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Statements

KRAS binds RAF1. 257 / 284
2 19 1 | 1 78 177 1
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"Interestingly, 1 demonstrates a 10-fold selectivity of binding to GCP-KRAS wt () which translates to a 4-fold selectivity of inhibition biochemically (inhibition of KRAS wt versus KRAS G12C binding to CRAF) ()."
31332011
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"To examine the potential effects of ACA-14 on KRAS functions, we used fluorescent-based biochemical assays to monitor the rates of nucleotide dissociation or exchange reactions of KRAS, as well as interaction of KRAS with the RBD of its effector c-Raf."
37101428
sparser
"Consistent with this report, when cells were treated with the RAF inhibitor SB590885, the interaction between Halo-KRAS Q61R and Nano-CRAF WT was increased approximately 35% ( xref )."
34158349
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"The interaction of HVR with the catalytic domain prevents low affinity Raf1 binding to K-Ras4B-GDP but has a small effect on GEF-catalyzed nucleotide exchange."
26682817
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"The crystal structure of the KRAS and RBDCRD complex and structural information obtained by comparing it with the autoinhibited structure of BRAF in complex with 14-3-3/MEK provide insights into the process of RAS mediated RAF activation."
33608534
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"The relatively modest effect of mutations at the KRAS-CRD interaction interface suggests that, overall, the interactions between KRAS and RBD play a more significant role in the formation of the KRAS and RBDCRD complex."
33608534
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"RAF1 (CRD) interacts with KRAS primarily via the inter-switch region and C-terminal helix alpha5."
33608534
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"The overall structure of CRD within the tandem RBDCRD present in the crystal structures of the KRAS and RBDCRD complex resembles the previously reported NMR structure of the CRD domain 20."
33608534
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"The structures of the KRAS and RBDCRD complex provide atomic details of the KRAS-CRD interaction interface, which is similar in size to the KRAS-RBD interface and also consists of nine hydrogen bonds (Supplementary Fig4a-c)."
33608534
sparser
"As expected, the co-immunoprecipitation analysis showed that C-RAF interacted with oncogenic KRAS under serum-starvation conditions, but this immunoprecipitated C-RAF was phosphorylated only upon EGF treatment ( xref B)."
36614192
sparser
"As expected, oncogenic KRAS was able to bind to C-RAF even in serum-starved conditions, but growth factor stimulation was necessary to allow phosphorylation and hence activation of this C-RAF bound to KRAS [ xref ]."
36614192
sparser
"For example, K42 demonstrated significant intolerance to any sidechain substitutions except positively charged arginine, which is in line with the KRAS-RAF1 RBD-CRD structure, where K42 forms two hydrogen bonds with CRD residues."
39484452
sparser
"Our results demonstrate that full-length K-Ras4B-GDP binds Raf-1 RBD with 52% efficiency when compared to the catalytic domain of K-Ras4B-GDP."
26682817
sparser
"At the same time, we observed an ∼27% increase in the efficiency of Raf-1 RBD binding to GTP- γ -S loaded full-length K-Ras4B compared to the catalytic domain, suggesting that the HVR can interact with Raf."
26682817
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"To investigate the role of the farnesyl group in the interaction between KRAS and RAF1 CRD and RBDCRD, we carried out SPR binding analysis using active fully processed KRAS-FMe and non processed KRAS (G-domain) on the chip surface and flowed CRD and RBDCRD over them."
33608534
sparser
"Finally, L23 has been previously reported to mediate KRAS-RAF1 interactions at the RAF1 cystine-rich domain (CRD) xref , and mutations here are likely to impact RAF1 binding."
39484452
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"KRAS interaction with RBD plays a major role in the high-affinity KRAS and RAF1 complex formation."
33608534
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"The structural superposition of the two crystal forms of the KRAS and RBDCRD complex shows conformational differences in the linker region, partly due to differentcrystal packing interactions."
33608534
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"The KRAS-RBD interface plays a dominant role in the formation of the KRAS and RAF1 complex, but the CRD is essential for RAS dependent RAF activation 10."
33608534
sparser
"In our KRAS-RAF1 RBD-CRD model, we find that D54R causes a direct charge repulsion with R67 of RAF1 ( xref )."
39484452
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"Structural alignment of MRAS (PDB : 1X1S) onto KRAS in the KRAS and RBDCRD complex suggests that among the interswitch region mutations, V45E might have the highest impact on RAF1 activation, as the glutamate side chain will likely clash with CRD."
33608534
sparser
"Similarly, E3K within the β1 strand also results in a local conformational change due to the E3K shifting the β2 strand, disrupting the KRAS-RAF1 interface ( xref )."
39484452
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"Furthermore, the KRAS and RBDCRD complex may undergo further reorganization driven by membrane association 31."
33608534
sparser
"As an application of the general model presented in this paper, we also investigate the formation of heterogeneous protein clusters, such as the KRAS-RAF1 clusters, and their lifetime through a simple extension of this model."
36518075
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"The drug disrupted the KRAS–CRAF complex with a rate constant of roughly 0.2 min for WT KRAS and an average rate constant of 0.05 ± 0.01 min for KRAS mutants (G12C/D/V, G13C/D and Q61H)."
37258666
sparser
"DRx-170 activity appears to correlate with KRAS–c-RAF dependency."
38637546
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"While in the first two minutes after TCR activation, recruitment of Raf-1 to either N-Ras or K-Ras in the cytoplasmic membrane was not different in RA patients and healthy controls, a sustained Raf-1 recruitment was characteristic for patients."
20007589
sparser
"Consistent with the strong interactions between KRas G12D and CRaf, the BiFC system yielded a clear signal when expressed in cells ( xref ; top panel)."
24963703
sparser
"These data confirm that the BiFC signal was due to specific interactions between KRas G12D and CRaf RBD."
24963703
reach
"Modeling KRAS and RBDCRD complex at the membrane."
33608534
sparser
"Cells expressing higher KRAS levels quickly convert GDP-KRAS to GTP-KRAS by increased upstream EGFR signaling and maintain the GTP-KRAS-CRAF interaction downstream by AURKA signaling, ultimately leading to cell cycle progression and escape from G0 [ xref ]."
32797215
sparser
"SPR experiments from Tran et al. that examined the KRAS-CRAF interaction found that the presence of the CRD increases the binding affinity to KRAS significantly. xref The authors conclude that the CRD is important for differentiating between the RAS GTPase superfamily, even though high sequence homology exists between the switch-I region and the RBDs of RAF. xref Interestingly, our results show that the CRD alone does not affect the affinity of BRAF towards HRAS or KRAS, with binding affinity values within the same nanomolar range for constructs NT2-4."
37163002
sparser
"Utilising our first in class cell-penetrating disruptor peptide therapy (DRx-170), we present proof-of-principle data which demonstrates that selective disruption of the pro-oncogenic c-RAF–PDE8A PPI is an encouraging and differentiated approach to inhibiting human KRAS–c-RAF dependent PDAC cell proliferation, adhesion, and migration (Fig.  xref )."
38637546
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"It is a mitotic serine/threonine kinase that exerts its inhibitory effect by blocking the interactions between CRAF and KRAS, suppressing ERK signalling, and inhibiting cell growth [54]."
