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Contents 1 Structural basis of B-Raf oncogenic mutations 1.1 Introduction 1.2 Catalytic function of B-Raf 1.3 Disease 1.4 Structural mechanism 1.4.1 Strongly activating mutations 1.4.2 Intermediate activating mutations 1.5 Known inhibitors 1.5.1 BAY43-9006 1.5.2 Vemurafenib

Structural basis of B-Raf oncogenic mutations

This page was created as a student project within a university course Structural biology of the cell (MB151P117) at Charles University in Prague.

A crystal structure of B-Raf kinase domain in complex with its inhibitor BAY43-9006 (PDB id 1uwh [1]) Show:Asymmetric Unit Biological Assembly Drag the structure with the mouse to rotate   Export Animated Image

Introduction

B-Raf (Rappidly Accelerated Fibrosarcoma) is a 766-amino acid serine/threonine protein kinase with the ability to switch tyrosine kinase signalling to serine/threonine phosphorylation and connect growth factor receptors with transcription factors. B-Raf is considered to be the most important kinase in RAF-MEK-ERK signal transduction pathway which involves a cascade of protein kinases and is essential for cellular proliferation and differentiation of species ranging from worms to humans. ^{[1] [2]}

B-Raf kinase belongs to Raf kinase family which in vertebrates has three members – A-Raf, B-Raf and C-Raf. All Raf proteins share three highly conserved regions embedded in variable sequences. CR1 (conserved region 1) containing a RAS binding domain and Cys rich domain, ^[3] CR2 (conserved region 2) rich in Ser and Thr residues, some of which are regulatory sites^[4] and CR3 (conserved region 3), a catalytic protein kinase domain. Raf kinase domain consists of N-lobe and C-lobe connected by a flexible hinge, the N-lobe is primarily responsible for ATP binding while the C-lobe is responsible for substrate binding. ^[5]

Catalytic function of B-Raf

B-Raf kinase acts to phosphorylate its downstream target MEK (Mitogen-activated protein kinase kinase) which in turn leads to phosphorylation of other kinases in the cascade. ^[6] B-Raf kinase domain switches between closed and opened conformation, however the active state is resctricted to the closed one. In B-Raf inactive conformation Phe594 of the DFG motif (Asp-Phe-Gly) at the N terminus of the activation loop blocks the ATP binding pocket. P loop(residues 464–471) forms hydrophobic interactions with the activation segment (residues 596–600) to stabilizes the inactive conformation. ^[7] A phosphorylation within the activation segment at residue Thr599 and Ser602 introduces negative charge which is unstable in the hydrophobic environment of the P-loop. ^[8] The change of conformation of DFG motif and activation segment opens the ATP binding pocket. ^[9]

Disease

Misregulation of cell-signalling pathways is often linked to cancer. In case of B-Raf over 30 mutations in its gene have been identified as oncogenic. A majority of these mutants are located within the kinase domain and enhance its kinase activity by maintaning the kinase domain in its active conformation. Increased B-Raf activity in turn leads to promoted tumor cell growth. ^[10]

Structural mechanism

Strongly activating mutations

Strongly activating mutations favour the active conformation without interfering with the catalytic process. These mutations mimic the conformational changes induced by activation segment phosphorylation. A Glu for Val substitution at position 600 accounts for 90 % of B-Raf oncogenic mutations. The aliphatic sidechain of Val600 interacts with the phenyl ring of Phe467 in the P-loop. Replacement of aliphatic Val sidechain with larger charged residue such as Glu, Asp, Lys, or Arg would destabilize the interaction that maintains DFG motif in the inactive conformation. In addition to that, it is also possible that acidic amino acid at the position 600 could interact with Lys507 of the αC helix and therefore further stabilize the active state. A Glu for Lys substitution at position 601 may also stabilize the active conformation by interacting with Lys507 and/or Lys570 of the αC and αE helices, respectively. ^[11]

Intermediate activating mutations

Intermediate activating mutations destabilize DFG inactive conformation but the overall catalytic activity is reduced compared to strongly activating mutations due to the fact that substitutive amino acids are in this case not optimal for catalysis. P-loop residue Gly468 contacts Leu597 in the inactive conformation therefore its substitution for Glu destabilizes the inactive conformation. However, it is known that in all protein kinases only Gly, Ala or Ser residues occur at this position. Bulky sidechain of Glu interferes with P-loop-ATP interactions which leads to decreased potential to elevate kinase activity of this mutant. ^[12] Another intermediate activating mutation is a substitution of Asn580 for Ser. Asn580 is a crucial metal-binding residue, a hydrogen bond between Asn580 and Phe595 stabilizes the inactive conformation of the DFG motif. Ser residue at position 580 therefore disrupts the inactive conformation. ^[13]

In some cases the mutations in B-Raf decrease its activity but activate RAF-MEK-ERK pathway by activating C-Raf.^[14]

Known inhibitors

BAY43-9006

BAY43-9006(also known as Sorafenib) inhibits the catalytic activity of the kinase domain by stabilizing it in its inactive conformation. ^[15] It interacts with kinase domain of both V600E and wild-type B-Raf.^[16] In fact, BAY43-9006 efficiently inhibits activity of several protein kinases such as Raf, VEGFR and PDGFR.^[17] BAY43-9006 is approved for treatment of renal cell carcinoma^[18] and hepatocellular carcinoma ^[19]

Vemurafenib

Vemurafenib (V600E MUtated B-RAF INhiBition) was designed to selectively target V600E mutant of B-Raf^[20] and unlike sorafenib inhibits the active conformation of B-Raf by blocking the ATP-binding site. It was the first drug designed using fragment-based lead discovery to be approved.^[21] Vemurafenib does not inhibit the activity of B-Raf without V600E/K mutation. It is commonly used in treatment of melanoma.^[22]