife

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

doi: 10.7554/eLife.37122

Figure Lengend Snippet: ( A ) BRET titration curves of KRAS mutants with full-length CRAF S257L (CRAF FL ). KRAS G12D interacts with GFP 2 -CRAF FL while it gives a low BRET ratio with CRAF FL -GFP 2 . The dominant negative KRAS S17N does not interact with GFP 2 -CRAF FL showing the accuracy and optimization of this biosensor. ( B ) Controls from . The expression level of the BRET pair was assessed by western blot with the GFP (for CRAF FL ) and pan-RAS (for RLuc8-KRAS G12D ) antibodies. iDAb expression was revealed using anti-FLAG antibody; anti-β-actin binding was used as the loading control. ( C ) Controls from . The expression level of the BRET pair was assessed with the GFP (for CRAF FL ) and pan-RAS (for RLuc8-KRAS G12D ) antibodies, anti-β-actin binding was used as the loading control. ( D–F ) Short-term incubation of the compounds (3 hr) on cells transfected with the KRAS G12D /CRAF FL biosensor. The BRET ratio was measured in the presence of an increasing dose of compound 3344 ( D ). This induces a dose-dependent decrease of MEK and ERK kinase phosphorylation ( E ) after cells expressing the KRAS G12D /CRAF FL biosensor pair were treated 3 hr with DMSO, 10 and 20 μM of Abd-2 and 3344 compounds or not treated (untreated lane). Quantification of the relative levels of pMEK1/2 and pERK1/2, normalized to total MEK1/2 and ERK1/2 respectively, are shown in panel F. ( G ) Controls from panel E. ( H ) Controls from . The expression level of each BRET pair was assessed with the GFP (for CRAF FL ) and pan-RAS (for RLuc8-KRAS G12X ) antibodies. One-way ANOVA followed by Dunnett’s post-hoc tests were used to determine the statistical significance of BRET, pERK and pMEK modulations induced by the compounds (*p<0.05, **p<0.01, ****p<0.0001). Each experiment was repeated twice ( A, E–F ) or three times ( D ). Where error bars are presented, they correspond to mean values ± SD of biological repeats ( A, D ) or correspond to mean ± SEM of biological repeats ( F ).

Article Snippet: The sample preparation is exemplified as follows: for a 5 μM GST-KRAS G12V sample: 55 μM of the 3344 compound (1.1 μL of a 10 mM solution in DMSO- d 6 ) was added to an Eppendorf before sequential addition of the D 2 O PBS buffer (194.0 μL) and GST-KRAS G12V (4.9 μL of a 205 μM solution, the protein is in an H 2 O buffer for stability reason).

Techniques: Titration, Dominant Negative Mutation, Expressing, Western Blot, Binding Assay, Control, Incubation, Transfection, Phospho-proteomics

Journal: eLife

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

doi: 10.7554/eLife.37122

Figure Lengend Snippet: 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 transfected into HEK293T cells and competed with iDAb expression as indicated; the BRET ratios are shown in A and western blot data in B . The iDAb RAS inhibition of phosphorylation of ERK and MEK signals are quantified in C . The β-actin loading control, iDAbs and BRET pair expression controls are shown in . In D , the BRET ratio of KRAS G12D /CRAF FL interaction was measured in the presence of an increasing dose of compound 3344. This induces a dose-dependent decrease of MEK and ERK kinase phosphorylation ( E ) after cells expressing the KRAS G12D /CRAF FL biosensor pair were treated 20 hr with DMSO, 10 and 20 μM of Abd-2 and 3344 compounds or not treated (untreated lane). The β-actin loading control and BRET pair expression controls are shown in . Quantification of the relative levels of pMEK1/2 and pERK1/2, normalized to total MEK1/2 and ERK1/2 respectively, are shown in F . The RAS biosensor toolkit includes KRAS G12A, G12C, G12V and G12R, in addition to KRAS G12D. In G , each was expressed with CRAF FL and BRET ratios determined at 0, 5, 10 and 20 μM Abd-2 or 3344. Statistical analyses in C were performed using a one-way ANOVA followed by Sidak’s post-hoc tests and in A , D , F and G using a one-way ANOVA followed by Dunnett’s post-tests (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001). Each experiment was repeated twice ( E–F ), three times ( B–D ), four times ( A ) or five times ( G ). Where error bars are presented, they correspond to mean values ± SD of biological repeats ( A, D, G ) or correspond to mean ±SEM of biological repeats ( C, F ). See also .

