IndraLab
Statements
sparser
"To test this hypothesis, we treated macrophages with the highly selective B-Raf inhibitor GDC-0879 ( xref ), because B-Raf inhibition with GDC-0879 in the presence of activated Ras induces B-Raf binding to c-Raf, leading to c-Raf hyperactivation and thereby elevated MEK/ERK signaling ( xref ; xref ; xref )."
sparser
"Interestingly, treatment with the BRAF inhibitor Vemurafenib decreased EphA2 levels in BRAF MT cells, but not in KRAS MT cells ( xref ); the sustained MAPK signaling following Vemurafenib or siBRAF in the KRAS MT CRC cells is consistent with previous studies showing that inhibition of BRAF in the presence of oncogenic RAS induces BRAF binding to CRAF, leading to CRAF hyper-activation and sustained MEK1/2-ERK1/2 activation ( xref )."
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"A recent study reported that a subset of MEK inhibitors that are inactive in RAS-mutant cancers (AZD6244, GDC-0973) promotes BRAF and CRAF heterodimer formation allowing feedback activation of MEK and ERK, whereas RAS active MEK inhibitors (GDC-0623, G-573) stabilize a nonproductive RAF and MEK complex preventing MEK feedback activation."
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"Targeting MEK downstream of BRAF and CRAF heterodimers using PD0325901 subsequent to RAF inhibitor treatment reduced ERK1/2 rebound and simultaneous treatment of tumor xenografts with both RAF and MEK inhibitors led to more pronounced tumor growth inhibition than either treatment alone."
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"While our data indicates B-Raf and c-Raf dimerization is likely a key mechanism promoting MAP Kinase pathway reactivation in response to dasatinib, we have also observed downregulation of negative regulators of the MAP Kinase pathway, including DUSP1, DUSP4, and SPRY1 (data not shown), as well as increased B-Raf protein levels in the BRAF-mutant, BCPAP, cell line in the dasatinib resistant cells (XREF_FIG)."
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"Interestingly, Braf-null MEFs reconstituted with BRAF S729A mutant showed similar basal ERK activity to WT BRAF expressing cells, suggesting that, in these cells dimerization of BRAF with CRAF is not critical for ERK activation and that the BRAF-KSR1 interaction is more important."
sparser
"Another drug resistance mechanism takes advantage of the fact that RAF kinases normally signal as homo- and hetero-dimers, and drug-bound BRAF in an inactive conformation is able to allosterically shift the associated non-drug bound wild-type BRAF or CRAF subunit into an activate conformation in a process called “transactivation” or “paradoxical activation” to promote MAPK signaling xref ."
sparser
"We have also used RNAi to examine the potential role of other proteins implicated in BRAF-CRAF complex formation or pathway activation, including the scaffold proteins KSR, Sprouty2 and RKTG and the small G protein RHEB, but our preliminary results have not revealed obvious roles for these proteins."
sparser
"This was the first study to show RAF dimerisation without using artificial dimerisation constructs and also showed that MEK1/2 activation by BRAF:CRAF heterodimers was caused by the RAS-mediated recruitment of RAF dimers to the plasma membrane, as a KRAS G12V variant (aa 1–166) lacking C-terminal membrane targeting residues failed to increase MEK1/2 phosphorylation despite co-immunoprecipitating with, and increasing the formation of, BRAF:CRAF heterodimers."
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"In addition, we find that C-Raf is critical for mutant H-Ras-driven signaling and that events stabilizing B-Raf and C-Raf dimerization, such as Raf inhibitor treatment or certain B-Raf mutations, can allow mutant H-Ras to engage B-Raf with increased affinity to promote tumorigenesis, thus revealing a previously unappreciated role for C-Raf in potentiating B-Raf function."
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"Phosphorylated Ksr can also function as a transactivator; however, since Raf binding to Ksr induces limited kinase activity XREF_BIBR, in quiescent cells the constitutive association of Ksr with B-Raf may serve to prevent C-Raf binding to B-Raf, safeguarding against undue activation of the pathway XREF_BIBR."
