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Dysregulation of Human ClpP Using Small Molecules with Piperazine-Based Scaffold for Diffuse Intrinsic Pontine Glioma
Morena Miciaccia1, Domenico Armenise1, Olga Maria Baldelli1
1Research Laboratory for Woman and Child Health, Department of Pharmacy - Pharmaceutical Sciences, University of Bari "Aldo Moro", 70125Bari, Italy.
Abstract:
Diffuse intrinsic pontine glioma (DIPG) is a dismal pediatric brainstem tumor with a median survival of approximately 12 months and no curative treatments. ONC201, the most clinically characterized small-molecule, selectively activates human caseinolytic protease P (hClpP). Guided by X-ray structure of the hClpP:ONC201 complex, piperazine-based derivatives were designed and identified compound 26 (DA29) as a lead. Structural studies confirmed its binding to the enzyme hydrophobic allosteric pocket, a finding consistent with its strong proteolytic activation. Permeability under dynamic flow conditions suggested the ability of 26 (DA29) to cross cellular barriers, indicating potential brain tumor penetration. Mechanistically, 26 (DA29) activates hClpP, displaces hClpX from the hClpXP complex, induces mitochondrial dysfunction and ROS accumulation, and exerts cytotoxic effects in patient-derived DIPG cells and organoids. Notably, unlike ONC201, it does not interact with D2/D3 dopamine receptors. Overall, these findings establish a new structural framework for hClpP-targeted therapies and support further development of 26 (DA29) for DIPG treatment.
Insights
A new drug candidate, compound 26 (DA29), shows promise for treating diffuse intrinsic pontine glioma (DIPG) by activating human caseinolytic protease P (hClpP) and exhibiting cytotoxicity in DIPG cells.
Area of Science:
- Oncology
- Molecular Biology
- Drug Discovery
Background:
- Diffuse intrinsic pontine glioma (DIPG) is a pediatric brainstem tumor with a poor prognosis and limited treatment options.
- ONC201, a small molecule, activates human caseinolytic protease P (hClpP), showing potential therapeutic effects.
Purpose of the Study:
- To design and identify novel hClpP-activating compounds for DIPG treatment.
- To investigate the mechanism of action and therapeutic potential of compound 26 (DA29).
Main Methods:
- Structure-guided design of piperazine-based derivatives based on the hClpP:ONC201 complex.
- Biochemical assays to confirm binding and proteolytic activation of hClpP.
- Cell permeability studies, mechanistic investigations in DIPG cells and organoids.
- Assessment of off-target interactions with dopamine receptors.
Main Results:
- Compound 26 (DA29) was identified as a lead compound, binding to the hClpP hydrophobic allosteric pocket.
- DA29 demonstrated strong proteolytic activation of hClpP, potential for brain tumor penetration, and induced mitochondrial dysfunction and ROS accumulation.
- DA29 exhibited cytotoxic effects in patient-derived DIPG cells and organoids, without interacting with D2/D3 dopamine receptors.
Conclusions:
- Compound 26 (DA29) represents a novel hClpP-targeted therapeutic agent for DIPG.
- These findings provide a new structural framework for hClpP-targeted therapies and support further clinical development of DA29.
