A new fluorescent sensor mitoferrofluor indicates the presence of chelatable iron in polarized and depolarized mitochondria.

Mitochondrial chelatable iron contributes to the severity of several injury processes, including ischemia/reperfusion, oxidative stress, and drug toxicity. However, methods to measure this species in living cells are lacking. To measure mitochondrial chelatable iron in living cells, here we synthesized a new fluorescent indicator, mitoferrofluor (MFF). We designed cationic MFF to accumulate electrophoretically in polarized mitochondria, where a reactive group then forms covalent adducts with mitochondrial proteins to retain MFF even after subsequent depolarization. We also show in cell-free medium that Fe2+ (and Cu2+), but not Fe3+, Ca2+, or other biologically relevant divalent cations, strongly quenched MFF fluorescence. Using confocal microscopy, we demonstrate in hepatocytes that red MFF fluorescence colocalized with the green fluorescence of the mitochondrial membrane potential (ΔΨm) indicator, rhodamine 123 (Rh123), indicating selective accumulation into the mitochondria. Unlike Rh123, mitochondria retained MFF after ΔΨm collapse. Furthermore, intracellular delivery of iron with membrane-permeant Fe3+/8-hydroxyquinoline (FeHQ) quenched MFF fluorescence by ∼80% in hepatocytes and other cell lines, which was substantially restored by the membrane-permeant transition metal chelator pyridoxal isonicotinoyl hydrazone. We also show FeHQ quenched the fluorescence of cytosolically coloaded calcein, another Fe2+ indicator, confirming that Fe3+ in FeHQ undergoes intracellular reduction to Fe2+. Finally, MFF fluorescence did not change after addition of the calcium mobilizer thapsigargin, which shows MFF is insensitive to physiologically relevant increases of mitochondrial Ca2+. In conclusion, the new sensor reagent MFF fluorescence is an indicator of mitochondrial chelatable Fe2+ in normal hepatocytes with polarized mitochondria as well as in cells undergoing loss of ΔΨm.

Design and Synthesis of Type-IV Inhibitors of BRAF Kinase That Block Dimerization and Overcome Paradoxical MEK/ERK Activation.

Despite the clinical success of BRAF inhibitors like vemurafenib in treating metastatic melanoma, resistance has emerged through "paradoxical MEK/ERK signaling" where transactivation of one protomer occurs as a result of drug inhibition of the other partner in the activated dimer. The importance of the dimerization interface in the signaling potential of wild-type BRAF in cells expressing oncogenic Ras has recently been demonstrated and proposed as a site of therapeutic intervention in targeting cancers resistant to adenosine triphosphate competitive drugs. The proof of concept for a structure-guided approach targeting the dimerization interface is described through the design and synthesis of macrocyclic peptides that bind with high affinity to BRAF and that block paradoxical signaling in malignant melanoma cells occurring through this drug target. The lead compounds identified are type-IV kinase inhibitors and represent an ideal framework for conversion into next-generation BRAF inhibitors through macrocyclic drug discovery.