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"In addition, CAV-1-mediated cross-talk between EphA2 and BRaf is required for response to dasatinib whereby dasatinib drives BRaf and EphA2 to CAV-1 at the plasma membrane, disrupting the BRaf and CRaf heterodimer and thus down-modulation of the mitogen activated protein kinase (MAPK) pathway in dasatinib sensitive cells, but not in dasatinib resistant cells (XREF_FIG)."
sparser
"Underlying the mechanism for the autocrine growth promotion, there are three indispensable molecular events disclosed in this study: enrichment of CRAF through inhibition of proteasomal degradation, CRAF stabilization effects of GSTP1 facilitation of the RAF dimer (CRAF-CRAF and CRAF-BRAF) formation, and activation of catalytic activity of CRAF."
sparser
"A recent study reported that a subset of MEK inhibitors that are inactive in RAS-mutant cancers (AZD6244, GDC-0973) promotes BRAF-CRAF heterodimer formation allowing feedback activation of MEK and ERK, whereas RAS active MEK inhibitors (GDC-0623, G-573) stabilize a nonproductive RAF-MEK complex preventing MEK feedback activation ( xref )."
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"We investigated the relationship between cRaf and BRaf and found that active Ras induced heterodimerization of cRaf and BRaf, an effect that was dependent on the serine residue at position 621 of cRAF : Moreover, we also found that cRaf COOH-terminus constitutively associated with BRaf, whereas the NH (2) terminus did not, even in the presence of active RAS : These data suggest that Ras induces the cRaf and BRaf complex formation through the exposure of 14-3-3 binding sites in the COOH-terminus of cRAF : Thus, Ras induced cRaf-Braf heterodimerization may explain the observed cooperativity of cRaf and BRaf in cells responding to growth factor signals."
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"In those cells without BRAF mutations, BRAF inhibitors have been shown to promote BRAF and CRAF heterodimer or CRAF-CRAF homodimer formation, in which one BRAF inhibitor bound RAF protein transactivates the adjacent CRAF subunit of the dimer, leading to downstream activation of MEK-ERK."
sparser
"In those cells without BRAF mutations, BRAF inhibitors have been shown to promote BRAF-CRAF heterodimer or CRAF-CRAF homodimer formation, in which one BRAF inhibitor bound RAF protein transactivates the adjacent CRAF subunit of the dimer, leading to downstream activation of MEK-ERK ( xref ; xref ; xref )."
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"Nonetheless, it has been suggested that selective BRAF inhibition induces RAS dependent dimerization of CRAF with WT BRAF, but not BRAF V600E, as well as the formation of CRAF homodimers, leading to subsequent activation of CRAF and downstream MEK-ERK signaling (XREF_FIG) [XREF_BIBR, XREF_BIBR, XREF_BIBR, XREF_BIBR]."
sparser
"This mutation can increase RAF dimer formation and promote ERK pathway signal expression. xref In the BRAF gene, a kind of splice variant that deletes a part of the exon that removes the RBD participates in the resistance to BRAF inhibitors by increasing the tendency to form homologous dimers and enhancing the MAPK signaling pathway. xref , xref , xref A study supported that the AGAP3‐BRAF fusion protein, which lacks the RBD, may be a novel resistance mechanism. xref Some researchers also demonstrated that BRAF, when inhibited, can be transformed into CRAF or ARAF through a kinase transformation that continues to activate MAPK, leading to resistance to BRAF inhibitors. xref In addition, in RAS mutations, BRAF can bind to CRAF when BRAF is inhibited, thereby promoting the MAPK pathway."
sparser
"G12C KRAS -mutant, xref , xref , xref so an alternative is to target downstream effectors in the RAF/MEK/ERK pathway, which has led to the development of RAF, MEK and ERK drugs. xref However, in KRAS -mutant cells, BRAF-selective drugs such as vemurafenib (PLX4032) and dabrafenib xref cause paradoxical hyperactivation of the RAF-ERK pathway through formation of BRAF-CRAF homo- and hetero-dimers. xref Unfortunately, targeting MEK downstream of RAF with drugs such as trametinib xref is ineffective in KRAS -mutant cancers because of feedback mechanisms xref and adverse side-effects, xref and therefore these drugs have been unsuccessful in KRAS -mutant PDAC, CRC and NSCLC. xref , xref "
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"The key experiments being replicated include Figure 1A, in which the original authors demonstrated that treatment of a subset of BRAF WT tumor cell lines with RAF small molecule inhibitors resulted in an increase in cell viability, Figure 2B, which reported that RAF inhibitor activation of the MAPK pathway was dependent on CRAF but not BRAF, and Figure 4A, where the dimerization of BRAF and CRAF was modulated by the RAF inhibitor PLX4720, but not GDC-0879."
sparser
"While BRAF inhibitors of both classes stimulate the formation of BRAF-CRAF dimers [ xref , xref ], type-II inhibitors are considerably less efficient than type-I inhibitors in stimulating the phosphorylation of downstream targets, i.e., of MEK kinases and, henceforth, in activating the MAPK pathway."
sparser
"Targeting the MEK5-ERK5 pathway using MEK5 inhibitor BIX02189 or treatment with RAF inhibitor LY3009120 blocked cell growth to a greater extent in A1847-T cells than parental, demonstrating the dependency of trametinib–resistant cells for MEK5 and CRAF-BRAF signaling ( xref – xref )."
