BRAF is a humangene that encodes aprotein called B-Raf. The gene is also referred to asproto-oncogene B-Raf andv-Raf murine sarcoma viral oncogene homolog B, while the protein is more formally known asserine/threonine-protein kinase B-Raf.[5][6]
The B-Raf protein is involved in sendingsignals inside cells which are involved in directingcell growth. In 2002, it was shown to bemutated in some humancancers.[7]
Certain other inheritedBRAF mutations cause birth defects.
Drugs that treat cancers driven byBRAF mutations have been developed. Two of these drugs,vemurafenib[8] anddabrafenib, are approved by FDA for treatment of late-stage melanoma. Vemurafenib was the first approved drug to come out offragment-based drug discovery.[9]
B-Raf is a member of theRaf kinase family of growthsignal transductionprotein kinases. This protein plays a role in regulating theMAP kinase/ERKssignaling pathway, which affectscell division,differentiation, and secretion.[10]
B-Raf is a 766-amino acid, regulatedsignal transductionserine/threonine-specific protein kinase. Broadly speaking, it is composed of three conserveddomains characteristic of theRaf kinase family: conserved region 1 (CR1), aRas-GTP-binding[11] self-regulatory domain, conserved region 2 (CR2), aserine-rich hinge region, and conserved region 3 (CR3), a catalyticprotein kinase domain thatphosphorylates aconsensus sequence on protein substrates.[12] In its active conformation, B-Raf forms dimers viahydrogen-bonding and electrostatic interactions of its kinase domains.[13]
Conserved region 1 (CR1)autoinhibits B-Raf's kinase domain (CR3) so that B-Raf signaling is regulated rather than constitutive.[12] Residues 155–227[14] make up theRas-binding domain (RBD), which binds to Ras-GTP'seffector domain to release CR1 and halt kinase inhibition. Residues 234–280 comprise aphorbol ester/DAG-bindingzinc finger motif that participates in B-Raf membrane docking after Ras-binding.[14][15]
Conserved region 2 (CR2) provides a flexible linker that connects CR1 and CR3 and acts as a hinge.[16]
Conserved region 3 (CR3), residues 457–717,[14] makes up B-Raf's enzymatic kinase domain. This largely conserved structure[17] is bi-lobal, connected by a short hinge region.[18] The smallerN-lobe (residues 457–530) is primarily responsible forATP binding while the largerC-lobe (residues 535–717) bindssubstrate proteins.[17] The active site is the cleft between the two lobes, and the catalyticAsp576 residue is located on the C-lobe, facing the inside of this cleft.[14][17]
TheP-loop of B-Raf (residues 464–471) stabilizes the non-transferablephosphate groups of ATP during enzyme ATP-binding. Specifically,S467,F468, andG469 backboneamides hydrogen-bond to the β-phosphate of ATP to anchor the molecule. B-Raf functional motifs have been determined by analyzing the homology ofPKA analyzed by Hanks and Hunter to the B-Raf kinase domain.[17]
V471,C532,W531,T529,L514, andA481 form a hydrophobic pocket within which the adenine of ATP is anchored through Van der Waals attractions upon ATP binding.[17][19]
Residues 574–581 compose a section of the kinase domain responsible for supporting the transfer of the γ-phosphate of ATP to B-Raf's protein substrate. In particular,D576 acts as aproton acceptor to activate thenucleophilic hydroxyl oxygen on substrate serine or threonine residues, allowing the phosphate transfer reaction to occur mediated bybase-catalysis.[17]
D594,F595, andG596 compose a motif central to B-Raf's function in both its inactive and active state. In the inactive state, F595 occupies the nucleotide-binding pocket, prohibiting ATP from entering and decreasing the likelihood of enzyme catalysis.[13][19][20] In the active state, D594chelates thedivalentmagnesiumcation that stabilizes the β- and γ-phosphate groups of ATP, orienting the γ-phosphate for transfer.[17]
Residues 596–600 form strong hydrophobic interactions with the P-loop in the inactive conformation of the kinase, locking the kinase in its inactive state until theactivation loop is phosphorylated, destabilizing these interactions with the presence of negative charge. This triggers the shift to the active state of the kinase. Specifically, L597 and V600 of the activation loop interact with G466, F468, and V471 of the P-loop to keep the kinase domain inactive until it is phosphorylated.[18]