34064232
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"Although the K-Ras/Raf-RBD interaction was readily detectable upon co-expression in a single cell line, or following lysis of co-cultured cell lines separately expressing K-Ras and RBD, bearing in mind the limitations of our assay, we were unable to detect the interaction in the intact, co-cultured cell lines or upon treatment of the Raf-RBD-expressing cells with exosomes containing K-Ras."
30192821
sparser
"These results strongly suggest that ACA-14 inhibits the binding of KRAS to its effector c-Raf, possibly by blocking KRAS residues involved in c-Raf binding (such as the effector-interacting E37, xref see xref D)."
37101428
sparser
"Previous data from our melting analysis and crystal structures highlighted the importance of position 151 on the melting temperature of both KRAS isoforms and the role of position 153 in KRAS’s interaction with RAF1."
38354232
sparser
"This suggests that although position 151 significantly influences KRAS’s thermal stability, it does not measurably affect KRAS interaction with RAF1 in cells, consistent with our structural data indicating that KRAS position 151 does not directly contact the RAF1 CRD ( xref )."
38354232
sparser
"Unexpectedly, in tumors induced by the K12D oncogene, the K-Ras mutant protein does not interact with Raf-1 nor activates the Erk canonical pathway."
16679305
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"Increasing levels of RIT1, but not RIT1 (a RAF-binding deficient mutant), enhanced the apparent KRAS:RAF1 binding affinity in a BRET assay (Fig. 8B), indicating that RIT1 promotes RAS:RAF interaction in a manner dependent on RIT1:RAF1 binding."
37450595
sparser
"This suggests that the substitutions lead to higher populations of KRAS-RAF1 complexes on the membrane without an increase in complex affinity."
38354232
sparser
"Treatment of KRAS G12D-harboring human pancreatic cancer cells with KRB-456 suppresses the cellular levels of KRAS bound to GTP and inhibits the binding of KRAS to RAF1."
38051103
sparser
"The relative lack of selectivity versus the KRAS G12D ::CRAF is expected due to the 5-fold weaker affinity of KRAS G12D for CRAF, while KRAS G12C and KRAS wt maintain the same affinity for the RAS binding domain of CRAF ( xref )."
31332011
sparser
"Using a variety of biophysical and cell biological assays, including FP, signaling, and pull-down assays, we have shown that ACA-14 impedes the interaction of KRAS with the RBD of its effector c-Raf and reduces the rates of intrinsic and GEF-mediated exchange of GDP to GTP."
37101428
reach
"Under conditions of serum supplementation or EGF stimulation, but not serum starvation, we observed an increase in CFP signal after YFP bleaching for both KRAS WT - and KRAS D154Q -expressing cells, s[MISSING/INVALID CREDENTIALS: limited to 200 char for Elsevier]"
29336889
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"Together, our observations support the conclusion that KRAS D154Q does not impair the interaction between KRAS and CRAF and that KRAS dimerization is not required for CRAF-KRAS interactions.Finally, w[MISSING/INVALID CREDENTIALS: limited to 200 char for Elsevier]"
29336889
sparser
"KRB-456 suppresses the cellular levels of KRAS bound to GTP, and inhibits binding of KRAS to RAF1 and signaling pathways downstream of RAS."
38051103
reach
"Binding of Raf-RBD to both KRas and NRas stabilizes switch I in a catalytically competent conformation as it does in HRas ( 9 , 13 , 53 )."
35794829
sparser
"KRB-456 Suppresses the Levels of KRAS Bound to GTP, Inhibits the Binding of KRAS to RAF1, and Deceases P-MEK and P-ERK Levels in Human Pancreatic Cancer Cells."
38051103
sparser
"We next examined the effects of KRB-456 on the levels of KRAS bound to GTP and on the binding of KRAS to RAF1 in intact Panc0203 and Panc1 human pancreatic cancer cells that harbor mt KRAS G12D. To this end, we first treated these cells with various concentrations of KRB-456 and processed the cells for viability assays, immunoprecipitation, GST-RBD pulldown, and western blotting as described in Materials and Methods. xref shows that KRB-456 inhibited the viability of Panc0203 and Panc1 cells in a concentration-dependent manner with IC 50 values of 4.9 ± 0.7 µmol/L and 14.8 ± 2.7 µmol/L, respectively."
38051103
sparser
"We next treated Panc0203 and Panc1 cells with KRB-456 for 15 minutes, lysed the cells, and determined the effects of KRB-456 on the levels of GTP-bound KRAS by GST-RBD pulldown assays and the binding of KRAS to RAF1 by co-immunoprecipitation assays as described in the Materials and Methods. xref shows that GST-RBD was able to pull down GTP-bound KRAS in Panc0203 and Panc1 cells treated with vehicle, and that treatment with KRB-456 significantly decreased the GTP-KRAS cellular levels."
38051103
sparser
"KRB-456 also inhibited the binding of KRAS to RAF1 in intact Panc0203 and Panc1 cells ( xref ) confirming the in vitro AlphaScreen and GST pulldown results of xref and xref ."
38051103
sparser
"KRB-456 binds with high affinity to KRAS G12D and KRAS G12V, decreases the cellular levels of GTP-bound KRAS, inhibits the binding of KRAS to RAF1 in pancreatic cancer cells, and inhibits in vivo tumor growth of subcutaneous and orthotopic xenografts from patients with pancreatic cancer."
38051103
sparser
"Decreased levels of the receptor for advanced glycation end products (RAGE) associate with lung carcinogenesis and metastasis regulating PI3K/AKT and KRAS-RAF1 signaling."
37941543
sparser
"KRB-456 binds KRAS (ITC and protein NMR data), inhibits the binding of KRAS to RAF1 in vitro (alpha screen and GST-RBD pulldown) as well as in intact cancer cells (co-immunoprecipitation with KRAS and blotting with RAF-1 and GST-RBD pulldown) and inhibits the immediate RAS/RAF downstream effector P-MEK clearly demonstrating that KRB-456 engages its target and suppresses KRAS oncogenic signaling in pancreatic cancer."
38051103
reach
"Using pharmacologic and genetic approaches, it has been shown that the cytosolic KRAS is still able to bind RAF-1 but cannot activate it, possibly due to inhibition of binding to the scaffold protein KSR, which plays an important role in RAS activation of RAF [29]."
| PMC
sparser
"In summary, we report here on the discovery of a novel natural product-inspired small molecule, KRB-456, that binds a dynamic allosteric binding pocket within the switch-I/II region of KRAS G12D, suppresses the levels of KRAS bound to GTP and inhibits the binding of KRAS to RAF1 in human pancreatic cancer cells that harbor KRAS G12D. The fact that KRB-456 thwarted the subcutaneous and orthotopic growth in vivo of PDXs from patients with pancreatic cancer that relapsed after chemotherapy and radiotherapy suggests that it may overcome drug resistance of these tumors."
38051103
sparser
"The extended beta sheet of the KRAS-RAF1 interaction does not provide any obvious pocket for direct inhibition of the interaction [ 35 ]."
34303932
sparser
"Binding of prenylated KRAS to nanodisc, and subsequent binding of the CRAF-RBD to KRAS, was shown to be dependent on PS [ xref ] and interfering with the PS content of the plasma membrane was shown to mislocalize KRAS and impact signaling [ xref , xref ]."