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"Other phosphorylation sites on CRAF and BRAF were diminished to some degree following treatment with KG5 which is not surprising considering the fact that KG5 is an allosteric inhibitor of RAF and blocks the dimerization of BRAF and CRAF XREF_BIBR thereby preventing co-activation of these molecules."
sparser
"It is noteworthy that mutation of BRAF Ser365, another known 14-3-3 binding site, to Ala in the context of Ser729 being still present dramatically reduces both basal and AMPK stimulated 14-3-3 binding ( xref ) and allows BRAF-CRAF dimerization and ERK activation to be maintained in the presence of AICAR ( xref and xref )."
sparser
"One intriguing phenomenon was that even a catalytically compromised B-Raf was capable of inducing kinase activity of Raf-1 in trans in a manner dependent on a physical interaction between B-Raf and Raf-1, suggesting that the underlying mechanism is independent of a simple transautophosphorylation route [ xref , xref - xref ]."
sparser
"Also, consistent with the idea that inhibitor-induced RAS−RAF association is not isoform- or allele-specific, we found that NRAS G12V , HRAS G12V , and KRAS Q61H all exhibited an increased propensity to interact with endogenous BRAF and CRAF upon GDC-0879 treatment (Fig. xref and Supplementary Fig. xref )."
sparser
"Successful strategies targeting this tunable-combinatorial signaling complex may include those inhibiting CRAF function (e.g., omni- or pan-RAF inhibitors), V600E BRAF-CRAF interaction, V600E BRAF- MUT MEK interaction/scaffolding, and MEK activation (e.g., phosphorylation by RAF)."
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"L779450 induced B-Raf and C-Raf dimerization was not reduced in MEFs expressing the R615H- and C809Y KSR1 mutants unable to bind B-Raf; whereas B-Raf and C-Raf dimerization was abolished in cells expressing S297A and S392A-KSR 1 that exhibits enhanced inhibitor induced B-Raf binding (XREF_FIG)."
sparser
"Oncogenic activation of this pathway can arise from mutations in any of the pathway components or promoters. xref Typically, the important downstream mediators in the MAPK cell signaling pathway are simulated by the activation of RAS which then signals downstream via the interaction between the serine–threonine kinases, BRAF and CRAF. xref , xref In BRAF wild-type cells, the BRAF kinase is activated by homo- or heterodimer formation with other RAF isoforms such as ARAF or CRAF. xref , xref In BRAF -mutated cells, the BRAF kinase remains constitutively activated in a monomeric state, with a >400-fold increase in kinase activity. xref – xref Upon activation of RAF, there is an interaction with downstream MEK kinases (MEK1 and MEK2) which initiates MEK phosphorylation that in turn facilitates an activating phosphorylation of ERK which promotes oncogenesis. xref , xref , xref Ultimately, the activation of ERK results in cellular proliferation along with migration and angiogenesis, accompanied by a deterrence of apoptosis, thereby promoting malignant cell growth. xref In the pathogenesis of melanoma, there is staunch reliance upon this signaling cascade."
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"The analysis of the BRAF V600E / NRAS melanoma mouse model showed an unexpected finding related to the effect of BRAF inhibitors in these cells : in fact, BRAF inhibitors induce RAS dependent binding of BRAF to CRAF, consequent activation first of CRAF and then of MEK-ERK signaling [XREF_BIBR]."
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"Selective inhibition of B-Raf drives oncogenic RAS dependent BRAF binding to C-Raf, CRAF activation and mitogen activated protein kinase kinase (MEK)-extracellular signal regulated kinase (ERK) signalling, revealing another paradigm of BRAF mediated signalling that promotes tumour progression [XREF_BIBR]."
