The mitogen-activated protein kinase (MAPK) pathway modulates cell proliferation through regulation of the communication between extracellular signaling and intranuclear transcription. BRAF, a Ser/Thr kinase in the MAPK pathway, is frequently mutated in cases of metastatic melanoma, most frequent of which is the phosphorylation mimicking V600E mutation. The FDA approved ATP-competitive inhibitor, vemurafenib, effectively inhibits signal transduction in the context of BRAF(V600E), but upon binding to BRAF(wt), paradoxical activation of the second RAF monomer within the dimer is induced, and drug resistance develops in the form of mutant RAS-driven tumorigenesis. The Brummer group rationalized that paradoxical activation identified the key residues for dimer formation and therefore peptides were designed to encompass this interface. The BRAF (wt) dimerization inhibiting linear has been tested and shown to inhibit downstream phosphorylation events in a dose-dependent manner in the presence of vemurafenib. The initial peptide exhibited a Kd = 3.84 µM in a direct binding assay and truncation of this sequence led to a 30-fold increase in potency, potentially due to electrostatic stabilization of a pseudo-cyclic conformation. An alanine scan was then completed to identify key binding residues and included those critical for dimerization in the full length protein context. Peptides were cyclized to decrease the entropic cost of binding by covalently stabilizing their bioactive β-turn conformation. Cyclic peptides exhibit sub-micromolar binding affinities and the ring size is currently being optimized to promote potent binding interactions. Lead molecules have been shown to inhibit paradoxical signaling in metastatic melanoma cell lines. The ultimate goal is to systematically replace segments of the optimized cyclic peptide and to generate a macrocyclic BRAF(wt) dimerization inhibitor which is cell permeable and proteolytically stable. This approach of inhibiting BRAF(wt) dimerization is a novel method of avoiding paradoxical activation of BRAF during treatment with vemurafenib in metastatic melanoma patients, while inhibiting mutant RAS-driven tumorigenesis.

Discovery and optimization of macrocyclic peptide dimerization inhibitors of BRAFwt

256th National Meeting (2018)

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