28384226
sparser
"MRTX-EX185 demonstrated time- and dose-dependent inhibition of the KRAS(G12D):CRAF(RBD) interaction (Supplementary Fig. xref ), providing support for functional disruption of MAPK signaling."
35314814
sparser
"Thus, evaluation of KRAS(G12D):CRAF interactions with endogenous proteins may be warranted to more accurately query the kinetics of pathway inhibition."
35314814
sparser
"A split luciferase biosensor xref was used to measure the interaction of KRAS with full length CRAF in live cells and to determine the effect of drug treatment over time (Fig. xref )."
37258666
sparser
"The drug disrupted the KRAS–CRAF complex with a rate constant of roughly 0.2 min −1 for WT KRAS and an average rate constant of 0.05 ± 0.01 min −1 for KRAS mutants (G12C/D/V, G13C/D and Q61H)."
37258666
sparser
"We tested the effect of treatment on the interaction of KRAS with the CRAF RBD in live cells, which is a proxy for KRAS activation (Fig. xref and Extended Data Fig. xref )."
39476862
sparser
"First, mutational effects are quantified for multiple phenotypes—here the binding of KRAS to RAF1 and the abundance of KRAS in the absence of RAF1."
38109937
sparser
"A recent structural and molecular dynamics simulation study of KRas4B-Raf1 RBDCRD (CRD, cysteine-rich domain) showed that monomeric KRas-Raf1 RBDCRD complex could use α4 and α5 to interact with membrane ( xref xref – xref )."
35522713
sparser
"Intriguingly, the previous observation that suppressed expression of SOS (a GEF for Ras) does not inhibit binding of K-Ras G12V to Raf-1 RBD can also be interpreted in the context of the “active-like”[MISSING/INVALID CREDENTIALS: limited to 200 char for Elsevier]"
35752212
sparser
"Similarly, we superposed the KRas-Raf1 RBDCRD structure (PDB: 6XI7) to the KRas dimer we observed in xref and found there is no obvious clash in the helical assembly either ()."
35522713
sparser
"Effects of compound TKR15Blocking K-Ras protein-Raf-1 protein interaction."
31851852
sparser
"This result can directly demonstrate that compound TKR15 can significantly target K-Ras protein and inhibit K-Ras protein-Raf-1 protein interaction."
31851852
sparser
"For example, it is likely that other SWI residues are important for mediating mTORC2 activation, such as P35 and I37 which correspond to residues P37 and I39 in Rheb previously shown to promote its binding to mTOR ( xref , xref ), as well as other residues in the allosteric domain such as R149, D153, and Y157 found to be involved in the interaction of human K-Ras with the cysteine-rich domain of Raf1 ( xref )."
38815864
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"The results also provide direct evidence in individual cells that post-translational modification of K-Ras and its localization at the plasma membrane (PM) were not essential for the interaction of K-Ras and Raf-1, whereas the interaction of Grb2 and K-Ras did depend on the PM localization of K-Ras."
24556667
sparser
"We focused on the role of CRD in the interaction of Raf-1 with K-Ras4B, the most oncogenic among the Ras isoforms, at the membrane."
29429878
sparser
"It also resembles one of the two observed conformations of KRas:Raf1 RBD-CRD on nanodiscs using PRE-NMR by Fang et al. ( xref B, left) [ xref ], although the folding of Raf1 CRD differs significantly between the two."
35205266
sparser
"We believe that, in the case of the complex of Raf-1 with K-Ras4B, this is the primary role of this Raf CRD domain."
29429878
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"Using this BEVL-BiFC system, we provided direct evidence in individual cells that post-translational modification of K-Ras and its PM localization were not essential for the interaction between K-Ras and Raf-1, while the interaction of Grb2 and K-Ras depended on PM localization of K-Ras."
24556667
reach
"Although these two bicyclic peptides also inhibited the interaction between wildtype K-Ras and Raf1-RBD, the IC values were more than 20-fold higher than those for K-Ras(G12D)."
33145412
reach
"Next, we determined whether everolimus treatment enhances the interaction of mt K-Ras and c-Raf for the Ras-Raf-ERK activation."
22342683
sparser
"Interestingly, the authors pointed out that this dimer model is present in the crystal (between two-unit cells) from which the KRas-GTP:Raf1 RBD-CRD structure was obtained [ xref ] ( xref D)."
35205266
sparser
"Indeed, although targeting the interaction between CRAF and its upstream activator KRAS can be a promising strategy, selectivity remains a pressing concern."
38111008
sparser
"In contrast, KRAS G12C binds RAF1 with affinities close to wild‐type KRAS."
34951114
sparser
"To further examine the inhibitory effect of BHP on the interaction of KRAS with Raf-1, MDA-MB231 cell lysates were incubated overnight at 4 °C with various concentration of BHP (10, 30 and 50 μM) and [MISSING/INVALID CREDENTIALS: limited to 200 char for Elsevier]"
23707634
sparser
"Co-precipitation of Kras with GST-Raf1-RBD was significantly decreased in cells expressing STAND-Y13-259 when compared to those expressing STAND-A36 or the mock vector control (Fig.  xref ), indicating that intracellular STAND-Y13-259 interferes with the interaction of activated Kras and Raf1 by binding to Kras."
31953402
sparser
"We optimized DoMY-Seq by taking advantage of the well-described and high-affinity interaction between KRAS and CRAF, and we provide high-resolution domain mapping on this and other protein-interacting pairs, including CRAF-MEK1, RIT1-RGL3, and p53-MDM2."
33410398
sparser
"Importantly, in BHP concentration-dependent manner, less amount of KRAS protein was co-precipitated with Raf-1 in cell lysate that treated with BHP, compared to treatment with DMSO, suggesting that BH[MISSING/INVALID CREDENTIALS: limited to 200 char for Elsevier]"
23707634
sparser
"Taken together, these results suggest that BHP suppresses breast cancer malignant phenotypes by inhibiting the interaction of KRAS with Raf-1."
23707634
reach
"However, CaM does not interfere with the binding of activated H- or K-Ras to Raf-1."
19666110
reach
"In KRAS mutant tumor cells, Aurora-A facilitates the interaction between KRAS and C-RAF and is associated with adaptive reactivation of KRAS after KRAS inhibitor treatment."
37679326
sparser
"Importantly, our study showed that treatment with BHP effectively interfered the interaction between KRAS and Raf-1, thereby attenuating ERK signaling."
23707634
sparser
"Consistently, again, we found that BHP inhibits the interaction of KRAS with Raf-1 in breast cancer cells."
23707634
isi
"We optimized DoMY-Seq by taking advantage of the well-described and high-affinity interaction between KRAS and CRAF, and we provide high resolution domain mapping on this and other protein interacting pairs, including CRAF-MEK1, RIT1-RGL3, and p53-MDM2."
33144325
sparser
"All three peptides inhibited the interaction between K-Ras(G12D)·GppNHp and Raf1-RBD at micromolar concentrations."
33145412
sparser
"In line with the hypothesis, we observed that treatment with BHP attenuated the interaction of KRAS with Raf-1 in concentration-dependent manner in cell lysates, indicating that BHP may interact physi[MISSING/INVALID CREDENTIALS: limited to 200 char for Elsevier]"
23707634
reach
"Targeting the GTP-bound state of KRAS with the tri-complex inhibitor RMC-4998 (0.1 μM) led to a faster disruption of the interaction between KRAS and CRAF as compared to inactive state selective inhibitors adagrasib (1 μM) and sotorasib (10 μM) (Fig. 3B)."