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"These effects are consistent with the ability of Raf-inhibitors to transactivate Raf-1 when a PKA activated Ras promotes Raf-1 and B-Raf heterodimerization, and are inhibited by interfering with cAMP and PKA signaling both in vitro and in vivo, as shown by the reduction of liver cysts in mice treated with combined octreotide and sorafenib."
sparser
"The difference between Raf inhibitor combination effects might be due to the preferred Raf binding conformation for each compound: AZD628 binds to the inactive conformation (DFG-out) of Craf, whereas both GDC0879 and PLX4720 bind to the active CRaf conformation (DFG-in) xref , with PLX4720 causing a c-helix shift that may block BRaf:CRaf heterodimer formation, as observed in our current study."
sparser
"Previously it was reported that BRAF V600E kinase activity was reduced when it heterodimerized with CRAF ( xref ), so we hypothesized that KIT could drive formation of BRAF V600E :CRAF complexes, which would have less activity than uncomplexed BRAF V600E . To test this, we used a bioluminescence resonance energy transfer (BRET) assay to measure the interaction between Renilla Luciferase-tagged CRAF (RLuc-CRAF) and Venus-tagged BRAF V600E (V-BRAF V600E )."
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"Our inability to demonstrate an obvious role for KSR in mediating BRAF binding to CRAF or CRAF activation by BRAF suggests that the mechanism underlying dimerization here may be different from those described in flies, but clearly additional studies are required to investigate further the role of scaffold proteins in mediating the phenomena we report."
sparser
"Packer et al. xref reported that the binding of weak RAF inhibitors (imatinib, nilotinib and dasatinib) to BRAF and CRAF drives BRAF:CRAF heterodimer as well as BRCF and CRAF homodimer formation in the presence of oncogenetic RAS, thus leading to paradoxical activation of both BRAF and CRAF, followed by the activation of MEK and ERK."
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"Interestingly, it was shown that in the TERTp mutant breast cancer cell line MDA-MB-231, small molecule mediated dimerization between BRAF and CRAF (a proposed mechanism of this paradoxical activation) was insufficient to recapitulate the activation These studies, and our analysis of CTRP data, suggest that paradoxical activation mechanisms may be less relevant in a subset of tumors, including TERTp mutants."
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"Interestingly, whereas we observed increased dimerization in our BRAF-mutant cell lines upon dasatinib treatment, a previous report did not observe dimerization in two BRAF-mutant melanoma cell lines upon treatment with dasatinib and concluded that B-Raf and c-Raf dimerization occurs in a Ras dependent manner."
sparser
"Our inability to demonstrate an obvious role for KSR in mediating BRAF binding to CRAF or CRAF activation by BRAF suggests that the mechanism underlying dimerization here may be different from those described in flies, but clearly additional studies are required to investigate further the role of scaffold proteins in mediating the phenomena we report."
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"In vitro studies have shown that in the presence of oncogenic RAS proteins, kinase impaired BRAF forms a complex with CRAF and leads to hyperactivation of the CRAF/MEK/ERK cascade, suggesting MEK inhibitors or CRAF inhibitors may benefit patients with concomitant kinase impaired BRAF mutation and activating RAS mutation [XREF_BIBR, XREF_BIBR]."
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"By the use of probes based on the principle of fluorescence resonance energy transfer, we found that this amino-terminal B-Raf-specific region is essential for homo-dimerization of B-Raf and hetero-dimerization of B-Raf and c-Raf at the plasma membrane, followed by phosphorylation of Thr118 in the amino-terminal B-Raf-specific region."
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"The additive effect between Raf and Mek inhibitors on CRaf PM translocation was observed when measuring CRaf and BRaf hetero-dimerization and CRaf kinase activation induced by GDC0879 in the KRasG12D and CRaf cells (XREF_FIG) as well as in H226 NSCLC cells where only endogenous proteins are expressed (XREF_SUPPLEMENTARY)."
sparser
"► BRAF activates ERK but in some circumstances BRAF inhibitors can induce tumor growth ► BRAF inhibitors drive BRAF-CRAF binding, activating ERK in cells with oncogenic RAS ► Kinase-dead mutants of BRAF have the same effect as BRAF inhibitors ► Oncogenic RAS and kinase-dead BRAF cooperate to induce melanoma in mice"
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"One possible explanation for this phenotype is that PLX8394, originally selected for its ability to prevent BRAF and CRAF heterodimer formation in the context of mutant or GTP bound RAS, is not able to disrupt homo-dimerization of BRAF fusion kinases that are stabilized by an additional dimerization domain encoded by the 5 ' partner."