Article Snippet: The sample preparation is exemplified as follows: for a 5 μM GST-KRAS G12V sample: 55 μM of the 3344 compound (1.1 μL of a 10 mM solution in DMSO- d 6 ) was added to an Eppendorf before sequential addition of the D 2 O PBS buffer (194.0 μL) and GST-KRAS G12V (4.9 μL of a 205 μM solution, the protein is in an H 2 O buffer for stability reason).

Techniques: Expressing, Transfection, Western Blot, Inhibition, Phospho-proteomics, Control

Journal: eLife

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

doi: 10.7554/eLife.37122

Figure Lengend Snippet: ( 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 . The expression level of the BRET pair was assessed with the GFP (for CRAF FL ) and pan-RAS (for RLuc8-KRAS WT ) antibodies. iDAb expression is revealed by the CMYC tag antibody; anti-β-actin binding was used as the loading control. ( C ) Controls from . The expression level of the BRET pair was assessed with the GFP (for CRAF FL ) and pan-RAS (for RLuc8-KRAS WT ) antibodies. Anti-β-actin binding was used as control. Panel D shows the short-term effect on BRET2 signal of compounds Abd-2 (grey bars) and 3344 (black bars) on KRAS WT /CRAF FL interaction after EGF treatment in a BRET competition experiment (3 hr incubation of the compounds). In panel E, HEK293T cells were transfected with the plasmids expressing the BRET pair KRAS WT /CRAF FL for 24 hr, serum starved 24 hr and then incubated for 3 hr with DMSO, 10 and 20 μM of Abd-2 and 3344 compounds. Cells were treated 5 min with EGF (50 ng/mL), lysed and analysed by western blot. Quantification of panel E is shown in panel F. ( G ) Controls from panel E. One-way ANOVA followed by Dunnett’s post-hoc tests were used to determine the statistical significance of BRET, pERK and pMEK modulations induced by the compounds (**p<0.01, ***p<0.001, ****p<0.0001). Each experiment was repeated twice ( A, E–F ) or three times ( D ). Where error bars are presented, they correspond to mean values ± SD of biological repeats ( A, D ) or correspond to mean ± SEM of biological repeats ( F ).

Article Snippet: The sample preparation is exemplified as follows: for a 5 μM GST-KRAS G12V sample: 55 μM of the 3344 compound (1.1 μL of a 10 mM solution in DMSO- d 6 ) was added to an Eppendorf before sequential addition of the D 2 O PBS buffer (194.0 μL) and GST-KRAS G12V (4.9 μL of a 205 μM solution, the protein is in an H 2 O buffer for stability reason).

Techniques: Titration, Expressing, Binding Assay, Control, Incubation, Transfection, Western Blot

Journal: eLife

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

doi: 10.7554/eLife.37122

Figure Lengend Snippet: 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 with or without iDAbs and stimulated by EGF (50 ng/mL). iDAb RAS shows an inhibition of KRAS WT /CRAF FL interaction after EGF treatment in a dose-dependent manner. B is a western blot of the transfected cells from panel A showing the effect of the iDAbs on EGF-stimulated RAS-RAF-MEK-ERK signaling pathway (pMEK and pERK signals are quantified in C ). β-actin loading control, iDAbs and BRET pair expression controls are shown in . The effect on BRET2 signal of compounds Abd-2 (grey bars) and 3344 (black bars) on KRAS WT /CRAF FL interaction after EGF treatment in a BRET competition experiment is shown in panel D. In panel E, HEK293T cells were transfected as in D with the plasmids expressing the BRET pair KRAS WT /CRAF FL for 24 hr and serum starved 20 hr in the presence of DMSO, 10 and 20 μM of Abd-2 and 3344 compounds. Cells were treated 5 min with EGF (50 ng/mL), lysed and analyzed by western blot. The expression level of the BRET protein pair is shown in as well as the loading control β-actin for the western blot. The western blot data are quantified in panel F. One-way ANOVA followed by Dunnett’s post-hoc tests were used to determine the statistical significance of BRET, pERK and pMEK modulations induced by the compound or the iDAb (*p<0.05, ***p<0.001, ****p<0.0001). Each experiment was repeated twice ( B–C ) or three times ( A, D–F ). Where error bars are presented, they correspond to mean values ± SD of biological repeats ( A, D ) or correspond to mean ±SEM of biological repeats ( C, F ). See also .

