Abstract B130: Differentiated pharmacology of the imipridone ONC201: the first selective DRD2/3 antagonist in clinical oncology
Authors: Madhukar Neel S. , Prabhu Varun Vijay , Anantharaman Lakshmi , Sulli Chidananda , Davidson Edgar , Deacon Sean , Rucker Joseph , Charter Neil , Doranz Benjamin , Oster Wolfgang , Elemento Olivier , Free R. Benjamin , Sibley David , Allen Joshua E. 
Published: August 2018

Abstract
Background: ONC201, founding member of the imipridone class of compounds, has demonstrated biologic activity and exceptional safety in advanced cancer patients. In this study, we identified and characterized a previously unknown binding target of ONC201. Methods: Binding targets for ONC201 were predicted by BANDIT–a machine learning-based drug target identification platform that uses clinical, genomics, chemical, and structural datasets for small molecules with validated binding targets. GPCR antagonism was evaluated using beta-arrestin recruitment and cAMP reporter assays in Chinese hamster ovary cells. KB values were determined with Schild analyses and DRD2 binding was confirmed with radio-labeled ligand competition assays. Shotgun mutagenesis across 350 amino acids of DRD2 was performed and dopamine-induced calcium flux assays were conducted in HEK-293T cells. Results: BANDIT predicted that ONC201 would bind with high selectivity to the G-protein coupled receptors (GPCRs) dopamine receptor D2(DRD2) and D3 (DRD3). DRD2 is overexpressed in glioblastoma, controls prosurvival signaling pathways, and its antagonism causes proapoptotic and antiproliferative effects in malignant cells. β-arrestin recruitment and cAMP assays determined that ONC201 selectively antagonizes DRD2 and DRD3. Consistent with BANDIT predictions and in contrast to other DRD2 antagonist antipsychotics, ONC201 did not antagonize other dopamine receptors or other closely related GPCRs. Schild analyses and radiolabeled ligand competition assays revealed DRD2 affinities that were consistent with those identified for ONC201 anticancer activity. In accordance with superior selectivity, ONC201 exhibited a wider therapeutic window compared to other antipsychotics. In support of the hypothesis that selectively targeting the Gi-coupled D2-like receptors yields superior anticancer efficacy, combined inhibition of DRD2 and DRD1, a Gs-coupled D1-like receptor, was found to be inferior to DRD2 inhibition alone. ONC201 exhibited a very slow association rate for DRD2 relative to antipsychotics, whereas the dissociation rate was similar to atypical antipsychotics that are better tolerated. Shotgun mutagenesis of DRD2 identified 8 residues that are critical for ONC201-mediated antagonism of dopamine-induced calcium flux via DRD2. Some of the residues were specific to DRD2 and DRD3, suggesting a potential role of these residues in conferring ONC201 selectivity. Consistent with competitive inhibition, several mutated residues were within the orthosteric binding site (OBS); however, two distal residues were identified outside of the OBS. The latter suggesting a secondary binding pocket and concurrent noncompetitive antagonism of DRD2. Conclusion: The receptor pharmacology of ONC201, the first selective DRD2/3 antagonist in clinical oncology, may underpin its unique selectivity, safety, and anticancer activity that has been observed in clinical trials.
Citation Format: Neel S. Madhukar, Varun Vijay Prabhu, Lakshmi Anantharaman, Chidananda Sulli, Edgar Davidson, Sean Deacon, Joseph Rucker, Neil Charter, Benjamin Doranz, Wolfgang Oster, Olivier Elemento, R. Benjamin Free, David Sibley, Joshua E. Allen. Differentiated pharmacology of the imipridone ONC201: the first selective DRD2/3 antagonist in clinical oncology [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B130.