37590355
reach
"Overall, this is consistent with an interaction of KRAS only with the active form of C-RAF, which requires dephosphorylation of S259 and unmasking of the RBD and CRD to allow KRAS binding to C-RAF at the membrane (reviewed in Matallanas et al [2011])."
38499327
sparser
"Using both oncogenic and wild type NanoLuc-KRAS as donors, this assay can identify increased association of oncogenic KRAS molecules with RAF1."
29432816
sparser
"The GTP-bound forms of non-modified (used as positive control in this assay) and lipid-modified K-Ras4B proteins bound Raf-1 RBD with high affinity in comparison to their GDP-bound forms (A)."
22569482
sparser
"Our observations that HPIP overexpression enhanced SOS1-KRAS and KRAS-RAF1 interactions and that HPIP KO reduced these interactions suggest that HPIP is critical for increased KRAS GTPase activity."
37294756
sparser
"It is a mitotic serine/threonine kinase that exerts its inhibitory effect by blocking the interactions between CRAF and KRAS, suppressing ERK signalling, and inhibiting cell growth [ xref ]."
34064232
reach
"In our simulation, the distinctive characteristics of the pKRAS-RAF1 complex can be attributed to the dynamics of the pKRAS switch regions."
34725361
reach
"KRAS bound to RAF-RBD was detected by western blotting using an anti-RAS antibody (Millipore #05-516, 1 : 2000)."
34725361
sparser
"Next, we determined whether everolimus treatment enhances the interaction of mt K-Ras and c-Raf for the Ras-Raf-ERK activation."
22342683
sparser
"The everolimus treated and untreated K- Ras mt PANC-1 cells were immunoprecipitated using c-Raf antibody and western blotting using K-Ras antibody revealed the increased interaction of mt K-Ras and c-Raf protein in the everolimus treated PANC-1 cells ( xref )."
22342683
sparser
"Furthermore, the BRET interaction signal between KRAS G12D and either PI3K (α or γ) or RALGDS was inhibited by Ch-3 ( xref ), demonstrating that the BRET data are not restricted to KRAS-CRAF interaction."
30683716
sparser
"Targeting the GTP-bound state of KRAS G12C with the tri-complex inhibitor RMC-4998 (0.1 μM) led to a faster disruption of the interaction between KRAS G12C and CRAF as compared to inactive state selective inhibitors adagrasib (1 μM) and sotorasib (10 μM) ( xref )."
37590355
sparser
"We have combined several different techniques – phosphoramidite synthesis, one-bead-one-compound library synthesis, fluorescence-activated bead sorting, and tandem mass spectrometry – to create a unique methodology for the selection of novel phosphoestamers that selectively inhibit protein–protein interactions between mutant KRAS G12D and RAF1."
39600501
sparser
"In this study, we constructed the ternary and quaternary complexes of KRas and CRaf based on crystal structures, denoted as (KRas) 2 ·CRaf and (KRas) 2 ·(CRaf) 2 , respectively."
40126127
sparser
"Additionally, MD simulations of the KRasG12D·CRaf complex revealed a stable and extended binding site at the KRas-CRaf interaction interface."
40126127
reach
"Following the transition to late endosomes, Raf1 bound K-Ras activates the protein complex that include adaptors p14 and MP1 that in turn recruits and binds MEK1, thereby facilitating ERK1/2 entry into the nucleus."
22875452
sparser
"We observed that PG abrogated the interaction of KRAS with RAF-1 ( Fig. 5 B)."
25843036
sparser
"We chose homogeneous time-resolved fluorescence (HTRF) as an initial biochemical screening assay since it has previously been shown to be a suitable format for the measurement of active KRAS/CRAF-RBD binding interactions xref ."
32587898
sparser
"In order to analyze if inhibition of CaM would exert similar effects on Raf-1 interacting with active K-Ras, we determined Raf-1 kinase activity ( Fig. 2 B) and Ser338 phosphorylation ( Fig. 2 C) in G[MISSING/INVALID CREDENTIALS: limited to 200 char for Elsevier]"
19666110
sparser
"This finding suggests that the interaction of RAF1 with KRAS depends on the GTP-bound state of KRAS rather than its localization and does not necessarily cause RAS pathway activation as has been previously reported ( xref )."
34021083
sparser
"Therefore, Ub likely occludes and thereby prevents C-RAF RBD binding to KRAS, consistent with experiments."
33929846
sparser
"We also analyzed the potential impact of ubiquitination at K147 on the interaction of KRAS with C-RAF CRD ( xref and xref , xref ). xref and shows that CRD-interacting residues of KRAS do not make direct contact with ubiquitin, suggesting that they are available for interaction with RAF CRD in mUbKRAS 147 ."
33929846
sparser
"While interaction based on Venus signal between K-Ras4B G12D and C-Raf in membrane was observed at 81.60 ± 13.24 of the cells, interaction between K-Ras4B G12D/K101D/R102E and C-Raf was observed at 28.63 ± 8.97 and interaction between K-Ras4B G12D/R41E/K42D and C-Raf was observed at 5.63 ± 1.43 of the transfected cells. ( xref – xref )."
31931009
sparser
"In KRAS G12C mutant tumor cells, Aurora-A facilitates the interaction between KRAS and C-RAF and is associated with adaptive reactivation of KRAS after KRAS G12C inhibitor treatment. xref Combination of an Aurora-A inhibitor and a KRAS G12C inhibitor shows synergistic effect in vitro and in vivo."
37679326
sparser
"However, CaM does not interfere with the binding of activated H- or K-Ras to Raf-1."
19666110
sparser
"Oxidative protein damage negatively affects protein-protein interaction: The case of KRAS-cRAF."
39378785
sparser
"However, in contrast to the dominant active mutant of Ki-Ras, which interacts with Raf-1, recruits it to the plasma membrane from the cytosol, and activates it, MAGUIN-1 neither activates Raf-1 nor recruits it to the plasma membrane."
10753660
sparser
"In cells coexpressing KRAS G12D and wild-type full-length cRaf, quenching of GFP fluorescence lifetime and hence KRAS G12D –cRaf interaction is significantly reduced upon compound treatment ( xref C)."
30842983
sparser
"Taken together, these findings indicate that C-Raf and K-Ras can bind with high affinity to all Ras or Raf family members respectively, whereas H-Ras displays preferential binding to C-Raf, and B-Raf exhibits a striking selectivity for K-Ras."
31606273
sparser
"We also used SPR to confirm that RRSP processing of KRAS reduced the interaction between RAF-RBD (residues 55–131) and KRAS."
30279169
reach
"Oncogenic KRAS binds and activates CRAF more efficiently and mediates KRAS signaling to ERK kinase in lung cancer cells, thus fulfilling an important role as an anti-apoptotic protein independent of BRAF."
25013473
sparser
"Binding of MRTX 1133 prevented the formation of KRAS G12D /GTP/RAF1 complex, inhibited signaling in cell lines and also showed in vivo tumor regression in 04.03 xenograft model of pancreatic cancer.[ xref ] Investigational New Drug application for MRTX 1133 is planned in the second half of 2022."
