Vimentin cytoskeleton collapses in response to the small-molecule inhibitor of FH2 domains (SMIFH2)-induced electrophilic stress

The type III intermediate filament protein vimentin plays an integral role in cell homeostasis and disease progression during fibrosis and cancer invasion. Previous work demonstrated that the pan-formin inhibitor small-molecule inhibitor of formin homology 2 domains (SMIFH2) induced a perinuclear collapse of the vimentin network, suggesting formins may regulate vimentin cytoskeleton organization. However, despite the designed function of SMIFH2 to inhibit formin homology 2 (FH2) domain-actin interactions, several major off-target effects of SMIFH2 have been reported, including inhibition of myosin family ATPase activity. SMIFH2 is also highly electrophilic, potentially reacting with nucleophilic residues within proteins other than formins. Therefore, we sought to determine the mechanism by which SMIFH2 disrupts the vimentin cytoskeleton. Depletion of specific formin proteins, targeted actin cytoskeleton disruption, or myosin family ATPase inhibition failed to replicate the SMIFH2 effect on the vimentin network. However, treatment with other electrophilic reagents, including prostaglandin A, reproduced the SMIFH2-mediated vimentin collapse, F-actin cytoskeletal changes, and activation of the NF-E2-related factor 2 stress sensory pathway. Additionally, fluorescence recovery after photobleaching analysis revealed that SMIFH2 inhibits vimentin filament dynamics, which was rescued by mutating the nucleophilic vimentin C328 residue. Thus, SMIFH2 disrupts the vimentin network due to its reactivity as an electrophilic species. This study reinforces the role of vimentin as a key stress sensor.