Molecular basis of atypicality in antipsychotic drug action

多巴胺D2受体（D2 R）的拮抗作用是所有临床使用的抗精神病药物（APD）治疗精神分裂症的疗效所必需的。不幸的是，D2 R的拮抗作用与锥体外系副作用（EPS）如帕金森综合征和迟发性运动障碍有关。一些较新的APD（称为非典型APD）被认为具有较低的副作用风险，但这些差异背后的机制尚不清楚。我们最近表明，结合率的APD预测其能力，导致EPS，虽然我们缺乏直接的实验证据，这种现象。此外，在另一项最近的研究中，我们已经揭示了一些但不是所有的APD可以作为药理学伴侣来增加D2 R的细胞表面表达。APD长期治疗后脑D2 R增加被认为是医源性精神病和精神分裂症患者随时间推移对药物治疗产生耐药性的原因。APD诱导的D2 R上调也与迟发性运动障碍有关，表明慢性D2 R上调可在D2 R表达神经元中产生永久性不良改变。然而，在与EPS相关的脑细胞中，既没有研究反向激动剂特性，也没有研究伴侣活性。假设：了解药物再结合的分子基础和APD在D2 R的药理学伴侣活性将揭示抗精神病药物作用的差异，预测其副作用概况。该项目将使用广泛的成像和生物物理技术，结合经典的药理学和生物化学方法来解决这一假设。我们已经使用荧光相关光谱（FCS）显示，药物与细胞膜和受体的相互作用驱动更高的局部浓度的药物接近细胞膜，并开发了一系列的荧光标记的D2 R拮抗剂，其结合动力学性质跨越的范围所表现出的APD。学生将研究药物再结合的分子决定因素。在表达不同水平SNAP-D2 R的模型细胞系中使用FCS与具有已知结合动力学的荧光D2 R配体组合，我们将评估不同水平的细胞表面D2 R表达如何驱动药物的局部浓度。然后，学生将这些研究扩展到纹状体和皮质神经元以及来自SNAP-D2 R小鼠的垂体催乳素的原代培养物，以了解这些现象如何影响疾病相关组织中内源性表达的D2 R的药物再结合。同时，学生将使用Tr-FRET结合来测量上述原代神经元培养物中天然表达的D2 R处的选定未标记APD的结合动力学。为了研究不同APD驱动D2 R细胞表面表达的能力，学生将使用BRET来量化D2 R运输。这些实验将与经典的共聚焦，TIRF显微镜和高内容成像。然后，我们将这些研究扩展到表达SNAP-D2 R的原代细胞培养物，以研究急性和长期应用APD对这些疾病相关细胞中D2 R表达和运输的影响。

Antagonism of the dopamine D2 receptor (D2R) is required for the efficacy of all clinically used antipsychotic drugs (APDs) for the treatment of schizophrenia. Unfortunately, antagonism of the D2R is associated with extrapyramidal side effects (EPS) such as Parkinsonism, and tardive dyskinesia. Some newer APDs (termed atypical APDs) are thought to have a lower side-effect risk but the mechanism behind these differences is unclear. We have recently shown that the binding association rate of APDs predicts their ability to cause EPS although we lack direct experimental evidence of this phenomenon. Furthermore, in another recent study we have revealed that some but not all APDs can act as pharmacological chaperones to increase cell surface expression of D2Rs. Increases in brain D2R after chronic treatment with APDs have been suggested as the reason for iatrogenic psychoses and for resistance of schizophrenia patients to pharmacotherapy over time. APD-induced upregulation of D2R has also been implicated in tardive dyskinesia, suggesting that chronic D2R upregulation can produce permanent adverse alterations in D2R-expressing neurons. However, neither the inverse agonist properties or the chaperone activity have been studied in brain cells relevant to EPS. Hypothesis: Understanding the molecular basis of drug rebinding and pharmacological chaperoning activity of APDs at the D2R will reveal differences in antipsychotic drug action that predict their side effect profile. This project will use a wide range of imaging and biophysical techniques in combination with classical pharmacology and biochemistry approaches to address this hypothesis. We have used Fluorescence Correlation Spectroscopy (FCS) to show that drug interactions with the cell membrane and receptors drives a higher local concentration of drug proximal to the cell membrane and developed a series of fluorescently-labeled D2R antagonists whose binding kinetics properties span the range of those exhibited by APDs. The student will investigate the molecular determinants of drug rebinding. Using FCS in model cell lines expressing different levels of SNAP-D2R in combination with fluorescent D2R ligands with known binding kinetics, we will assess how different levels of cell surface D2R expression can drive local concentrations of the drug. Then the student will extend these studies to primary cultures of striatal and cortical neurons as well as pituitary lactotrophs derived from the SNAP-D2R mouse to understand how these phenomena might impact drug rebinding at endogenously expressed D2Rs in disease-relevant tissues. In parallel, the student will use Tr-FRET binding to measure the binding kinetics of selected unlabelled APDs at natively expressed D2Rs in the above primary neuronal cultures. To investigate the ability of different APDs to drive D2R cell surface expression the student will use BRET to quantify D2R trafficking. These experiments will be complemented with classical confocal, TIRF microscopy and high content imaging. Then we will extend these studies to primary cell cultures expressing the SNAP-D2R to investigate the impact of the acute and longer-term application of APDs on D2R expression and trafficking in these disease-relevant cells.