36638742
sparser
"Since the GTP-bound, active form of KRAS can interact with RAF-1, KRAS activation was assessed by quantifying the amount of KRAS that interacts with RAF-1."
25633745
reach
"KRB-456 suppresses the cellular levels of KRAS bound to GTP, and inhibits binding of KRAS to RAF1 and signaling pathways downstream of RAS."
38051103
sparser
"The c-Raf-RBD normally binds to KRas when KRas is GTP-bound (KRas GTP )."
32511232
sparser
"To perform these computational designs, we first made a homology model of c-Raf-RBD bound to KRas GTP (see xref for details)."
32511232
reach
"Treatment of KRAS G12D-harboring human pancreatic cancer cells with KRB-456 suppresses the cellular levels of KRAS bound to GTP and inhibits the binding of KRAS to RAF1."
38051103
reach
"This Switch I conformation in this structure is different from the lower resolution structure (PDB: 6E6F) despite both being in the State 1 conformation (the dynamic Switch I is captured in two different snapshots as observed in two different crystal forms) and is clearly different from the State 2 conformation of KRAS bound to RAF1-RBD (Fig. 4b)."
37268708
sparser
"Next, we used fries / EWAK * for prospective design and discovered a novel point mutation that improves binding of c-Raf-RBD to KRas in its active, GTP-bound state (KRas GTP )."
32511232
sparser
"RMC-7977 exhibited similar activity for wild-type and mutant RAS variants in biochemical assays, and in the live-cell nano-BRET assay the cellular potency for inhibition of CRAF (RBD) binding to wild-type KRAS was only modestly lower than that for the oncogenic variants."
38589574
reach
"First, mutational effects are quantified for multiple phenotypes—here the binding of KRAS to RAF1 and the abundance of KRAS in the absence of RAF1."
38109937
sparser
"Of those, point mutants R67L, N71R, and V88I were predicted to improve the intermolecular interactions between c-Raf-RBD and KRas GTP ."
32511232
sparser
"In contrast to [ xref ], EWAK * accurately predicted that these mutations decrease binding of c-Raf-RBD to KRas GTP ."
32511232
sparser
"We found decreased binding to the Raf1 RBD by KRAS G12D/K104Q and reduced cell growth, invasion and migration."
33060649
sparser
"This allowed us to quickly obtain a qualitative probe of c-Raf-RBD variant binding to KRas."
32511232
sparser
"We determined that c-Raf-RBD(RKY) binds to KRas GTP roughly 36 times more tightly than wild-type c-Raf-RBD, making it the tightest known c-Raf-RBD variant binding partner of KRas GTP ."
32511232
sparser
"Oncogenic KRAS binds and activates CRAF more efficiently and mediates KRAS signaling to ERK kinase in lung cancer cells, thus fulfilling an important role as an anti-apoptotic protein independent of BRAF ( xref , xref )."
25013473
reach
"KRB-456 Suppresses the Levels of KRAS Bound to GTP, Inhibits the Binding of KRAS to RAF1, and Deceases P-MEK and P-ERK Levels in Human Pancreatic Cancer Cells."
38051103
reach
"We next examined the effects of KRB-456 on the levels of KRAS bound to GTP and on the binding of KRAS to RAF1 in intact Panc0203 and Panc1 human pancreatic cancer cells that harbor mt KRAS G12D."
38051103
sparser
"To evaluate the effectiveness of compound 1 in disrupting KRAS G12D and RAF1 binding, we used a biochemical protein–protein interaction (PPI) Homogeneous Time–Resolved Fluorescence (HTRF) assay."
39642212
reach
"We next treated Panc0203 and Panc1 cells with KRB-456 for 15 minutes, lysed the cells, and determined the effects of KRB-456 on the levels of GTP-bound KRAS by GST-RBD pulldown assays and the binding of KRAS to RAF1 by co-immunoprecipitation assays as described in the Materials and Methods."
38051103
reach
"Using the POI-IP model, KRAS/RAF1, we showed that engineering FSY at the footprint-assigned KRAS residue resulted in a KRAS variant that can bind irreversibly to RAF1."
38684118
reach
"KRB-456 also inhibited the binding of KRAS to RAF1 in intact Panc0203 and Panc1 cells (Fig. 5C) confirming the in vitro AlphaScreen and GST pulldown results of Figs."
38051103
sparser
"To understand how the tandem domains, RBD and CRD, in the N-terminal regulatory part of RAF1 interact with KRAS, and their roles in RAS-mediated RAF activation, we attempted to crystallize and solve the structure of KRAS in complex with the RBDCRD region of RAF1 (Fig.  xref )."
33608534
sparser
"Measurement of binding affinities by surface plasmon resonance (SPR) showed high-affinity interaction ( K D ~ 356 nM) between KRAS and RAF1(RBD), as shown previously xref ."
33608534
reach
"KRB-456 binds with high affinity to KRAS G12D and KRAS G12V, decreases the cellular levels of GTP-bound KRAS, inhibits the binding of KRAS to RAF1 in pancreatic cancer cells, and inhibits in vivo tumor growth of subcutaneous and orthotopic xenografts from patients with pancreatic cancer."
38051103
sparser
"KRAS interaction with RBD plays a major role in the high-affinity KRAS-RAF1 complex formation."
33608534
sparser
"Structural superposition of KRAS-RAF1(RBD) (hereinafter referred to as KRAS-RBD) with KRAS-RBDCRD using KRAS residues shows no significant differences at the KRAS-RBD interaction interface in the two structures (Fig.  xref )."
33608534
sparser
"To this end, we treated MiaPaCa2 cells with FGTI-2734 as described above, and processed the cells for RAF-1 immunoprecipitation, followed by immunoblotting of KRAS and KSR, a scaffolding protein that binds RAF-1 and mediates RAS activation of RAF-1. xref shows that in the absence or presence of FGTI-2734, RAF-1 was able to bind KRAS, suggesting that both membrane and cytosolic KRAS are able to bind RAF-1."
31227505
sparser
"Analysis of the binding affinity shows that R59A, N64A, and Q66A mutants in RBDCRD resulted in a 3–12-fold reduction in the binding affinity with KRAS, whereas mutation of R89L resulted in almost complete loss of interaction between RBDCRD and KRAS."
33608534
sparser
"Mapping the KRAS interaction interface on the RBDCRD surface shows that both RBD and CRD contribute significantly to RAF1 interaction with KRAS (Fig.  xref , lower panel)."
33608534
sparser
"RAF1(CRD) interacts with KRAS primarily via the inter-switch region and C-terminal helix α5."
33608534
sparser
"To investigate the role of the farnesyl group in the interaction between KRAS and RAF1 CRD/RBDCRD, we carried out SPR binding analysis using active fully processed KRAS-FMe and non-processed KRAS (G-domain) on the chip surface and flowed CRD and RBDCRD over them."
33608534
sparser
"AURKA binds newly produced KRAS-G12C, which in turn stabilizes the interaction between CRAF and KRAS and subsequently mediates ERK effectors."
35267628
sparser
"Our pharmacological and genetic approaches show that cytosolic KRAS is still able to bind one of its major effectors RAF-1 but is not able to activate it, possibly due to inhibition of binding to the scaffolding protein KSR (see xref ) which plays a major role in RAS activation of Raf ( xref )."
