Chemical inhibition of mitochondrial fission via targeting the DRP1-receptor interaction

Mitochondrial fission is critical for mitochondrial dynamics and homeostasis. The dynamin superfamily GTPase DRP1 is recruited by three functionally redundant receptors, MFF, MiD49, and MiD51, to mitochondria to drive fission. Here, we exploit high-content live-cell imaging to screen for mitochondrial fission inhibitors and have developed a covalent compound, mitochondrial division inhibitor (MIDI). MIDI treatment potently blocks mitochondrial fragmentation induced by mitochondrial toxins and restores mitochondrial morphology in fusion-defective cells carrying pathogenic mitofusin and OPA1 mutations. Mechanistically, MIDI does not affect DRP1 tetramerization nor DRP1 GTPase activity but does block DRP1 recruitment to mitochondria. Subsequent biochemical and cellular characterizations reveal an unexpected mechanism that MIDI targets DRP1 interaction with multiple receptors via covalent interaction with DRP1-C367. Taken together, beyond developing a potent mitochondrial fission inhibitor that profoundly impacts mitochondrial morphogenesis, our study establishes proof of concept for developing protein-protein interaction inhibitors targeting DRP1.