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"Here, we demonstrate for the first time that activation of Rheb is associated with decreased B-Raf and C-Raf phosphorylation at residues Ser 446 and Ser 338, respectively, concomitant with a decrease in the activities of both kinases and decreased heterodimerization of B-Raf and C-Raf."
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"It is unclear why PLX4720 only induces weak binding of BRAF to CRAF, but this may stem from its unique property of displacing the alpha-C helix of BRAF when it binds and suggests that this helix is important for BRAF binding to CRAF, something that will only be resolved when the BRAF : CRAF crystal structure is solved."
sparser
"To determine whether the NtA motif was required on both the “activator” and the “receiver”, as is implied by current models, we tested whether a CRAF “receiver” mutant that cannot be phosphorylated on residues 338–341 (AAFF substituted for SSYY), could be activated by activator forms of BRAF and CRAF ( xref )."
sparser
"While mutant BRAF V600E is constitutively active and has a limited role in dimerization, xref the BRAF-CRAF heterodimer is believed to be
the primary species in both native signaling and paradoxical activation. xref , xref , xref Genetic and biochemical results
have repeatedly implicated CRAF as the primary species responsible
for phosphorylating MEK in paradoxical activation and native signaling. xref − xref , xref − xref Specifically, inhibitor-bound
BRAF is implicated in promoting heterodimerization with unbound CRAF,
causing transactivation of CRAF through an allosteric mechanism at
the protein–protein interface between protomer kinase domains. xref , xref "
sparser
"Nevertheless, peptides generated from the dimerization interface of BRAF have been shown to prevent formation of homodimers and heterodimers of BRAF–CRAF in a BRAF-mutant or RAS-mutant cellular context xref , suggesting the possibility of an alternative strategy for developing inhibitors of RAF dimerization."
sparser
"It is unclear why PLX4720 only induces weak binding of BRAF to CRAF, but this may stem from its unique property of displacing the α-C helix of BRAF when it binds ( xref ) and suggests that this helix is important for BRAF binding to CRAF, something that will only be resolved when the BRAF:CRAF crystal structure is solved."
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"Moreover, BRAF inhibitors drive RAS dependent BRAF binding to CRAF, CRAF activation, and MEK-ERK signaling in oncogenic RAS [XREF_BIBR] revealing a paradigm of BRAF mediated signaling that promotes tumour progression with clinical implications [XREF_BIBR] and highlighting the importance of understanding pathway signaling in clinical practice and of genotyping tumours prior to administering BRAF selective drugs, to identify patients who are likely to respond and also to identify patients who may experience adverse effects such as increased invasion [XREF_BIBR]."
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"Meanwhile, our IP analysis showed that dasatinib induced the heterodimer of BRaf and CRaf in Ishikawa cells, but not in SKUT-2 cells (XREF_FIG), indicating that co-localization between BRaf and CAV-1 induced by dasatinib in SKUT-2 cells interferes with the formation of the BRaf and CRaf heterodimer and therefore can inactivate signaling downstream of BRaf and CRaf (MAPK pathway)."
sparser
"Among the mechanisms promoting BRAFi resistance that affect the MAPK signalling pathway, several lead to the dysregulation of RAF1 activity: hyperactivation of RTKs, loss of NF1, mutations in NRAS, increased expression of RAF1, RAF1-BRAF heterodimers, and loss of ERK negative feed-back loops [ xref , xref , xref ]."
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"However, the administration of sorafenib to mice with conditionally knocked-out Pkd2 caused an unexpected increase in liver cyst area, cell proliferation and expression of pERK, possibly due to the ability of Raf-inhibitors to transactivate Raf-1 when a PKA activated Ras promotes Raf-1 and B-Raf heterodimerization [XREF_BIBR]."
sparser
"Packer et al. [42] reported that the binding of weak RAF inhibitors (imatinib, nilotinib and dasatinib) to BRAF and CRAF drives BRAF:CRAF heterodimer as well as BRCF and CRAF homodimer formation in the presence of oncogenetic RAS, thus leading to paradoxical activation of both BRAF and CRAF, followed by the activation of MEK and ERK."
sparser
"In addition, we reported that DGKη interacted with C-Raf and B-Raf (mitogen-activated kinase (MAPK) kinase kinase (MAPKKK)) in response to epidermal growth factor (EGF) stimulation and regulated the Raf–MAPK/extracellular signal-regulated kinase (ERK) kinase (MEK)–ERK pathway [ xref ]."
