A Fluorescence-EnhancedProbe Enables Rapid Detectionof BRAF(V600E) Mutant Activity in Living Cells and Tumor Tissues

Abstract BRAF is the most frequently mutated protein kinase in human cancers, and in particular, the V600E mutation in the kinase domain confers a constitutively active conformation on the protein, thereby promoting oncogenesis. To date, several BRAF kinase inhibitors have been approved for treating cancer patients with the BRAF(V600E) mutation. However, the benefits of BRAF kinase inhibitors in clinical practice are limited, with an objective response rate of less than ∼50%. Therefore, novel diagnostic tools enabling the visualization of BRAF-mutant activity in an activity-based manner are quite desirable to determine BRAF-mutant status and guide treatment decisions for BRAF inhibitors. Here, we describe the design, synthesis, and application of a new fluorescence-enhanced probe HX50 by introducing an environment-sensitive fluorophore to the pharmacophore of BRAF inhibitors to visualize BRAF kinase activity in living cancer cells and tumor tissues (mouse models and human tumor biopsy samples). Interestingly, we found that the probe enables distinguishing the BRAF(V600E) mutation from the wild type not only in living cancer cells but also in xenograft mouse models. Furthermore, the fluorescence-enhanced probe was also able to visualize BRAF activity with high signal contrast in human tumors from lung cancer patients carrying the BRAF(V600E) mutation, but not BRAF wild-type. These results demonstrate that the strategically designed drug-based fluorescence-enhanced probe enables assessment of BRAF-mutant activity in tumor samples and holds promise as a useful diagnostic tool for predicting drug sensitivity and informing therapeutic strategy decisions for BRAF inhibitors.