Dysregulation of human ClpP using small molecules with piperazine-based scaffold for diffuse intrinsic pontine glioma therapy validated by patient-derived tumor organoids

Diffuse intrinsic pontine glioma (DIPG) is an aggressive brainstem tumor affecting children, with median survival under 12 months and no curative treatments. Current therapeutic development centers on ONC201, an imipridone that exhibits clinical activity by selectively activating human caseinolytic protease P ( h ClpP). X-ray analysis of the h ClpP:ONC201 complex offers insights into optimizing the chemical structure of ONC201. Based on this, we designed and synthesized simplified piperazine-based analogs, identifying the polyfunctional chemotype 26 (DA29) through the systematic screening of five new series featuring piperazine-, piperazine-1-one (exocyclic carbonyl), piperazine-1,4-dione (exocyclic carbonyls), piperazine-2-one (endocyclic carbonyl), and piperazine-2,5-dione (endocyclic carbonyls) core scaffolds. The X-ray structure of the h ClpP: 26 (DA29) complex demonstrated a direct binding to the enzyme’s hydrophobic pocket, present at the apical domain, consistent with its strong proteolytic activation. Permeability studies under dynamic flow conditions indicated that 26 (DA29) can cross cellular barriers modeling drug transport, suggesting potential brain tumor penetration. Mechanistic studies showed that 26 (DA29) activates h ClpP, displaces h ClpX from the h ClpXP complex, induces mitochondrial dysfunction and causes ROS accumulation, exerts cytotoxicity and alters lipid profile in patient-derived DIPG cells with validation extended to patient-derived DIPG organoids for personalized medicine. 26 (DA29) does not interact with D2/D3 dopamine receptors as ONC201 does and then is deprived of extrapyramidal collateral effects. Overall, these findings establish a new structural framework for h ClpP-targeted therapy, representing a valuable step towards the development of effective treatments for DIPG.