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. 2021 Nov 19;3(1):vdab169. doi: 10.1093/noajnl/vdab169

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© The Author(s) 2021. Published by Oxford University Press, the Society for Neuro-Oncology and the European Association of Neuro-Oncology.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

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Figure 2. — In vitro DIPG apoptosis is dependent on SDHA degradation following either ONC201 or German-sourced ONC201 (GsONC201) treatment. (A) Protonated ONC201 and free base German-sourced ONC201 (GsONC201) showed similar antiproliferative effects in DIPG cell lines: SU-DIPG-IV, SU-DIPG-XXI, SU-DIPG-XXXVI, SU-DIPG-VI, SU-DIPG-XIII, and SU-DIPG-XVII. (Bi) Cytotoxicity was assessed via annexin V/PI staining and flow cytometry following 72-h treatment with 5 μM ONC201 and GsONC201 (Bii) and compared with vehicle controls (Student’s t-test). (C) Both ONC201 (O; pink) and GsONC201 (Gs; black), activated CLPP to sequester SDHA, and modulate the activity of the mTOR/AKT signaling pathways. DIPG cell lines sensitive (S; aqua) to ONC201 or GsONC201 showed increased TRAIL expression and induced cell death, cleaved PARP and CASP3, whereas resistant DIPG cells lines (R; orange) showed no change in the activity in programmed cell death proteins (FC = fold change, yellow = increased phosphorylation, blue = decreased phosphorylation, red = increased protein expression or cleavage (Cl), green = decreased protein expression, ND = not determined; Student’s t-test used to determine significance between treated vs untreated samples, *P < .05, **P < .01, ***P < .001). (Di) Docking of both the implicit protonated ONC201 (pink structure) and free base ONC201 (black structure) into the defined binding pocket of CLPP (co-crystal structure of CLPP and ONC201 [PB: 6DL7; ONC201]), revealed that protonated ONC201 species (at N1, pKa = 8.1) is likely to have enhanced binding, indicated by the higher “fitness” scores calculated, in comparison to free base ONC201. This enhanced binding is facilitated by an H-bond interaction with the protonated N1 of ONC201 and the tyrosine 118 residue in the binding pocket. Docking of protonated ONC201 (pink) and GsONC201 (black) into the defined binding pocket of DRD2 did not show significant variation. (Dii) Quantification of unbiased docking scores of protonated and free base forms of ONC201 into CLPP and DRD2 (Student’s t-test used to determine the significance of ONC201 vs GsONC201 docking scores based on n = 4 top docking poses, *P < .05, **P < .01).