PROJECT SUMMARY The activating V600E mutation in the protein kinase BRAF is a well-established driver of human cancer. The effectiveness of current BRAF inhibitors developed to target V600E BRAF is limited by pharmacological resistance that unavoidably develops only months after initial treatment. It has been discovered that these inhibitors effectively suppress the activity of V600E monomers, but do not inhibit V600E BRAF dimers, and instead elevate their activity through a phenomenon called paradoxical activation (PA). Thus, molecular lesions promoting the formation of V600E BRAF dimers are primary mechanisms leading to resistance, and highlights the inability of current drugs to inhibit V600E BRAF dimers as a severe clinical problem. The exact structural mechanisms that cause PA and make V600E BRAF dimers impervious to inhibition, however, are not well understood. The goal of this proposal is to dissect the natural allosteric mechanisms of regulation of the V600E BRAF dimer, and determine how inhibitors modulate these structural networks in order to understand the molecular basis of PA. The central hypothesis is that The V600E BRAF dimer is intrinsically asymmetric due to allosteric coupling across the dimer interface, and inhibitors affect this coupling to drive the formation of catalytically active dimers that are only occupied by one inhibitor molecule. The rationale for this work is that understanding the relationship between BRAF dimer allostery and inhibitor-induced allostery can inform future drug development efforts in creating inhibitors that can tune these structural effects to completely inhibit the V600E BRAF dimer. The central hypothesis will be tested through two specific aims: 1.) Quantify BRAF dimerization affinity in the presence of RAF inhibitors to establish the thermodynamic factors of PA, and 2.) Measure the allosteric mechanisms of BRAF dimerization and determine their response to inhibitor binding. These aims will be achieved through the use of a Förster resonance energy transfer (FRET) assay that can quantify BRAF dimerization in the presence of inhibitors, and double electron-electron resonance (DEER) to directly measure conformational rearrangements of the BRAF kinase domain in response to dimerization and inhibitor binding. The expected results from this proposal will further the understanding of the structural mechanisms of BRAF-inhibitor interactions and how they lead to PA. This knowledge will thus lay the groundwork for the rational design of next-generation inhibitors and improved therapeutic strategies to effectively inhibit the V600E BRAF dimer and overcome resistance.