Date Approved
9-21-2023
Embargo Period
9-26-2025
Document Type
Dissertation
Degree Name
Doctor of Philosophy
Department
Chemistry and Biochemistry
College
College of Science & Mathematics
Advisor
Zhihong Wang, PhD
Committee Member 1
Zhiwei Liu, PhD
Committee Member 2
Hao Zhu, PhD
Committee Member 3
Subash Jonnalagadda, PhD
Committee Member 4
John Tomsho, PhD
Keywords
Allosteric inhibitor, Braftide, CID-BRAF, Phosphorylation, RAF kinase
Subject(s)
Biochemistry--Research
Disciplines
Biochemistry, Biophysics, and Structural Biology | Chemistry
Abstract
RAF kinases (ARAF/BRAF/CRAF or Raf-1) are serine/threonine kinases in the MAPK (RAS-RAF-MEK-ERK) pathway. The MAPK pathway is crucial in development and normal cell function. These same roles are dysregulated in cancer; BRAF is the most frequently mutated isoform in cancers and in particular, activation loop mutants BRAF V600E/K mutants have the highest mutational incidence. Three FDA approved inhibitors potently inhibit BRAF V600E/K mutants. However, these same inhibitors fail to address the non-V600E/K mutations and result in paradoxical activation of the MAPK pathway. Two strategies were employed to address the limitations of the FDA-approved inhibitors: provide novel regulatory details of RAF kinase and identify the mechanism of action of Braftide, a lead peptide that circumvents paradoxical activation. Both strategies use a chemo-proteomic approach and validation in a physiologically relevant environment with cell-based assays. Using mutational analysis of an identified phosphorylation site of BRAF, we identify a negative regulatory switch of BRAF that decreases downstream MEK phosphorylation by dissociating the known 14-3-3 scaffolding protein interaction and RAF dimerization. Furthermore, crosslinking-mass spectrometry revealed the binding regions of Braftide that disrupt known regulatory mechanisms of BRAF and the CDC37, co-chaperone. Together, these reports resolve foundational mechanisms for next generation inhibitor development for MAPK pathway attenuation
Recommended Citation
Yu, Alison, "MECHANISTIC INVESTIGATIONS OF A NOVEL BRAF REGULATORY SWITCH AND BRAFTIDE, AN ALLOSTERIC PEPTIDE INHIBITOR OF BRAF" (2023). Theses and Dissertations. 3160.
https://rdw.rowan.edu/etd/3160