Article Snippet: The sample preparation is exemplified as follows: for a 5 μM GST-KRAS G12V sample: 55 μM of the 3344 compound (1.1 μL of a 10 mM solution in DMSO- d 6 ) was added to an Eppendorf before sequential addition of the D 2 O PBS buffer (194.0 μL) and GST-KRAS G12V (4.9 μL of a 205 μM solution, the protein is in an H 2 O buffer for stability reason).

Techniques: Transfection, Inhibition, Western Blot, Control, Expressing

Journal: eLife

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

doi: 10.7554/eLife.37122

Figure Lengend Snippet: ( 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 the BRET pair but not KRAS S17N . The optimal BRET signal is obtained with the following pair: RLuc8-KRAS G12D /PI3Kα FL -GFP 2 . ( B ) Controls from . The expression level of the BRET pair was assessed with the GFP (for PI3Kα FL ) and pan-RAS (for RLuc8-KRAS G12D ) antibodies. iDAb and p85α FL expression was revealed by the CMYC tag antibody, β-actin was used as the loading control. ( C ) Controls from . The expression level of the BRET pair was assessed with the GFP (PI3Kα FL ) and pan-RAS (RLuc8-KRAS G12D ) and CMYC (p85α FL ) antibodies. Anti-β-actin was used as the loading control. In panel D, HEK293T cells transfected with the BRET biosensor KRAS G12D /PI3Kα FL were treated for 3 hr with DMSO (white bar), 5, 10 and 20 μM of Abd-2 (grey bars) and 3344 (black bars) compounds and the BRET signal of the biosensor was assessed. In panel E, the cells were transfected and treated as in panel D but with 10 and 20 μM of Abd-2 and 3344 compounds. 3 hr after the treatment, cells were lysed and analyzed by western blot using anti-pAKT (Ser 473) or anti-pan-AKT antibody. The signal in the western blot is quantitated in panel F. ( G ) Controls from panel E. One-way ANOVA followed by Dunnett’s post-hoc tests were used to determine the statistical significance of BRET and pAKT modulations induced by the compounds (**p<0.01, ****p<0.0001). Each experiment was repeated twice ( A, E–F ) or three times ( D ). Where error bars are presented, they correspond to mean values ± SD of biological repeats ( A, D ) or correspond to mean ± SEM of biological repeats ( F ).

Article Snippet: The sample preparation is exemplified as follows: for a 5 μM GST-KRAS G12V sample: 55 μM of the 3344 compound (1.1 μL of a 10 mM solution in DMSO- d 6 ) was added to an Eppendorf before sequential addition of the D 2 O PBS buffer (194.0 μL) and GST-KRAS G12V (4.9 μL of a 205 μM solution, the protein is in an H 2 O buffer for stability reason).

Techniques: Titration, Expressing, Control, Transfection, Western Blot

Journal: eLife

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

doi: 10.7554/eLife.37122

Figure Lengend Snippet: 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 co-transfected with the biosensor and increasing levels of competitor plasmids encoding iDAbs RAS (black striped bars) or iDAb control (grey striped bars) or biosensor alone (white bar). iDAb RAS impedes KRAS G12D /PI3Kα FL interaction and this inhibition causes a decrease of pAKT at serine 473 as shown by western blot in B and its quantification in C . UT is for untransfected cells. In D, HEK293T cells transfected with the BRET biosensor KRAS G12D /PI3Kα FL were treated for 20 hr with DMSO (white bar), 5, 10 and 20 μM of Abd-2 (grey bars) and 3344 (black bars) compounds and the BRET signal of the biosensor was assessed. In panel E, the cells were transfected and treated as in D but with 10 and 20 μM of Abd-2 and 3344 compounds. 20 hr after the treatment, cells were lysed and analysed by western blot using anti-pAKT (Ser 473) or anti-pan-AKT antibody. The signal in the western blot is quantitated in F . Related controls are shown on . One-way ANOVA followed by Dunnett’s post-hoc tests were used to determine the statistical significance of BRET and pAKT modulations induced by the compound or the iDAb (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001). Each experiment was repeated twice ( E–F ) or three times ( A–D ). Where error bars are presented, they correspond to mean values ± SD of biological repeats ( A, D ) or correspond to mean ±SEM of biological repeats ( C, F ). See also .

