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

## TL;DR

Researchers developed a new drug targeting a brain tumor enzyme, showing promise in treating a deadly childhood brain cancer using patient-derived organoids.

## Contribution

A new piperazine-based compound targeting hClpP is developed and validated for DIPG therapy using patient-derived organoids.

## Key findings

- Compound 26 (DA29) binds to hClpP's hydrophobic pocket and activates proteolysis.
- 26 (DA29) crosses cellular barriers and induces mitochondrial dysfunction in DIPG cells.
- The compound does not interact with dopamine receptors unlike ONC201.

## Abstract

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 (hClpP). X-ray analysis of the hClpP: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 hClpP: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 hClpP, displaces hClpX from the hClpXP 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. The compound does not interact with D2/D3 receptors as ONC201 does, with validation extended to patient-derived DIPG organoids for personalized medicine. Overall, these findings establish a new structural framework for hClpP-targeted therapy, representing a valuable step toward developing effective treatments for DIPG.