31227505
sparser
"The KRAS-RBD interface plays a dominant role in the formation of the KRAS-RAF1 complex, but the CRD is essential for RAS-dependent RAF activation xref ."
33608534
sparser
"This suggests that in addition to the high-affinity binding of RAF1 to KRAS provided by RBD, proper CRD association with KRAS is required for robust RAF1 activation."
33608534
sparser
"These mutants showed no significant changes in KRAS-RAF1 binding (Fig.  xref )."
33608534
sparser
"Oncogenic mutants at G12, G13, and Q61 do not affect KRAS-RAF1(RBDCRD) interaction."
33608534
sparser
"Structural overlay of the KRAS-CRAF and KRAS-p110α shows that the central β-sheet of the two RBDs is rotated by 38 degrees relative to one another xref ."
39788953
sparser
"Molecular Dynamics Investigation into the Stability of KRas and CRaf Multimeric Complexes."
40126127
sparser
"Extending published mutagenesis, NMR and molecular dynamic (MD) simulation studies, we propose how the KRAS-RAF1(RBDCRD) complex (hereinafter referred to as KRAS-RBDCRD) might interact with the membrane."
33608534
sparser
"Additionally, there are more H-bond and salt-bridge interactions at the KRAS-CRAF RBD interface compared to the KRAS-p110α RBD interface, providing a basis for the stronger binding affinity of KRAS for CRAF compared to PI3Kα."
39788953
sparser
"Model showing the KRAS-RAF1(RBDCRD) complex at the membrane."
33608534
reach
"We optimized DoMY-Seq by taking advantage of the well described and high-affinity interaction between KRAS and CRAF, and we provide high resolution domain mapping on this and other protein interacting pairs, including CRAF-MEK1, RIT1-RGL3, and p53-MDM2."
33144325
reach
"Importantly, our study showed that treatment with BHP effectively interfered the interaction between KRAS and Raf-1, thereby attenuating ERK signaling."
23707634
reach
"KRAS activates downstream pathways by binding to its classical downstream effector RAF1, whether the ubiquitination modification affects their binding."
40240366
reach
"We hypothesized that steric hindrance might be generated when KRAS is modified by polyubiquitin chains, potentially affecting the binding between KRAS and its downstream effector RAF1."
40240366
trips
"KRAS interaction with RAF1 RAS-binding domain and cysteine-rich domain provides insights into RAS-mediated RAF activation."
33608534
sparser
"Recent data on the binary KRas4B–Raf-1 complex suggested that Raf-1 CRD not only executes membrane anchorage, but also supports the high-affinity interaction of Raf-1 RBD with KRas4B catalytic domain."
32257057
sparser
"In addition, MRTX1133 was able to prevent binding of a RAF1 RBD peptide to KRAS G12D preloaded with the nonhydrolyzable GTP analog GMPPCP with an IC50 of 9 n m ."
36383067
reach
"SOS1-KRAS complex (PDB: 6EPO), KRAS-RAF1 complex (PDB: 6VJJ), MEK1-BRAF complex (PDB: 6Q0J), MEK1 (PDB: 3WIG), and ERK1 (PDB: 4QTB) were accessed from the RCSB PDB protein data bank."
37162909
sparser
"As shown in xref , BBO-8520 started to inhibit the RAF1(RBD):KRAS G12C (ON) interaction within the first 2 minutes of treatment."
39642212
reach
"As expected, oncogenic KRAS was able to bind to C-RAF even in serum-starved conditions, but growth factor stimulation was necessary to allow phosphorylation and hence activation of this C-RAF bound to KRAS [48]."
36614192
reach
"In the BiFC system, the K-Ras and Raf1 complexes were mainly located in the cell membrane, while the Raf1 control was uniformly distributed in the cytoplasm."
23012550
reach
"In contrast, an apparent cytosolic distribution pattern was observed in cells co-transfected with mcerulean-Raf1 and EGFP-K-RasC185S, suggesting that the membrane localization of K-Ras and Raf1 complexes is not entirely dependent on K-Ras, and that other factors, such as the irreversible conformation formed between K-Ras and Raf1 may play a role."
23012550
reach
"This study sheds light on the interaction between K-Ras and Raf1 and provides a practical method to elucidate the mechanism underlying K-Ras and Raf1 binding to the cell membrane."
23012550
reach
"Recently, detailed interactions between K-Ras and Raf1 have been elucidated including the specifics of the conformational change which Raf1 undergoes upon binding to K-Ras [XREF_BIBR]."
23012550
reach
"However, there are still some unresolved issues regarding their interactions such as where and how the activation of Raf1 and K-Ras occurs in cells, whether K-Ras and Raf1 simply traffic together or are part of a larger multicomponent signaling complexes, as well as whether the ultimate cellular localization of the K-Ras and Raf1 complexes is independent of the original Raf1 and K-Ras locations."
23012550
sparser
"Although the main role of Raf-1 CRD is membrane anchorage, their study showed it is also important for the KRas–Raf-1 interaction."
33978412
sparser
"Differential binding of Raf-1 domains to KRas4B can explain the reduced flexibility of the KRas4B interface residues."
33978412
sparser
"To obtain a complete mechanistic picture of the Ras–Raf interaction, we model the farnesylated/methylated KRas4B-GTP dimer interacting with two Raf-1 conserved region 1 segments at both sides of the KRas4B dimer."
32257057
sparser
"Concomitantly, Ras dimerization further increases the affinity of KRas4B interaction with Raf-1 RBD-CRD."
32257057
reach
"Results above show that the BiFC system composed of Raf1 and K-Ras is sensitive for EGF stimulation, and the trend of BiFC fluorescence change is consistent with previous report on the K-Ras response to EGF stimulation [XREF_BIBR], indicating that the activation of K-Ras can promote its complex formation with Raf1, and the Raf1 and K-Ras interaction in BiFC system has not been interfered by the fluorescent protein fragments they connected, and this system is suitable for further studies of Raf1 and K-Ras interaction."
23012550
sparser
"By phosphorylating mutant Kras-bound cRaf, growth factors can potently engage the ras-Raf signaling cascade, which deactivates slowly due to decreased GTPase activity of mutant Kras [ xref , xref ]."
21699731
reach
"As shown in XREF_FIG, sub-cellular localization of the K-Ras and Raf1 complexes which was mainly distributed in the cell membrane (XREF_FIG) varied from the K-Ras-C185S and Raf1, which was mainly localized in the cytoplasm (XREF_FIG), and these distinct subcellular localizations were further confirmed in different imaging depth (XREF_FIG), indicating that the C-terminal CAAX site is the determinant for sub-cellular localization of K-Ras and Raf1 complexes, which is consistent with previous reports [XREF_BIBR]."
23012550
reach
"Moreover, significant co-localized signal between K-Ras and Raf1 was found in the cell membrane (XREF_FIG), suggesting that K-Ras affects the cellular localization of Raf1, and the cellular interaction between K-Ras and Raf1 is likely to facilitate the membrane binding ability of Raf1."
23012550
reach
"This suggests that the Raf1 and K-Ras interaction in BiFC system has not been destructed by their respectively connected fluorescent protein, and such system is well suited for the study of Raf1 and K-Ras interaction."
