Discovery of FRAG-i: a small molecule inhibitor of Drp1 that modulates mitochondrial fission

Mitochondrial dynamics, regulated by the balance of fission and fusion, are essential for cellular homeostasis, and their disruption—particularly via excessive Drp1-dependent fission—contributes to cancer and other pathologies. Current Drp1 inhibitors lack specificity or exhibit off-target toxicity, highlighting the need for safer alternatives. Here, we identify FRAG-i, a small-molecule Drp1 inhibitor discovered through multi-stage virtual screening and molecular dynamics refinement of a 3.5-million-compound library. FRAG-i binds the Drp1 GTPase domain with high affinity (Kd = 732.4 ± 4.2 nM) and increases Drp1 thermal stability in recombinant and cellular assays. Functionally, FRAG-i selectively suppresses mitochondrial fission in A549 lung carcinoma cells while sparing the fused networks of non-cancerous BEAS-2B epithelial cells. FRAG-i preserves mitochondrial membrane potential, ATP levels, and redox balance without cytotoxicity, and modulates Drp1, MiD49, and Mfn2 expression in a context-dependent manner. These results establish FRAG-i as a selective, non-toxic Drp1 inhibitor with potential for further therapeutic development.