A solid understanding of the mechanisms governing ligand binding is crucial for rational design of therapeutics targeting the dopamine D₂ receptor (D₂R). Here, we use G protein-coupled inward rectifier potassium (GIRK) channel activation in Xenopus oocytes to measure the kinetics of D₂R antagonism by a series of aripiprazole analogues, as well as the recovery of dopamine (DA) responsivity upon washout. The aripiprazole analogues comprise an orthosteric and a secondary pharmacophore and differ by the length of the saturated carbon linker joining these two pharmacophores. Two compounds containing 3- and 5-carbon linkers allowed for a similar extent of recovery from antagonism in the presence of 1 or 100 μM DA (>25 and >90% of control, respectively), whereas recovery was less prominent (∼20%) upon washout of the 4-carbon linker compound, SV-III-130, both with 1 and 100 μM DA. Prolonging the coincubation time with SV-III-130 further diminished recovery. Curve-shift experiments were consistent with competition between SV-III-130 and DA. Two mutations in the secondary binding pocket (V91A and E95A) of D₂R decreased antagonistic potency and increased recovery from SV-III-130 antagonism, whereas a third mutation (L94A) only increased recovery. Our results suggest that the secondary binding pocket influences recovery from inhibition by the studied aripiprazole analogues. We propose a mechanism, supported by in silico modeling, whereby SV-III-130 initially binds reversibly to the D₂R, after which the drug-receptor complex undergoes a slow transition to a second ligand-bound state, which is dependent on secondary binding pocket integrity and irreversible during the time frame of our experiments.

中文翻译：

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与多巴胺D2受体有两种相互作用方式的配体-不可克服的拮抗作用的诱导拟合机制。

对控制配体结合的机制的深入了解对于合理设计靶向多巴胺D ₂受体（D ₂ R）的疗法至关重要。在这里，我们使用非洲爪蟾卵母细胞中的G蛋白偶联的内向整流钾（GIRK）通道激活来测量D ₂的动力学一系列阿立哌唑类似物的R拮抗作用，以及洗脱后多巴胺（DA）响应性的恢复。阿立哌唑类似物包含正构和次级药效基团，并且区别在于连接这两个药效基团的饱和碳接头的长度不同。在1或100μMDA（分别为对照的> 25和> 90％）的存在下，两种含有3和5个碳原子的化合物可以从拮抗作用中恢复相似程度（分别为对照的25％和> 90％） ）分别用1和100μMDA冲洗4-碳连接基化合物SV-III-130。用SV-III-130延长共孵育时间会进一步降低回收率。曲线平移实验与SV-III-130和DA之间的竞争一致。D ₂的次级结合口袋中的两个突变（V91A和E95A）R降低了拮抗效力，并增加了从SV-III-130拮抗作用的恢复，而第三个突变（L94A）仅提高了恢复。我们的结果表明，次级结合口袋会影响阿立哌唑类似物抑制作用的恢复。我们提出了一种在计算机模拟中支持的机制，其中SV-III-130最初可逆地与D ₂ R结合，此后，药物-受体复合物经历缓慢过渡至第二个配体结合状态，这取决于次级在我们实验的时间范围内，绑定袋的完整性和不可逆转性。