Date of Award

Spring 2021

Document Type

Open Access Dissertation


Pharmacology, Physiology and Neuroscience

First Advisor

Campbell McInnes


In cases of metastatic melanoma, BRAF is frequently mutated to the V600E oncoprotein causing uncontrolled cell proliferation driven by the MAPK-ERK pathway. There are several BRAF inhibitors, such as vemurafenib, which are FDA approved, but patients treated with these Type-I kinase inhibitors frequently observe relapse under mutant RAS and BRAF-wt conditions due to paradoxical activation. The mechanism of this resistance occurs through binding of the inhibitor to BRAF-wt initiating conformational changes which leads to BRAF dimerization. Once in the dimerized state, the inhibited monomer induces allosteric transactivation of the second monomer. This drug-induced activation of BRAF in cells with mutant RAS leads to uncontrolled cellular proliferation. In the context of mutant RAS/BRAF-wt cells treated with Type-I inhibitors, the MAPK/ERK pathway continually signals for initiation of cell proliferation, leading to mutant RAS-driven tumorigenesis. Currently there are no FDA approved treatments on the market for inhibiting RAS-driven tumorigenesis directly due to RAS family members having picomolar affinity for GDP/GTP. Recently there has been some progress in clinical trials of AMG510 (sotorasib), which binds outside of the catalytic GDP/GTP binding site. In a small cohort of 13 patients with KRAS-G12C-driven tumors, 7 patients observed partial responses to the target dose and 6 had stable disease.1,64 Though this clinical trial is exciting there is still a need for therapies targeted toward preventing paradoxical activation in melanoma patients and for alternative therapies for patients suffering from mutant RAS-driven tumorigenesis. Herein we discuss the linear design of potent Type-IV BRAF inhibitors which have been seen to inhibit paradoxical activation of mutant RAS/BRAF-wt driven tumorigenesis.

Initially, the linear native sequence of peptides from the BRAF dimer interface (DIF) and variations of this were tested for direct binding using an intrinsic tryptophan fluorescence assay. Contributions of residue sidechains was further assessed through an alanine-scan of the truncated, linear sequence. Linear data combined with the crystal structure (PDB 4E26) contributed to the design of a 6-residue macrocyclic peptide which possessed enhanced binding. These alterations enhanced binding interactions giving a peptide with Kd=0.06µM compared to the native sequence with Kd=3.84 µM.

Cyclic peptides were then optimized to include physiochemical properties which agree with the beyond the rule of 5 guidelines for passive cell permeability of macrocycles larger than 500 Da. Further modifications consisted of REPLACEment of exocyclic sequences with more drug-like analogs which are uncharged and lipophilic in nature. Additional derivatization included N-methylation of the peptide backbone. The macrocyclic peptidomimetics described herein represent potential next generation BRAF therapeutics which have potent binding and have anti-tumor activity under paradoxical activation conditions.