B-Raf is aserine/threonine-specific protein kinase. As such, it catalyzes the phosphorylation ofserine andthreonine residues in aconsensus sequence on target proteins byATP, yieldingADP and a phosphorylated protein as products.[17] Since it is a highly regulatedsignal transductionkinase, B-Raf must first bindRas-GTP before becoming active as an enzyme.[15] Once B-Raf is activated, a conserved protein kinase catalytic core phosphorylates protein substrates by promoting the nucleophilic attack of the activated substrate serine or threoninehydroxyl oxygen atom on theγ-phosphate group of ATP throughbimolecular nucleophilic substitution.[17][21][22][23]
The kinase (CR3) domain of humanRaf kinases is inhibited by two mechanisms:autoinhibition by its ownregulatoryRas-GTP-binding CR1 domain and a lack ofpost-translational phosphorylation of key serine andtyrosine residues (S338 and Y341 for c-Raf) in the CR2 hinge region. During B-Raf activation, the protein'sautoinhibitory CR1 domain first binds Ras-GTP'seffector domain to the CR1 Ras-binding domain (RBD) to release the kinase CR3 domain like other members of the human Raf kinase family. The CR1-Ras interaction is later strengthened through the binding of thecysteine-rich subdomain (CRD) of CR1 to Ras andmembranephospholipids.[12] UnlikeA-Raf andC-Raf, which must be phosphorylated on hydroxyl-containing CR2 residues before fully releasing CR1 to become active, B-Raf is constituitively phosphorylated on CR2 S445.[24] This allows the negatively charged phosphoserine to immediately repel CR1 through steric and electrostatic interactions once the regulatory domain is unbound, freeing the CR3 kinase domain to interact with substrate proteins.
After the autoinhibitory CR1 regulatory domain is released, B-Raf's CR3kinase domain must change to itsATP-binding activeconformer before it can catalyzeprotein phosphorylation. In the inactive conformation, F595 of the DFG motif blocks thehydrophobicadenine binding pocket whileactivation loop residues form hydrophobic interactions with the P-loop, stopping ATP from accessing its binding site. When the activation loop is phosphorylated, the negative charge of the phosphate is unstable in the hydrophobic environment of the P-loop. As a result, the activation loop changesconformation, stretching out across the C-lobe of thekinase domain. In this process, it forms stabilizingβ-sheet interactions with the β6 strand. Meanwhile, the phosphorylated residue approaches K507, forming a stabilizingsalt bridge to lock the activation loop into place. The DFG motif changes conformation with the activation loop, causing F595 to move out of the adenine nucleotide binding site and into a hydrophobic pocket bordered by theαC and αE helices. Together, DFG and activation loop movement upon phosphorylation open the ATPbinding site. Since all other substrate-binding and catalytic domains are already in place, phosphorylation of the activation loop alone activates B-Raf's kinase domain through a chain reaction that essentially removes a lid from an otherwise-prepared active site.[18]
To effectively catalyze protein phosphorylation via the bimolecular substitution of serine and threonine residues withADP as aleaving group, B-Raf must first bind ATP and then stabilize thetransition state as the γ-phosphate of ATP is transferred.[17]
B-Raf binds ATP by anchoring the adenine nucleotide in anonpolar pocket (yellow, Figure 1) and orienting the molecule through hydrogen-bonding and electrostatic interactions with phosphate groups. In addition to the P-loop and DFG motif phosphate binding described above, K483 and E501 play key roles in stabilizing non-transferable phosphate groups. The positive charge on the primaryamine of K483 allows it to stabilize the negative charge on ATP α- and β-phosphate groups when ATP binds. When ATP is not present, the negative charge of the E501carboxyl group balances this charge.[17][18]
Once ATP is bound to the B-Raf kinase domain, D576 of the catalytic loop activates a substrate hydroxyl group, increasing its nucleophilicity to kinetically drive the phosphorylation reaction while other catalytic loop residues stabilize the transition state (Figure 2). N581 chelates the divalent magnesium cation associated with ATP to help orient the molecule for optimal substitution. K578 neutralizes the negative charge on the γ-phosphate group of ATP so that the activated ser/thr substrate residue will not experience as much electron-electron repulsion when attacking the phosphate. After the phosphate group is transferred, ADP and the new phosphoprotein are released.[17]