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"Other phosphorylation sites on CRAF and BRAF were diminished to some degree following treatment with KG5 which is not surprising considering the fact that KG5 is an allosteric inhibitor of RAF and blocks the dimerization of BRAF and CRAF 20 thereby preventing co-activation of these molecules."
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"We have also used RNAi to examine the potential role of other proteins implicated in BRAF and CRAF complex formation or pathway activation, including the scaffold proteins KSR, Sprouty2 and RKTG and the small G protein RHEB, but our preliminary results have not revealed obvious roles for these proteins."
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"As our analysis revealed that mutations at MLK3 are most probably functionally relevant, occurring almost exclusively in MSI carcinomas, and that MLK3 is described as a component of the multiprotein BRAF and RAF1 complex, we decided to analyse how mutations in MLK3 (all types) correlated with mutations in KRAS and/or BRAF in our series."
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"Finally, B-Raf and c-Raf dimerization is an off-target effect that does not appear to be conserved amongst all Src inhibitors, as the Src inhibitor, saracatinib, does not strongly induce B-Raf and c-Raf dimerization in either the BRAF-mutant (BCPAP) or RAS-mutant (Cal62) cell lines (XREF_SUPPLEMENTARY), which is consistent with lack of phospho-ERK1/2 induction in response to c-Src knockdown (XREF_SUPPLEMENTARY)."
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"In contrast, equivalent C-Raf and B-Raf dimerization was observed in KSR -/- and WT-KSR1 MEFs treated with PLX4720, which does not promote KSR1 and B-Raf binding, and PLX4720 induced C-Raf and B-Raf dimerization was significantly increased in both cells lines when Ras V12 was expressed (XREF_FIG)."
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"The dimerization between C-Raf and B-Raf promotes ERK signaling; however, complex formation of KSR and B-Raf actually limits ERK activation [XREF_BIBR], which suggests that KSR in cells being treated with Raf kinase inhibitors may actually limit paradoxical, rebound ERK activation in response to inhibitor induced Raf dimerization and activation."
sparser
"Phosphorylated Ksr can also function as a transactivator; however, since Raf binding to Ksr induces limited kinase activity xref , in quiescent cells the constitutive association of Ksr with B-Raf may serve to prevent C-Raf binding to B-Raf, safeguarding against undue activation of the pathway xref ."
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"In further support that KSR1 can compete with C-Raf for inhibitor induced binding to B-Raf, C-Raf and B-Raf dimerization was consistently reduced in WT-KSR1 MEFs versus KSR -/- MEFs when cells were treated with any of the Raf inhibitors capable of promoting KSR1 and B-Raf binding (XREF_FIG), and C-Raf and B-Raf dimerization was still reduced in L779450 treated WT-KSR1 MEFs even when Ras V12 was expressed (XREF_FIG)."
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"In addition, upon BRAF inhibition, there is ERK-dependent activation of EGFR and subsequent reactivation of the MAPK pathway via CRAF, as well as dimerization between CRAF and BRAF ensuring pathway resignalling, and this renders BRAF inhibitors that act to sequester a BRAF mutant monomer, ineffective [24, 136]."
sparser
"Recent research has indicated
the potential for selective inhibition of CRAF-driven signaling to
be a more effective and tolerated therapeutic approach for mutant
RAS tumors. xref , xref Identifying selective CRAF kinase
inhibitors that would block BRAF-CRAF heterodimer signaling could
thus be a valuable drug for the treatment of mutant RAS-driven tumors,
but would present a major challenge for medicinal chemistry."
sparser
"CR1 contains two key regions, the RAS‐binding domain (RBD) and the cysteine‐rich domain (CRD), which combine with RAS‐GTP and are indispensable for cell membrane recruitment. xref , xref , xref An important function of CR1 is to inhibit CR3 and hence keep BRAF inactive. xref , xref When CR1 and RAS‐GTP are combined, this inhibition is relieved, and BRAF is activated (phosphorylation of the activation segment (AS) is also required). xref CR3 contains several key regions, namely, the P‐loop (also known as the glycine‐rich loop, located in the N‐region), an αC helix (important for the formation of BRAF‐CRAF dimers), a dimerization interface (DIF), a catalytic loop, a DFG motif, and the AS."