BRAF is the most frequently mutated kinase in human cancers and is one of the major effectors of oncogenic RAS, making BRAF a target of considerable interest for anti-cancer drug development. Wild-type BRAF and a variety of oncogenic BRAF mutants are dependent on dimerization of the kinase domain, which also emerges as a culprit of drug resistance and side effects of current BRAF therapies. Thus, allosteric BRAF inhibitors capable of disrupting BRAF dimers could abrogate hyperactivated MAPK (mitogen-activated protein kinase) signaling driven by oncogenic BRAF or RAS and overcome the major limitations of current BRAF inhibitors. To establish this, we applied an in silico approach to design a series of peptide inhibitors targeting the dimer interface of BRAF. One resulting inhibitor was found to potently inhibit the kinase activity of BRAF homo- and heterodimers, including oncogenic BRAFG469A mutant. Moreover, this inhibitor synergizes with FDA-approved, ATP-competitive BRAF inhibitors against dimeric BRAF, suggesting that allosteric BRAF inhibitors have great potential to extend the application of current BRAF therapies. Additionally, targeting the dimer interface of BRAF kinase leads to protein degradation of both RAF and MEK, uncovering a novel scaffolding function of RAF in protecting large MAPK complexes from protein degradation. In conclusion, we have developed a potent lead peptide inhibitor for targeting the dimer interface of BRAF in cancer cells. The dual function of this peptide inhibitor validates the strategy for developing allosteric BRAF inhibitors that specifically dissociate RAF dimers and destabilize the MAPK signaling complex.

Class 3 mutations in B-Raf proto-oncogene, Ser/Thr kinase (BRAF), that result in kinase-impaired or kinase-dead BRAF have the highest mutation frequency in BRAF gene in lung adenocarcinoma. Several studies have reported that kinase-dead BRAF variants amplify mitogen-activated protein kinase (MAPK) signaling by dimerizing with and activating WT C-Raf proto-oncogene, Ser/Thr kinase (CRAF). However, the structural and functional principles underlying their activation remain elusive. Herein, using cell biology and various biochemical approaches, we established that variant BRAFD594G, a kinase-dead representative of class 3 mutation-derived BRAF variants, has a higher dimerization potential as compared with WT BRAF. Molecular dynamics simulations uncovered that the D594G substitution orients the αC-helix toward the IN position and extends the activation loop within the kinase domain, shifting the equilibrium toward the active, dimeric conformation, thus priming BRAFD594G as an effective allosteric activator of CRAF. We found that B/CRAF heterodimers are the most thermodynamically stable RAF dimers, suggesting that RAF heterodimers, and not homodimers, are the major players in determining the amplitude of MAPK signaling in cells. Additionally, we show that BRAFD594G:CRAF heterodimers bypass autoinhibitory P-loop phosphorylation, which might contribute to longer duration of MAPK pathway signaling in cancer cells. Last, we propose that the dimer interface of the BRAFD594G:CRAF heterodimer may represent a promising target in the design of novel anticancer therapeutics.

The most prevalent BRAF mutation, V600E, occurs frequently in melanoma and other cancers. Although extensive progress has been made toward understanding the biology of RAF kinases, little in vitro characterization of full‐length BRAFV600E is available. Herein, we show the successful purification of active, full‐length BRAFV600E from mammalian cells for in vitro experiments. Our biochemical characterization of intact BRAFV600E together with molecular dynamics (MD) simulations of the BRAF kinase domain and cell‐based assays demonstrate that BRAFV600E has several unique features that contribute to its tumorigenesis. Firstly, steady‐state kinetic analyses reveal that purified BRAFV600E is more active than fully activated wild‐type BRAF; this is consistent with the notion that elevated signaling output is necessary for transformation. Secondly, BRAFV600E has a higher potential to form oligomers, despite the fact that the V600E substitution confers constitutive kinase activation independent of an intact side‐to‐side dimer interface. Thirdly, BRAFV600E bypasses inhibitory P‐loop phosphorylation to enforce the necessary elevated signaling output for tumorigenesis. Together, these results provide new insight into the biochemical properties of BRAFV600E, complementing the understanding of BRAF regulation under normal and disease conditions.

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In normal cells, BRAF is tightly regulated by upstream stimulus and negative feedback regulation loops such as inhibitory P‐loop phosphorylation; however, the mutant BRAFV600E has increased activity/oligomerization and bypasses inhibitory P‐loop phosphorylation to support the growth of cancer cells. Firstly, purified full‐length BRAFV600E has higher kinase activity than fully active wild‐type BRAF in vitro. Secondly, BRAFV600E has a higher potential to form oligomers despite the fact it has increased kinase activity independent from the side‐to‐side dimer interface. Lastly, BRAFV600E evades inhibitory P‐loop phosphorylation to impose increased activity required for tumorigenesis. More information can be found in the full paper by Z. Wang et al. on page 2850 in Issue 22, 2019 (DOI: 10.1002/cbic.201900266).

Luminescence is the more suitable assay for assay of plant natural product extracts. The sensitivities of the luminescence and fluorescence assays are comparable. A Z'-factor of 0.58 for the luminescence assay makes it suitable for medium-to-high throughput screening efforts.

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