Since constitutively active B-Raf mutants commonly cause cancer (see Clinical Significance) by excessively signaling cells to grow, inhibitors of B-Raf have been developed for both the inactive and active conformations of the kinase domain as cancer therapeutic candidates.[18][19][20]
BAY43-9006 (Sorafenib, Nexavar) is a V600Emutant B-Raf andC-Raf inhibitor approved by theFDA for the treatment of primaryliver andkidney cancer. Bay43-9006 disables the B-Rafkinase domain by locking the enzyme in its inactive form. The inhibitor accomplishes this by blocking the ATP binding pocket through high-affinity for the kinase domain. It then binds key activation loop andDFG motif residues to stop the movement of the activation loop and DFG motif to the active conformation. Finally, a trifluoromethyl phenyl moiety sterically blocks the DFG motif and activation loop active conformation site, making it impossible for the kinase domain to shift conformation to become active.[18]
The distalpyridyl ring of BAY43-9006 anchors in the hydrophobic nucleotide-binding pocket of the kinase N-lobe, interacting with W531, F583, and F595. The hydrophobic interactions with catalytic loop F583 and DFG motif F595 stabilize the inactive conformation of these structures, decreasing the likelihood of enzyme activation. Further hydrophobic interaction of K483, L514, and T529 with the center phenyl ring increase the affinity of the kinase domain for the inhibitor. Hydrophobic interaction of F595 with the center ring as well decreases the energetic favorability of a DFG conformation switch further. Finally, polar interactions of BAY43-9006 with the kinase domain continue this trend of increasing enzyme affinity for the inhibitor and stabilizing DFG residues in the inactive conformation. E501 and C532 hydrogen bond theurea and pyridyl groups of the inhibitor respectively while theureacarbonyl accepts a hydrogen bond from D594's backboneamide nitrogen to lock the DFG motif in place.[18]
The trifluoromethyl phenyl moiety cements the thermodynamic favorability of the inactive conformation when the kinase domain is bound to BAY43-9006 by sterically blocking the hydrophobic pocket between the αC and αE helices that the DFG motif and activation loop would inhabit upon shifting to their locations in the active conformation of the protein.[18]
PLX4032 (Vemurafenib) is a V600mutant B-Raf inhibitor approved by theFDA for the treatment of late-stagemelanoma.[13] UnlikeBAY43-9006, which inhibits the inactive form of the kinase domain, Vemurafenib inhibits the active "DFG-in" form of the kinase,[19][20] firmly anchoring itself in the ATP-binding site. By inhibiting only the active form of the kinase, Vemurafenib selectively inhibits the proliferation of cells with unregulated B-Raf, normally those that causecancer.
Since Vemurafenib only differs from its precursor, PLX4720, in aphenyl ring added forpharmacokinetic reasons,[20] PLX4720's mode of action is equivalent to Vemurafenib's. PLX4720 has good affinity for the ATP binding site partially because its anchor region, a 7-azaindolebicyclic, only differs from the natural adenine that occupies the site in two places where nitrogen atoms have been replaced by carbon. This enables strong intermolecular interactions like N7 hydrogen bonding to C532 and N1 hydrogen bonding to Q530 to be preserved. Excellent fit within the ATP-binding hydrophobic pocket (C532, W531, T529, L514, A481) increases binding affinity as well.Ketone linker hydrogen bonding to water and difluoro-phenyl fit in a second hydrophobic pocket (A481, V482, K483, V471, I527, T529, L514, and F583) contribute to the exceptionally high binding affinity overall. Selective binding to active Raf is accomplished by the terminal propyl group that binds to a Raf-selective pocket created by a shift of the αC helix. Selectivity for the active conformation of the kinase is further increased by a pH-sensitive deprotonatedsulfonamide group that is stabilized by hydrogen bonding with the backbone peptide NH of D594 in the active state. In the inactive state, the inhibitor's sulfonamide group interacts with the backbonecarbonyl of that residue instead, creating repulsion. Thus, Vemurafenib binds preferentially to the active state of B-Raf's kinase domain.[19][20]
Mutations in theBRAF gene can cause disease in two ways. First, mutations can be inherited and cause birth defects. Second, mutations can appear later in life and cause cancer, as anoncogene.