23012550
reach
"In this study, we compared the localization of K-Ras and Raf1 complexes, and co-expressed K-Ras and Raf1 in living COS-7 cells."
23012550
reach
"Thus, the comparison of the effect of K-Ras and CAAX mutated K-Ras protein K-RasC185S on the cellular localization of Raf1 may provide useful information regarding the cellular interaction between K-Ras and Raf1."
23012550
reach
"Finally, our results suggest that the combination and translocation of Raf1 and K-Ras to the membrane occurs as follows : the combination between Raf1 and K-Ras on the membrane alters the conformation of Raf1 and K-Ras complexes, and enhances its membrane binding capacity."
23012550
reach
"In other words, the Raf1 and K-Ras complexes will not lose their positioning in the cell membrane while association occurs in the cell membrane, and unless they are separated."
23012550
reach
"Although the combination of Raf1 and K-RasC 185S in BiFC may not occur under physiological conditions, investigating this interaction under the special conditions of BiFC may provide new insights into the understanding of the cellular interaction between Raf1 and K-Ras."
23012550
sparser
"Further, BRET analysis revealed that NS1 disrupts CRAF interaction with K-RAS(G12V) but not N-RAS(G12V) ( xref )."
27820802
reach
"In summary, the intercellular interaction of Raf1 and K-Ras was investigated using BiFC assay and a co-expression system with fluorescent protein fusions."
23012550
reach
"The membrane localization of Raf1 is not entirely dependent on K-Ras, whereas the K-Ras and Raf1 complexes formation plays a key role in this process."
23012550
sparser
"Our simulations showed that these quaternary complexes were highly stable with confined KRas4B membrane orientation, supported by the Raf-1 interactions with both KRas4B and membrane."
32257057
sparser
"In addition, we modeled the interaction of RIT1 with the CRD domain of RAF1 using the solved KRAS:RAF1(RBD-CRD) crystal structure (fig."
37450595
sparser
"Specifically, when JOSD2 removes the polyubiquitin chains from KRAS, the steric hindrance is reduced, thereby promoting the KRAS-RAF1 interaction, which will be further investigated in our future study."
40240366
reach
"For example, KRAS binds to the RAF1-RBD approximately fivefold less strongly than wild-type RAS does ."
36028717
sparser
"Together with the entropic-driven binding event of KRAS to the RBD of RAF1, our data show a clear correlation between the binding affinity of KRAS and its altered conformational dynamics."
35213144
reach
"C-RAF binding by K-RAS was more similar to wild-type K-RAS, and K-RAS failed to interact with C-RAF-RBD at all (Figure 4F)."
35202574
sparser
"Raf-1 bound KRas4B has the lowest fluctuations in α 1-L2 and interswitch regions but the highest in Switch-II region."
33978412
sparser
"The mutant selectivity of JDQ443 translated in cellular systems, with JDQ443 potently and selectively inhibiting effector recruitment to KRAS G12C , but not to RAS WT paralogs as demonstrated in the mechanistic KRAS:cRAF NanoBiT recruitment assay in HEK293 cells ( xref )."
35404998
reach
"The mutant selectivity of JDQ443 translated in cellular systems, with JDQ443 potently and selectively inhibiting effector recruitment to KRAS , but not to RAS WT paralogs as demonstrated in the mechanistic KRAS:cRAF NanoBiT recruitment assay in HEK293 cells (Table 1)."
35404998
sparser
"GTP-bound active RAS recruits RAF proteins (e.g., RAF1 and BRAF) to the plasma membrane to orchestrate MAPK signaling xref . xref shows PPIs of both KRAS-RAF1 and KRAS-BRAF constructed in one structure complex."
37162909
sparser
"ITC experiments showed that these salt bridge-forming inhibitors bound to both GDP and GTP-bound KRASG12D and effectively disrupted KRAS-CRAF interaction, but did not bind to wild-type or G12C mutant KRAS."
38734764
reach
"Thereafter, nucleotide association and KRAS/RAF-RBD interaction were separated."
32106676
sparser
"We next examined whether high RIT1 expression levels, akin to those resulting from pathogenic RIT1 mutations, modulate KRAS:RAF1 binding."
37450595
reach
"Next, we tested the functionality of the assay in a real-time measurement by monitoring Eu -GTP association and KRAS/RAF-RBD interaction."
32106676
sparser
"Moreover, aurora kinase A (AURKA) was shown to promote drug inhibition escape by interacting with KRAS G12C and c-Raf ( xref )."
33777798
reach
"A notable difference between K-RAS and K-RAS was that K-RAS was capable of binding C-RAF (Figure 5B)."
35202574
reach
"These studies revealed a dynamic picture of the assembly of the KRAS and CRAF complex via multivalent and dynamic interactions between KRAS, CRAF RBD-CRD, and the membrane."
32414921
sparser
"In contrast, the recent MoA characterization of BBO-8956, an early-stage covalent KRAS G12C inhibitor, leads to less clear conclusions since RAF1-RBD can still bind to the KRAS G12C-BBO-8956 complex, and 31 P NMR spectra are complex and allow several interpretations."
39664928
reach
"Nucleotide association and KRAS/RAF-RBD interaction were launched by 10 nM SOS (2 μL), and signals were monitored during a 60 min incubation at RT."
32106676
sparser
"PIONEER-predicted interface mutations of KRAS-RAF1 N-terminal, such as p."
37162909
sparser
"Despite significant advances in understanding KRAS biology, the structural and dynamic mechanisms of KRAS binding and allostery by which oncogenic mutations enhance KRAS-RAF1 binding and signaling remain incompletely understood."
39975035
sparser
"Mutational scanning of KRAS-RAF1 complexes identified key binding affinity hotspots, including Y40, E37, D38, and D33, and together with the MM-GBSA analysis revealed the hotspots leverage synergistic electrostatic and hydrophobic binding interactions in stabilizing the KRAS-RAF1 complexes."
39975035
sparser
"The identification of binding hotspots and allosteric communication routes offers new opportunities for developing targeted therapies to disrupt KRAS-RAF1 interactions and inhibit oncogenic signaling."
39975035
sparser
"Supporting the validity of these predictions, many indirect targets uncovered in our networks have already demonstrated success in pre-clinical models (e.g., HIF1A-EGLN1, KRAS-RAF1, and MYC-CDK7)."
33328249
sparser
"Increasing levels of RIT1, but not RIT1 E55G (a RAF-binding deficient mutant), enhanced the apparent KRAS:RAF1 binding affinity in a BRET assay ( xref ), indicating that RIT1 promotes RAS:RAF interaction in a manner dependent on RIT1:RAF1 binding."
37450595
reach
"We optimized DoMY-Seq by taking advantage of the well described and high-affinity interaction between KRAS and CRAF, and we provide high-resolution domain mapping on this and other protein interacting pairs, including CRAF-MEK1, RIT1-RGL3, and p53-MDM2."
33410398
sparser
"Following the transition to late endosomes, Raf1-bound K-Ras activates the protein complex that include adaptors p14 and MP1 ( xref ) that in turn recruits and binds MEK1, thereby facilitating ERK1/2 entry into the nucleus ( xref )."
22875452
sparser
"KRAS-Q61H preferentially binds to C-Raf, due to altered conformation of the Switch II region, which stabilises the switch I region."
