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

Abstract

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.