Novel hClpP protease activators with potential anticancer activity in DIPG patients

Human (h)ClpXP is a soluble mitochondrial complex, formed by the protease ClpP and the chaperone ClpX, which degrades misfolded or aggregated proteins to maintain organelle function and integrity. ClpP is targeted by ONC201, the first compound to show promise in patients with Diffuse Intrinsic Pontine Glioma (DIPG), through hyperactivation of the protease and disruption of mitochondrial metabolism with cytotoxic effects. Despite initial clinical responses, ONC201 showed limited efficacy and resistance; however, its mechanism of action revealed that ClpP hyperactivation plays a pivotal role in mitochondrial quality control, refocusing research to develop more potent and selective ClpP activators as a novel therapeutic strategy. Aiming to uncover the structural determinants for potent hClpP activation, we generated novel scaffolds designed to engage the allosteric site of the protease and to enhance its catalytic activity. Among the tested compounds, we identified two novel molecules, THX6 and DA29, which show sub-micromolar hClpP activation and cytotoxicity in cell lines exhibiting different degrees of sensitivity to ONC201. We investigated the ability of both compounds to bind hClpP and to dysregulate key mitochondrial processes, such as oxidative phosphorylation, organelle biogenesis and mitophagy, in cultured DIPG cells. Our findings support the conclusion that mitochondrial dysfunction could contribute to the cellular toxicity triggered by either THX6 or DA29, which therefore stand as promising candidates to tackle this rare aggressive paediatric glioma.