39372214
reach
"Recruitment of Grb2 and Raf1 to KRas positive LEs."
19289794
reach
"These 3D coculture results, coupled with the in vivo PDX results showing that KRB-456 is effective against tumors derived from relapsed patients (two of the biopsies were resected from patients who relapsed after treatment with 5-FU, gemcitabine, capecitabine, and/or radiation), suggest that KRB-456 can overcome resistance to chemotherapy and radiotherapy.KRB-456 binds KRAS (ITC and protein NMR data), inhibits the binding of KRAS to RAF1 in vitro (alpha screen and GST-RBD pulldown) as well as in intact cancer cells (co-immunoprecipitation with KRAS and blotting with RAF-1 and GST-RBD pulldown) and inhibits the immediate RAS/RAF downstream effector P-MEK clearly demonstrating that KRB-456 engages its target and suppresses KRAS oncogenic signaling in pancreatic cancer."
38051103
sparser
"High-resolution crystal structures of active KRAS G13D and KRAS G13D -RAF1 RBD complex provide snapshots of the State 1 and 2 conformations, respectively."
37268708
sparser
"C-RAF binding by K-RAS A59T was more similar to wild-type K-RAS, and K-RAS A59Tp failed to interact with C-RAF-RBD at all ( xref )."
35202574
sparser
"Because K-RAS A59E binds C-RAF-RBD, but K-RAS A59Tp does not, we examined the dynamics of MAPK signaling in more detail."
35202574
reach
"Therefore, at least in the case of pancreatic cancer where the tumors appear to be highly dependent on energy from autophagy especially when KRAS or its downstream effectors are inhibited, combination of KRB-456 with autophagy inhibitors may also prove to be efficacious.In summary, we report here on the discovery of a novel natural product-inspired small molecule, KRB-456, that binds a dynamic allosteric binding pocket within the switch-I/II region of KRAS G12D, suppresses the levels of KRAS bound to GTP and inhibits the binding of KRAS to RAF1 in human pancreatic cancer cells that harbor KRAS G12D."
38051103
sparser
"We used two complementary cell-based assays to examine whether the recombinant CPP-RBDs inhibited RAS, namely (1) immunoblotting that measured phosphorylation of RAS downstream proteins (AKT and ERK) [MISSING/INVALID CREDENTIALS: limited to 200 char for Elsevier]"
33964212
sparser
"Zhang and others described that K-RAS4A interacts with Raf-1 with higher affinity than K-RAS4B, leading to increased RAF1-1MEK-ERK signalling cascade, and that K-RAS4A showed increased anchorage-independent growth in assays that compared K-RAS4A- and K-RAS4B-transformed NIH 3T3 cells ( xref )."
34604308
sparser
"A study of the KRAS-CRAF interface indicates that four switch I residues form polar bonds with six CRAF residues: E31-K84, D33-K84, E37 with R59 and R67, D38 with T68 and R89, respectively."
34450320
sparser
"Although most of these investigations studied the interaction of KRAS with CRAF, it is plausible that RAS nanoclusters also regulate the interaction with other effectors in the PM."
33562401
sparser
"Here, we report NMR studies of the KRAS:CRAF RBD-CRD complex."
32414921
sparser
"These studies revealed a dynamic picture of the assembly of the KRAS-CRAF complex via multivalent and dynamic interactions between KRAS, CRAF RBD-CRD, and the membrane."
32414921
sparser
"We optimized DoMY-Seq by taking advantage of the well-described and high-affinity interaction between KRAS and CRAF, and we provide high resolution domain mapping on this and other protein interacting pairs, including CRAF-MEK1, RIT1-RGL3, and p53-MDM2."
33144325
sparser
"Also, phloroglucinol effectively decreased the active form of KRAS that could interact with RAF-1 (Fig. xref )."
25456733
sparser
"The results also provide direct evidence in individual cells that post-translational modification of K-Ras and its localization at the plasma membrane (PM) were not essential for the interaction of K-Ras and Raf-1, whereas the interaction of Grb2 and K-Ras did depend on the PM localization of K-Ras."
24556667
sparser
"Overall, this is consistent with an interaction of KRAS only with the active form of C-RAF, which requires dephosphorylation of S259 and unmasking of the RBD and CRD to allow KRAS binding to C-RAF at the membrane (reviewed in xref )."
38499327
sparser
"In terms of KRAS binding, F141 and K179 are critical for the interaction between KRAS and C-RAF during the activation process ( xref )."
38499327
sparser
"This study sheds light on the interaction between K-Ras and Raf1 and provides a practical method to elucidate the mechanism underlying K-Ras and Raf1 binding to the cell membrane."
23012550
sparser
"Recently, detailed interactions between K-Ras and Raf1 have been elucidated including the specifics of the conformational change which Raf1 undergoes upon binding to K-Ras [ xref ]."
23012550
sparser
"In summary, the intercellular interaction of Raf1 and K-Ras was investigated using BiFC assay and a co-expression system with fluorescent protein fusions."
23012550
sparser
"Moreover, significant co-localized signal between K-Ras and Raf1 was found in the cell membrane ( xref ), suggesting that K-Ras affects the cellular localization of Raf1, and the cellular interaction between K-Ras and Raf1 is likely to facilitate the membrane-binding ability of Raf1."
23012550
sparser
"Thus, the comparison of the effect of K-Ras and CAAX mutated K-Ras protein K-RasC185S on the cellular localization of Raf1 may provide useful information regarding the cellular interaction between K-Ras and Raf1."
23012550
sparser
"This finding is similar with previous reports on the interactions between Raf1 and K-Ras [ xref , xref ]."
23012550
sparser
"Although the combination of Raf1/K-RasC185S in BiFC may not occur under physiological conditions, investigating this interaction under the special conditions of BiFC may provide new insights into the understanding of the cellular interaction between Raf1 and K-Ras."
23012550
sparser
"For example, KRAS G12D binds to the RAF1-RBD approximately fivefold less strongly than wild-type RAS does xref ."
36028717
sparser
"The role of D154 and α4-α5 helices in RAS dimerization also becomes problematic when one takes into account the modeling of the KRAS-CRAF (RBD-CRD) complex on the membrane using previously identified CRD membrane-interacting residues into the lipid bilayer which places KRAS helices α4 and α5 in membrane contact xref , consistent with an earlier study using FRET vectors xref ."
36990093
reach
"After washing the agarose beads three times with Pierce IP Lysis Buffer, the activated KRAS (GTP-RAS) bound to RAF-1 RBD-agarose beads was released by the addition of SDS lysis buffer."
33122197
sparser
"Using this BEVL-BiFC system, we provided direct evidence in individual cells that post-translational modification of K-Ras and its PM localization were not essential for the interaction between K-Ras and Raf-1, while the interaction of Grb2 and K-Ras depended on PM localization of K-Ras."
24556667
sparser
"Herein, we demonstrate how a novel cell-penetrating peptide disruptor (DRx-170) of the c-RAF–PDE8A protein–protein interaction (PPI) represents a differentiated approach to exploiting the c-RAF–cAMP/PKA signaling axes and treating KRAS–c-RAF dependent PDAC."
38637546
sparser
"This proof-of-concept study supports the development of DRx-170 as a novel and differentiated strategy for targeting c-RAF activity in KRAS–c-RAF dependent PDAC."
38637546