Inherited mutations in this gene causecardiofaciocutaneous syndrome, a disease characterized by heart defects, mental retardation and a distinctive facial appearance.[25]
Mutations in this gene have been found in cancers, includingnon-Hodgkin lymphoma,colorectal cancer,melanoma,papillary thyroid carcinoma,non-small-cell lung carcinoma,adenocarcinoma of the lung,neuroendocrine carcinoma,[26]brain tumors includingglioblastoma, andpleomorphic xanthoastrocytoma as well as inflammatory diseases likeErdheim–Chester disease.[10]
The V600E mutation of the BRAF gene has been associated withhairy cell leukemia in numerous studies and has been suggested for use in screening forLynch syndrome to reduce the number of patients undergoing unnecessaryMLH1 sequencing.[27][28]
More than 30 mutations of theBRAF gene associated with human cancers have been identified. The frequency of BRAF mutations varies widely in human cancers, from more than 80% inmelanomas andnevi, to as little as 0–18% in othertumors, such as 1–3% in lung cancers and 5% incolorectal cancer.[29] In 90% of the cases, thymine is substituted with adenine at nucleotide 1799. This leads to valine (V) being substituted for by glutamate (E) at codon 600 (now referred to asV600E) in the activation segment that has been found in human cancers.[30] This mutation has been widely observed inpapillary thyroid carcinoma, colorectal cancer, melanoma andnon-small-cell lung cancer.[31][32][33][34][35][36][37] BRAF-V600E mutation are present in 57% of Langerhans cell histiocytosis patients.[38] The V600E mutation is a likely driver mutation in 100% of cases ofhairy cell leukaemia.[39] High frequency of BRAF V600E mutations have been detected inameloblastoma, a benign but locally infiltrative odontogenic neoplasm.[40] The V600E mutation may also be linked, as a single-driver mutation (a genetic 'smoking gun') to certain cases of papillarycraniopharyngioma development.[41]
Other mutations which have been found are R461I, I462S, G463E, G463V, G465A, G465E, G465V, G468A, G468E, G469R, N580S, E585K, D593V, F594L, G595R, L596V, T598I, V599D, V599E, V599K, V599R, V600K, A727V, etc. and most of these mutations are clustered to two regions: the glycine-rich P loop of the N lobe and the activation segment and flanking regions.[18] These mutations change the activation segment from inactive state to active state, for example in the previous cited paper it has been reported that the aliphatic side chain of Val599 interacts with the phenyl ring of Phe467 in the P loop. Replacing the medium-sized hydrophobic Val side chain with a larger and charged residue as found in human cancer(Glu, Asp, Lys, or Arg) would be expected to destabilize the interactions that maintain the DFG motif in an inactive conformation, so flipping the activation segment into the active position. Depending on the type of mutation the kinase activity towardsMEK may also vary. Most of the mutants stimulate enhanced B-Rafkinase activity toward MEK. However, a few mutants act through a different mechanism because although their activity toward MEK is reduced, they adopt a conformation that activates wild-type C-RAF, which then signals toERK.
As mentioned above, some pharmaceutical firms are developing specific inhibitors of mutated B-raf protein foranticancer use because BRAF is a well-understood, high yield target.[19][43]Vemurafenib (RG7204 or PLX4032) was licensed by the USFood and Drug Administration (FDA) as Zelboraf for the treatment of metastatic melanoma in August 2011 based on Phase III clinical data. Improved survival was seen, as well as a response rate to treatment of 53%, compared to 7–12% with the former best chemotherapeutic treatment,dacarbazine.[44]Dabrafenib in combination withtrametinib was approved byFDA in June 2022 for treatment of all metastatic BRAF V600E mutated solid tumors.[45] In clinical trials, B-Raf increased metastatic melanoma patient chance of survival. In spite of the drug's high efficacy, 20% of tumors still develop resistance to the treatment. In mice, 20% of tumors become resistant after 56 days.[46] While the mechanisms of this resistance are still disputed, some hypotheses include the overexpression of B-Raf to compensate for high concentrations of Vemurafenib[46] and upstream upregulation of growth signaling.[47]
More generalB-Raf inhibitors include GDC-0879, PLX-4720,Sorafenib,dabrafenib andencorafenib.
Belvarafenib is classified as a panRAF inhibitor. A panRAF inhibitor blocks the catalytic function of both proteins in the dimer.[48]
BRAF (gene) has been shown tointeract with:
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This article incorporatespublic domain material fromDictionary of Cancer Terms.U.S. National Cancer Institute.This article incorporates text from theUnited States National Library of Medicine, which is in thepublic domain.
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