Mitigating Excessive Mitochondrial Fission Through the Development of a Selective Macrocyclic Inhibitor Targeting Fis1/Mid51 Signaling

Mitochondrial fission protein 1 (Fis1) and mitochondrial dynamics protein of 51 kDa (Mid51) regulate stress-induced mitochondrial fragmentation implicated in cardiovascular disease. Using homologous sequence analysis and structure-guided design, we identified a linear peptide inhibitor (CVP-240) targeting the Fis1/Mid51 protein–protein interaction (PPI) and optimized it into a macrocyclic derivative (CVP-764). Both compounds bind Mid51 with high affinity, selectively disrupt Fis1/Mid51 signaling over Drp1-dependent interactions, and exhibit nanomolar binding in fluorescence polarization assays using FAM-conjugated tracers. In H9c2 cardiomyocytes, CVP-240 and CVP-764 preserve mitochondrial membrane potential, reduce reactive oxygen species, maintain mitochondrial network integrity, and improve cell viability under stress. Macrocyclization enhances proteolytic and serum stability and confers intrinsic cell permeability without the need for a cell-penetrating sequence. In silico ADMET profiling and preliminary in vivo toxicity studies support a favorable safety profile, establishing CVP-764 as a promising lead for targeting pathological mitochondrial fission.