Challenges and Opportunities in the Crusade of BRAF Inhibitors: From 2002 to 2022

Abstract

Serine/threonine-protein kinase B-Raf (BRAF; RAF = rapidly accelerated fibrosarcoma) plays an important role in the mitogen-activated protein kinase (MAPK) signaling cascade. Somatic mutations in the BRAF gene were first discovered in 2002 by Davies et al., which was a major breakthrough in cancer research. Subsequently, three different classes of BRAF mutants have been discovered. This class includes class I monomeric mutants (BRAF^V600), class II BRAF homodimer mutants (non-V600), and class III BRAF heterodimers (non-V600). Cancers caused by these include melanoma, thyroid cancer, ovarian cancer, colorectal cancer, nonsmall cell lung cancer, and others. In this study, we have highlighted the major binding pockets in BRAF protein, their active and inactive conformations with inhibitors, and BRAF dimerization and its importance in paradoxical activation and BRAF mutation. We have discussed the first-, second-, and third-generation drugs approved by the Food and Drug Administration and drugs under clinical trials with all four different binding approaches with DFG-IN/OUT and αC-IN/OUT for BRAF protein. We have investigated particular aspects and difficulties with all three generations of inhibitors. Finally, this study has also covered recent developments in synthetic BRAF inhibitors (from their discovery in 2002 to 2022), their unique properties, and importance in inhibiting BRAF mutants.

Figure 1

BRAF signaling pathway. (green arrow) Normal BRAF pathway. (red arrow) Oncogenic BRAF pathway.

Figure 2

Global cancer statistics for new cases and deaths for 2020 and BRAF^V600E (%) mutation in different cancer types.

Figure 3

BRAF protein structure and binding pocket. (a) Regulatory (CR1 & CR2) and kinase (CR3) domain between N and C-terminal in BRAF kinase protein. (b) BRAF protomer (PDB ID:6P7G) with different orientations. (c) BRAF dimer (PDB ID:2FB8). Different binding pockets (p-loop, αC-helix, DIF, DFG, catalytic loop, activation segment, and hinge region) are labeled in specific colors.

Figure 4

Dimerization of Raf in cell signaling. Note: (A) In the normal RAS-dependent signaling pathway, Raf dimerization is required for Raf kinase activation and signaling to MEK for further cell proliferation and regulation. (B) In oncogenic states, it is required for MEK/ERK signaling, which is upregulated by Raf dimerization. These include: (1) mutant c-Raf protein with b-Raf in dimerization impaired kinase activity from normal to oncogenic, (2) RTKs and RasGTPases induced by mutation, (3) in the context of active Ras, treatment with ATP-competitive Raf inhibitors, and (4) Raf inhibitor resistance is mediated by self-homodimerizing BRAF^V600E splice variants.

Figure 5

On the basis of DFG (purple color) and αC (cyan color) movement, four different binding conformations are shown. (a) PDB ID: 4MNF (GDC-0879); type I inhibitors (αC-IN/DFG-IN) (b) PDB ID: 5HI2 (Sorafenib); type II inhibitors (αC-IN/DFG-OUT) (c) PDB ID: 4RZV (Vemurafenib); type I_1/2 or type III (αC-OUT/DFG-IN) (d) PDB ID: 5CSX (BI 882370); type_I/II or type IV (αC-OUT/DFG-OUT).

Figure 6

First-generation BRAF inhibitors.

Figure 7

Second-generation BRAF inhibitors.

Figure 8

Third-generation BRAF inhibitors.

Figure 9

Paradoxical activation. Note: Mechanism of autoinhibition: (1) In this case, inhibition of BRAF in the presence of a mutant or growth factor-activated RAS leads to abrogation of BRAF autoinhibition, so that it homodimerizes with BRAF and becomes hyperactivated. Conformational changes: (2, 3) At low doses, the drug binds only one RAF protomer and causes the other to transactivate. (4) At high doses, it binds to and inhibits both RAF dimers, effectively knocking down the signaling complex.

Figure 10

Various synthesized compounds with parents’ scaffolds as BRAF mutant inhibitors. Note: (*) Cpd No.: Compound number; (**) Parent scaffold: Different derivatives of the parent scaffold were synthesized, but only the most potent compounds based on cell line or enzyme kinase activity were chosen.

Figure 10

Various synthesized compounds with parents’ scaffolds as BRAF mutant inhibitors. Note: (*) Cpd No.: Compound number; (**) Parent scaffold: Different derivatives of the parent scaffold were synthesized, but only the most potent compounds based on cell line or enzyme kinase activity were chosen.

Figure 10

Various synthesized compounds with parents’ scaffolds as BRAF mutant inhibitors. Note: (*) Cpd No.: Compound number; (**) Parent scaffold: Different derivatives of the parent scaffold were synthesized, but only the most potent compounds based on cell line or enzyme kinase activity were chosen.

Figure 10

Various synthesized compounds with parents’ scaffolds as BRAF mutant inhibitors. Note: (*) Cpd No.: Compound number; (**) Parent scaffold: Different derivatives of the parent scaffold were synthesized, but only the most potent compounds based on cell line or enzyme kinase activity were chosen.

Figure 10

Various synthesized compounds with parents’ scaffolds as BRAF mutant inhibitors. Note: (*) Cpd No.: Compound number; (**) Parent scaffold: Different derivatives of the parent scaffold were synthesized, but only the most potent compounds based on cell line or enzyme kinase activity were chosen.

Figure 10

Various synthesized compounds with parents’ scaffolds as BRAF mutant inhibitors. Note: (*) Cpd No.: Compound number; (**) Parent scaffold: Different derivatives of the parent scaffold were synthesized, but only the most potent compounds based on cell line or enzyme kinase activity were chosen.