Article Snippet: The sample preparation is exemplified as follows: for a 5 μM GST-KRAS G12V sample: 55 μM of the 3344 compound (1.1 μL of a 10 mM solution in DMSO- d 6 ) was added to an Eppendorf before sequential addition of the D 2 O PBS buffer (194.0 μL) and GST-KRAS G12V (4.9 μL of a 205 μM solution, the protein is in an H 2 O buffer for stability reason).

Techniques: Produced, Transfection, Control, Inhibition, Western Blot

Journal: eLife

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

doi: 10.7554/eLife.37122

Figure Lengend Snippet: 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 ( C , D ). These were treated with 5, 10 and 20 μM of Abd-2 (grey bars) or 3344 (black bars) compounds for 20 hr. DMSO (white bar) was used as the negative control. Statistical analyses were performed using a one-way ANOVA followed by Dunnett’s post-tests (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001). Each experiment was repeated at least four times. Where error bars are presented, they correspond to mean values ± SD of biological repeats ( A–D ). See also .

Article Snippet: The sample preparation is exemplified as follows: for a 5 μM GST-KRAS G12V sample: 55 μM of the 3344 compound (1.1 μL of a 10 mM solution in DMSO- d 6 ) was added to an Eppendorf before sequential addition of the D 2 O PBS buffer (194.0 μL) and GST-KRAS G12V (4.9 μL of a 205 μM solution, the protein is in an H 2 O buffer for stability reason).

Techniques: Transfection, Expressing, Negative Control

Journal: eLife

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

doi: 10.7554/eLife.37122

Figure Lengend Snippet: 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. The compound is shown binding in the hydrophobic pocket near switch I (shown in red) and switch II (shown in blue). The electron density map of the compound (2Fo-Fc) is shown as green mesh, and contoured at 1.0 rms. ( B ) We have modeled the potential interactions that could prevent 3344 and a RAS effector binding simultaneously to the same RAS molecule by overlaying our structure of the KRAS-3344 complex onto the published structures of top panel: HRAS-CRAF RBD (PDB 4G3X), middle panel: HRAS-RALGDS RA (PDB 1LFD), bottom panel: HRAS-PI3Kγ RBD (PDB 1HE8). ( C, D ) Two human mutant KRAS expressing lines (C: DLD-1 and D: H358) were serum-starved for 24 hr and treated 3 hr with different concentrations of 3344 (2, 5, 10 and 20 μM) before stimulation with EGF (50 ng/mL) for 10 min. Cells were harvested, proteins extracted and separated by SDS-PAGE for western blot analysis. Western membranes were treated with anti-pAKT S473; anti-pan AKT; anti-pERK1/2 and anti-ERK1/2 as indicated. Statistical analyses of pERK/ERK and pAKT/AKT quantifications were performed using a one-way ANOVA followed by Dunnett’s post-tests (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001). Where error bars are presented, they correspond to mean values ± SEM of biological repeats ( C–D ). Each experiment was performed twice ( C–D ).

Article Snippet: The sample preparation is exemplified as follows: for a 5 μM GST-KRAS G12V sample: 55 μM of the 3344 compound (1.1 μL of a 10 mM solution in DMSO- d 6 ) was added to an Eppendorf before sequential addition of the D 2 O PBS buffer (194.0 μL) and GST-KRAS G12V (4.9 μL of a 205 μM solution, the protein is in an H 2 O buffer for stability reason).

Techniques: Mutagenesis, Binding Assay, Expressing, SDS Page, Western Blot

Journal: eLife

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

doi: 10.7554/eLife.37122

Figure Lengend Snippet: Data processing and refinement statistics.

Article Snippet: The sample preparation is exemplified as follows: for a 5 μM GST-KRAS G12V sample: 55 μM of the 3344 compound (1.1 μL of a 10 mM solution in DMSO- d 6 ) was added to an Eppendorf before sequential addition of the D 2 O PBS buffer (194.0 μL) and GST-KRAS G12V (4.9 μL of a 205 μM solution, the protein is in an H 2 O buffer for stability reason).

Techniques: