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. 2017 Jan;13(1):62-68.
doi: 10.1038/nchembio.2231. Epub 2016 Nov 7.

Inhibition of RAS Function Through Targeting an Allosteric Regulatory Site

Free PMC article

Inhibition of RAS Function Through Targeting an Allosteric Regulatory Site

Russell Spencer-Smith et al. Nat Chem Biol. .
Free PMC article


RAS GTPases are important mediators of oncogenesis in humans. However, pharmacological inhibition of RAS has proved challenging. Here we describe a functionally critical region, located outside the effector lobe of RAS, that can be targeted for inhibition. We developed NS1, a synthetic binding protein (monobody) that bound with high affinity to both GTP- and GDP-bound states of H-RAS and K-RAS but not N-RAS. NS1 potently inhibited growth factor signaling and oncogenic H-RAS- and K-RAS-mediated signaling and transformation but did not block oncogenic N-RAS, BRAF or MEK1. NS1 bound the α4-β6-α5 region of RAS, which disrupted RAS dimerization and nanoclustering and led to blocking of CRAF-BRAF heterodimerization and activation. These results establish the importance of the α4-β6-α5 interface in RAS-mediated signaling and define a previously unrecognized site in RAS for inhibiting RAS function.


Figure 1
Figure 1. RAS-specific monobody targets H-RAS and K-Ras
a, Binding titration of NS1 displayed on yeast cells with the RAS isoforms bound to nucleotide using flow cytometry detection. Affinity values (KD) are as follows: H-RAS GTPγS, 13.5±1.9 nM; H-RAS GDP, 15.7±2.7 nM; K-RAS GTPγS, 67±16 nM; K-RAS GDP, 60±11 nM; N-RAS GTPγS, not detectable; N-RAS GDP, not detectable. Error bars represent s.d. from N=3. The errors for the KD values are s.d. from three independent measurements. b & c, Assessment of NS1 specificity in cells. b, Co-localization of CFP-NS1 (pseudo-colored red) co-localizes with YFP-tagged RAS isoforms (each pseudo-colored green). Scale bars, 10 um. c, Co-immunoprecipation of HA-tagged RAS isoforms by CFP-NS1. Full blot images for Fig. 1c are shown in Supplementary Fig. 2
Figure 2
Figure 2. NS1 inhibits RAS-mediated signaling and transformation
a, Effect of CFP-NS1 expression on EGF-stimulated ERK activation in HEK293 cells. CFP-NS1 and MYC-tagged ERK were co-expressed, and phosphorylation of MYC-tagged ERK was detected by Western blot with phosphospecific ERK antibodies. b, Cells transfected with the indicated oncogene along with CFP or CFP-NS1 were analyzed for ERK activation as in a. c, Effect of NS1 on AKT activation by RAS. Cells were transfected and analyzed as in b except HA-AKT was used in place of MYC-ERK. Quantification of results from b and c is provided in Supplementary Fig. 4. d, NIH/3T3 transformation assay. Quantification of relative foci number for each oncogene is shown in the graphs. Results represent the ratio of foci number in presence of CFP-NS1 vs CFP alone and are the average of three independent experiments performed in triplicate +/− s.d. p values were determined by a Student’s t-test between CFP and CFP-NS1 for each oncogene. **, p<0.01, *, p<0.05. Effects of NS1 on oncogenic HER2/Neu, BRAF, and MEKK1 are shown in Supplementary Fig. 5a. e & f. Effects of NS1 on signaling and proliferation of human tumor cells. e. Titration of CFP-NS1 effects on ERK activation and proliferation (graph) in T24 bladder carcinoma cells (e) and A375 melanoma cells (f). Results in the graphs are the average of triplicate wells +/− s.e.m. shown by bars. Western blots in e and f are representative of at least 3 independent experiments. Full blot images for Figs 2a–c, e and f and Supplementary Fig. 5b are shown in Supplementary Figs. 3 and 6, respectively.
Figure 3
Figure 3. NS1 targets the α4-α5 interface in RAS distal to switch regions
a, Crystal structure of NS1:H-RAS-GDP. b, Chemical shift changes in H-RAS upon NS1 binding. Amino acids undergoing strong chemical shifts are highlighted red and weaker reacting residues highlighted orange in the H-RAS:RAF RBD structure (PDB ID: 4G0N). The most significant changes occurred in the β5, α4, β6, and α5 regions of H-RAS, which lie on the surface of RAS in opposition to the switch regions which bind effectors such as RAF, indicated in the figure. c, Amino acid sequences of the α4-β6-α5 region (residues 122–166) of the RAS isoforms. Asterisks mark amino acids whose mutation affects affinity to NS1. Dots mark amino acid side chains within 5 angstroms of amino acid side chains in NS1. d, Recognition of R135 in H-RAS by NS1. Polar interactions between R135 and surrounding amino acids and water (green sphere) are highlighted by dashed yellow lines. e. Effect of Arg135 to Lys mutation on H-RAS binding NS1 in vitro. Results are the average of three independent binding experiments +/− s.d. WT, 165.3 +/−2.9 AU; R135K, 0.44+/−0.29 AU.
Figure 4
Figure 4. NS1 targets a putative RAS dimerization interface
a, Comparison of H-RAS crystal structures in the Protein Data Bank (PDB). Seventy-four of 80 active state H-RAS structures contained α4-α5 dimers but no α4-α5 dimers were present in inactive state structures. A protomer (shown in gray) of the α4-α5 dimers from six active state H-RAS structures (PDB ID, 5P21, 1AGP, 4L9W, 2C5I, 3K9L, and 3KUD) were superimposed using Pymol. SW1 and SW2 regions are highlighted yellow. b, NS1 monobody binds the α4-α5 dimer interface as highlighted in brown. The dimer interface shown is from PDB ID: 5P21. These regions overlap with the presumed dimerization interface identified in N-RAS. c. Comparison of NS1 binding surface (highlighted red) and α4-α5 dimer interface (denoted with black boarder) on H-RAS structure.
Figure 5
Figure 5. NS1 blocks RAS dimerization/nanoclustering and subsequent RAF activation
a, Model for NS1 inhibition of RAS-mediated signaling. b, The extent of nanoclustering of GFP-H-RAS(G12V) or GFP-K-RAS(G12V) in BHK plasma membrane sheets immunolabeled with α-GFP-4.5nm gold gold particles was evaluated in K-functions summarized as Lmax. Data represent mean Lmax values ± s.e.m. (Number of samples analyzed: H-RAS+RFP, n=19; H-RAS+RFP-NS1, n=23; K-RAS+RFP, n=16; K-RAS+RFP-NS1, n=24.; * indicates p < 0.05, bootstrap tests). c, The dimer/monomer ratio estimated from data in b. Analysis of the point patterns from b allowed calculation of dimer/monomer ratio shown as mean ± s.e.m. (The number of samples analyzed was the same as in b; * indicates p < 0.05, one-way ANOVA). d, Effect of NS1 on K-RAS(G12V) or N-RAS(G12V) dimerization measured by BRET. Each experiment was performed at least three times with values representing the average of three independent transfections +/− s.d. Additional controls for these experiments are provided in Supplementary Fig. 11e. e, Plasma membrane localization of GFP-K-RAS(G12V) and GFP-H-RAS(G12V) determined by counting total number of gold-labelled GFP particles per 1µm2 area of intact plasma membrane sheets. Values are shown as mean ± s.e.m. (n>15; * indicates p < 0.05, one-way ANOVA).
Figure 6
Figure 6. Effect of NS1 on oncogenic RAS activation of RAF
a, Effect of CFP-NS1 vs CFP on binding of endogenous BRAF to either H-RAS(G12V) or K-RAS(G12V). Results are representative of at least 3 independent experiments. b, Effects of CFP-NS1 vs CFP on oncogenic H-RAS(Q61L)-induced heterodimerization of CRAF and BRAF measured by co-immunoprecipitation in HEK-293T cells. c, K-RAS(G12V) induced heterodimerization of BRAF and CRAF measured by BRET. Each experiment was performed at least three times with values representing the average of three independent transfections +/− s.d. Additional controls are provided in Supplementary Fig. 19. d, Effect of NS1 on in vitro RAF kinase activity. Quantification of results from three independent experiments is shown in Supplementary Fig. 21. Full blot images for Figs 6a, b, and d are shown in Supplementary Fig. 15.

Comment in

  • RAS signaling: Divide and conquer.
    Holderfield M, Morrison DK. Holderfield M, et al. Nat Chem Biol. 2016 Dec 16;13(1):7-8. doi: 10.1038/nchembio.2264. Nat Chem Biol. 2016. PMID: 27984577 No abstract available.

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