Elucidating the Molecular Impact of DA29‐Induced ClpP Chemoactivation on Mitochondrial Function

Cellular function relies on tightly regulated mitochondrial proteolysis, which controls organelle protein quality and specific mitochondrial functions, including gene expression and oxidative phosphorylation. In human mitochondria, one of the main proteolytic machineries is the soluble ClpXP complex, consisting of the protease ClpP and its chaperone ClpX, which degrades misfolded or aggregated proteins to preserve organelle function and integrity. As hyperactivation of ClpP has been shown to inhibit tumor cell growth in various types of cancer, this protease has become an important target for the development of small-molecule therapeutics. A piperazine-based compound, DA29, has proven effective in inducing cytotoxicity in diffuse intrinsic pontine glioma (DIPG) patient-derived cells and organoids, causing mitochondrial dysfunction and ROS accumulation. In this study, we employed in cellulo systems to conduct molecular and mechanistic analyses of ClpP hyperactivation mediated by DA29. Our data show that DA29 impairs mitochondrial function by specifically activating ClpP. Interestingly, degradation of ClpP itself is promoted when its proteolytic activity exceeds a certain threshold or is sustained for prolonged periods, suggesting a complex regulatory network that tunes protease levels within mitochondria.