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: Recombinant DNA reagent , pBABEpuro-CRAF S257L FL plasmid , Addgene , Addgene#51125 , , .

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: Recombinant DNA reagent , pBABEpuro-CRAF S257L FL plasmid , Addgene , Addgene#51125 , , .

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:

Article Snippet: Recombinant DNA reagent , pBABEpuro-CRAF S257L FL plasmid , Addgene , Addgene#51125 , , .

Techniques: Transfection, Construct, Plasmid Preparation, Control, Sequencing, Recombinant, Software

Journal: Scientific Reports

Article Title: Discovery of Potent VEGFR-2 Inhibitors based on Furopyrimidine and Thienopyrimidne Scaffolds as Cancer Targeting Agents

doi: 10.1038/srep24460

Figure Lengend Snippet: Percent inhibition of multiple kinases enzymatic activity exhibited by the target compounds (16e, 21b, 21c, 21e) at 10 μM.

Article Snippet: Poly (Glu, Tyr) sodium salt, (4:1, Glu:Tyr) (Sigma#P7244) served as the standardized substrate for c-Kit, c-Scr kinases, while Inactive MEK1 (BPS Bioscience) and IGF-1Rtide (Anaspec) is the standardized substrate for c-Raf and RET kinases respectively.

Techniques: Inhibition, Activity Assay

Journal: Biophysical Journal

Article Title: Anionic Lipids Impact RAS-Binding Site Accessibility and Membrane Binding Affinity of CRAF RBD-CRD

doi: 10.1016/j.bpj.2020.06.021

Figure Lengend Snippet: Modeling of membrane-anchored RBD-CRD. (A) Domain structure of CRAF. (B) One of the sampled conformations of CRAF RBD-CRD in the simulations. All simulations were started with the CRD (orange cartoons) anchored to a membrane patch but with the RBD (green cartoons) initially positioned away from the the membrane. The membrane patch is shown here using atom-based coloring (blue for carbon, red for oxygen, orange for phosphorus, darker blue for nitrogen, and light gray for hydrogen). The conformation shown here corresponds to pose GH5 in (24), in which the RBD has approached the membrane surface while keeping the main RAS-interacting β-strand (red cartoons) accessible for binding to the RAS G domain. The two zinc ions coordinated with the CRD are shown as gray spheres.

Article Snippet: Cloning, protein expression, and protein purification Gateway Entry clones for CRAF constructs were synthesized using optimization for Escherichia coli or insect cells (ATUM, Newark, CA) that incorporate an upstream tobacco etch virus protease cleavage site (ENLYFQ/G) followed by the appropriate CRAF (human) sequences: CRAF RBD-CRD (52–192) wild-type (WT) and mutants (mts), CRAF RBD (52–131), and CRAF CRD (136–188).

Techniques: Binding Assay

Journal: Biophysical Journal

Article Title: Anionic Lipids Impact RAS-Binding Site Accessibility and Membrane Binding Affinity of CRAF RBD-CRD

doi: 10.1016/j.bpj.2020.06.021

Figure Lengend Snippet: Membrane orientations of the RBD from CG simulations of CRAF RBD-CRD. (A) Free energy surface map for RBD orientations relative to the membrane surface from the CG RBD-CRD simulations. The reaction coordinates along the two axes are described in the text. Two major basins can be seen from this map. (B) Representative snapshots of CG RBD-CRD configurations for the two basins identified in (A). Both CG frames were backmapped to show the secondary structure of RBD-CRD. The RBS of the RBD, comprising a β-strand at residues K65–N71 and the adjacent α-helix at residues K84–R89, are colored red. The left and right panels show the RBS located near the membrane surface or away from it, respectively. Also highlighted in the right panel is a CRAF loop homologous to an ARAF loop that was previously shown to associate with membranes in the presence of KRAS (32). (C) The relative ASA of the RBS in each CG frame is projected onto the surface map of both reaction coordinates from (A). (D) Percentage of total CG simulations frames that fall under particular ranges of relative ASA for the RBS.

Article Snippet: Cloning, protein expression, and protein purification Gateway Entry clones for CRAF constructs were synthesized using optimization for Escherichia coli or insect cells (ATUM, Newark, CA) that incorporate an upstream tobacco etch virus protease cleavage site (ENLYFQ/G) followed by the appropriate CRAF (human) sequences: CRAF RBD-CRD (52–192) wild-type (WT) and mutants (mts), CRAF RBD (52–131), and CRAF CRD (136–188).

Techniques:

Journal: Biophysical Journal

Article Title: Anionic Lipids Impact RAS-Binding Site Accessibility and Membrane Binding Affinity of CRAF RBD-CRD

doi: 10.1016/j.bpj.2020.06.021

Figure Lengend Snippet: Limited accessibility of the RBS on the RBD from AA simulations of CRAF RBD-CRD. (A) Free energy surface map for RBD orientations relative to the membrane surface from the AA RBD-CRD simulations, using the same two reaction coordinates as in Fig. 2A. The AA frames were filtered to include only those showing a z distance of 2 nm or less between the RBD COM and the membrane surface. Black vertical dashed line at x = 3.5 nm separates the two observed basins. (B) Normalized probability distribution for percentage volume overlap between KRAS4b G domain and the membrane, based on structural alignment of a homology model of the KRAS4b G domain-CRAF RBD complex and the AA RBD-CRD simulations. The x axis is binned with widths of 5%. A majority of the frames show volume overlaps of greater than 5% (orange columns); however, a separate peak occurs for small volume overlaps between 0 and 5% (blue column). (C) Normalized probability distributions of the z distance between the RAS-interacting β-strand COM and the membrane COM for AA frames showing low (<5%) volume overlap of RAS with the membrane (blue columns) and for AA frames showing high (>5%) volume overlap (orange columns). Black dashed line indicates that a z distance cutoff value of 3.5 nm can effectively distinguish between conformations that have the RBS accessible (low overlap) or inaccessible (high overlap). Error bars in (B) and (C) give SEM over 30 AA simulations.

Article Snippet: Cloning, protein expression, and protein purification Gateway Entry clones for CRAF constructs were synthesized using optimization for Escherichia coli or insect cells (ATUM, Newark, CA) that incorporate an upstream tobacco etch virus protease cleavage site (ENLYFQ/G) followed by the appropriate CRAF (human) sequences: CRAF RBD-CRD (52–192) wild-type (WT) and mutants (mts), CRAF RBD (52–131), and CRAF CRD (136–188).

Techniques: