ONC201 is a first-in-class imipridone compound that is in clinical trials for the treatment of high-grade gliomas and other advanced cancers. Recent studies identified that ONC201 antagonizes D2-like dopamine receptors at therapeutically relevant concentrations. In the current study, characterization of ONC201 using radioligand binding and multiple functional assays revealed that it was a full antagonist of the D2 and D3 receptors (D2R and D3R) with low micromolar potencies, similar to its potency for antiproliferative effects. Curve-shift experiments using D2R-mediated β-arrestin recruitment and cAMP assays revealed that ONC201 exhibited a mixed form of antagonism. An operational model of allostery was used to analyze these data, which suggested that the predominant modulatory effect of ONC201 was on dopamine efficacy with little to no effect on dopamine affinity. To investigate how ONC201 binds to the D2R, we employed scanning mutagenesis coupled with a D2R-mediated calcium efflux assay. Eight residues were identified as being important for ONC201's functional antagonism of the D2R. Mutation of these residues followed by assessing ONC201 antagonism in multiple signaling assays highlighted specific residues involved in ONC201 binding. Together with computational modeling and simulation studies, our results suggest that ONC201 interacts with the D2R in a bitopic manner where the imipridone core of the molecule protrudes into the orthosteric binding site, but does not compete with dopamine, whereas a secondary phenyl ring engages an allosteric binding pocket that may be associated with negative modulation of receptor activity.

中文翻译：

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亚胺吡酮抗癌药物 ONC201 的药理学表征揭示了 D2 多巴胺受体的负变构作用机制

ONC201是一种一流的亚胺吡酮化合物，正在临床试验中用于治疗高级别胶质瘤和其他晚期癌症。最近的研究确定 ONC201 在治疗相关浓度下拮抗 D2 样多巴胺受体。在当前的研究中，使用放射性配体结合和多功能分析对 ONC201 进行表征表明，它是 D2 和 D3 受体（D2R 和 D3R）的完全拮抗剂，具有低微摩尔效力，与其抗增殖作用的效力相似。使用 D2R 介导的β 的曲线移动实验-arrestin 招募和 cAMP 分析表明 ONC201 表现出一种混合形式的拮抗作用。变构的操作模型用于分析这些数据，这表明 ONC201 的主要调节作用是对多巴胺功效的影响，对多巴胺亲和力几乎没有影响。为了研究 ONC201 如何与 D2R 结合，我们采用了扫描诱变和 D2R 介导的钙外流测定。8 个残基被确定为对 ONC201 对 D2R 的功能拮抗作用很重要。这些残基的突变随后在多个信号分析中评估 ONC201 拮抗作用，突出显示了参与 ONC201 结合的特定残基。连同计算建模和仿真研究，