Reverse engineering of BNIP3 identifies a mitochondrial protective peptide

Abstract Recent advances in mitochondrial network dynamic and signalling highlight mitochondria as key therapeutic targets across diverse diseases. Yet, high drug development failure rates reflect an incomplete understanding of upstream molecular regulators of mitochondrial fate. Here, we address this gap by reverse engineering of the BH3-only protein BNIP3. Structural modelling and sequence–function analyses of its N-terminus identify a critical functional domain and amino acid hotspots that directly activate BCL-2 executioner proteins, triggering mitochondrial cell death. Leveraging these insights, we develop a BNIP3 antagonist peptide (B-017) that disrupts interactions between BNIP3 and BCL-2 executioner proteins, preserving mitochondrial integrity. B-017 demonstrates target specificity, a favourable safety profile, and robust suppression of cell death signalling in human cells. In clinically relevant animal models, it reduces tissue damage in the heart, brain, and liver. Together, these findings position B-017 as a promising therapeutic candidate targeting mitochondrial dysfunction.