Mitofusins reside on the outer mitochondrial membrane and regulate mitochondrial fusion, a physiological process that impacts diverse cellular processes. Mitofusins are activated by conformational changes and subsequently oligomerize to enable mitochondrial fusion. Here, we identify small molecules that directly increase or inhibit mitofusins activity by modulating mitofusin conformations and oligomerization. We use these small molecules to better understand the role of mitofusins activity in mitochondrial fusion, function, and signaling. We find that mitofusin activation increases, whereas mitofusin inhibition decreases mitochondrial fusion and functionality. Remarkably, mitofusin inhibition also induces minority mitochondrial outer membrane permeabilization followed by sub-lethal caspase-3/7 activation, which in turn induces DNA damage and upregulates DNA damage response genes. In this context, apoptotic death induced by a second mitochondria-derived activator of caspases (SMAC) mimetic is potentiated by mitofusin inhibition. These data provide mechanistic insights into the function and regulation of mitofusins as well as small molecules to pharmacologically target mitofusins.

Unique to targeting the C-terminal domain of Hsp90 (C-Hsp90) is the ability to uncouple the cytotoxic and cytoprotective outcomes of Hsp90 modulation. After the identification of novobiocin as a C-Hsp90 interacting ligand a diverse gamut of novologues emerged, from which KU-32 and KU-596 exhibited strong neuroprotective activity. However, further development of these ligands is hampered by the difficulty to obtain structural information on their complexes with Hsp90. Using saturation transfer difference (STD) NMR spectroscopy, we found that the primary binding epitopes of KU-32 and KU596 map at the ring systems of the ligands and specifically the coumarin and biphenyl structures, respectively. Based on both relative and absolute STD effects, we identified KU-596 sites that can be explored to design novel third-generation novologues. In addition, chemical shift perturbations obtained by methyl-TROSY reveal that novologues bind at the cryptic, C-Hsp90 ATP-binding pocket and produce global, long-range structural rearrangements to dimeric Hsp90.