ID #398 Drug-candidates targeting mitochondrial protease HsClpP developed for paediatric diffuse midline glioma/diffuse intrinsic pontine glioma treatment

Abstract Background Human protease ClpP (HsClpP) with AAA+ unfoldase (ClpX) form ClpXP complex responsible of “mitochondrial quality control”. hClpP hyperactivation by small-molecule activators is a recent strategy in cancer therapy. Most known HsClpP activator is imipridone ONC201. ONC201 is FDA approved and is in worldwide phase III clinical trial to treat high grade gliomas, including paedriatic DIPG, which has no effective treatment. Materials and Methods ONC201 is effective in very few DIPG cases. Radiotherapy is the gold standard treatment. Stabilizes symptoms without changing the prognosis of patients with a median overall survival of approximately twelve months. A program to identify new drug candidates for DIPG patients is therefore essential. HsClpP dysregulation was achieved by developing two series (THX and DA) of small molecules with tetrahydropyridopyrimidindione- and piperazine-based scaffolds validated by patient-derived tumor organoids. Results THX and DA series were designed and produced by identifying a suitable synthetic methodology for their preparation. Among these two series of compounds, THX6 and DA29 were found to activate HsClpP with EC50 = 1.18 and 0.85 mM, respectively. ONC201 EC50 is 5.9 mM. THX6 and DA29 are more cytotoxic than ONC201 in DIPG patients-derived cell lines, included those resistant to ONC201. The two compounds altered the lipid profile in the same cell. In addition, THX6 and DA29 inhibit the expression of mitochondrial-related proteins (such as parkin, TFAM, NRF1, SDHA), leading to impaired mitochondrial function. Conclusion The identified THX6 and DA29 have a chemical structure different from ONC201, are HsClpP activators, and are highly cytotoxic in DIPG cell lines, capable to cross the blood-brain barrier, meaning they would penetrate the brain and reach the tumor area. 1. Perrone MG, Ruggiero A, Centonze A, Carrieri A, Ferorelli F, Scilimati A. Diffuse Intrinsic Pontine Glioma (DIPG): breakthrough and clinical perspective. Curr. Med. Chem. 2021, 28:17, 3287-3317. 2. Armenise D, Baldelli OM, Liturri A, Cavallaro G, Fortuna CG, Ferorelli S, Miciaccia M, Perrone MG, Scilimati A. Mitochondrial serine protease ClpP: Cancer Marker and Drug Target. Pharmaceuticals 2025, 18, 1443 (1-29). 3. Maitra A, Mandorino, Armenise D, Baldelli OM, Miciaccia M, Ferorelli S, Papusha L, Druy A, Perrone MG, Scilimati A. Decoding Gene Expression Changes in Pediatric Cerebral Tumors: Before and After Radiotherapy. MedResRev 2025, 1-11. 4. Miciaccia M, Rizzo F, Centonze A, Cavallaro G, Contino M, Armenise D, Baldelli OM, Solidoro R, Ferorelli S, Scarcia P, Agrimi G, Zingales V, Cimetta E, Ronsisvalle S, Sipala FM, Loguercio Polosa P, Fortuna CG, Perrone MG, Scilimati A. Harmaline to Human Mitochondrial Caseinolytic Serine Protease Activation for Pediatric Diffuse Intrinsic Pontine Glioma Treatment. Pharmaceuticals 2024, 17, 135 (1-17). 5. Miciaccia M, Baldelli OM, Fortuna CG, Cavallaro G, Armenise D, Liturri A, Ferorelli S, Muñoz DP, Bonifazi A, Rizzo A, Cormio A, Filieri S, Micalizzi G, Dugo P, Mondello L, Sardanelli AM, Bruni F, Loguercio Polosa P, Perrone MG, Scilimati, A. ONC201-derived Tetrahydropyridopyrimidindiones as powerful ClpP protease activators to tackle diffuse midline glioma. J. Med. Chem. 2025, 68, 5190 − 5210. 6. Miciaccia M, Armenise D, Baldelli OM, Liturri A, Mammone M, Graziano G, Contino M, Rizzo F, Loguercio Polosa P, Bruni F, Deligne C, Broutier L, Cavallaro G, Fortuna CG, Tolomeo A, Green C, Bonifazi A, Majaesic E, Barghash MM, Nok Lee PJ, Houry WA, Gratteri C, Talarico C, Filieri S, Micalizzi G, Donnarumma D, Dugo P, Mondello L, Sardanelli AM, Nazarian J, Ferorelli S, Perrone MG, Scilimati A. Dysregulation of human ClpP using small molecules with piperazine-based scaffold for diffuse intrinsic pontine glioma therapy validated by patient-derived tumor organoids. Nature Communications 2026, submitted for publication. 7. Filieri S, Miciaccia M, Armenise D, Baldelli OM, Liturri A, Ferorelli S, Sardanelli AM, Perrone MG, Scilimati A. Can Focused Ultrasound overcome the failure of chemotherapy in treating pediatric Diffuse intrinsic pontine glioma due to BBB obstacle? Pharmaceuticals 2025, 18, 525 (1-15).