Project 3: Elucidation of Biochemical and Behavioral Efficacies of Antipsychotics

在经典药理学中，激动剂只激活单一的线性信号转导通路，而拮抗剂则阻断激动剂的作用，并且没有内在活性。在过去的几年里，人们已经清楚地看到，信号转导通路不仅仅是信号的线性级联。相反，它们被组织成复杂的信令网络，需要高水平的监管才能产生准确和独特的细胞响应。因此，一个给定的受体，通过不同的配体诱导的功能构象，可以通过与不同的信号伙伴相互作用而参与多种形式。通过这种方式，给定的配体可以与受体结合，在一条信号通路上充当拮抗剂，同时在另一条或另一条信号通路上充当激动剂 对白。这一特性建立在几个G蛋白偶联受体(GPCRs)--治疗干预的最重要目标--上。重要的是，没有一种临床使用的药物是在考虑到这些多重信号因素的情况下开发出来的。此外，激动剂和拮抗剂很少是完全选择性的，对于给定的受体，可以通过影响受体介导的各种过程来改变信号转导，如与G蛋白的相互作用、脱敏、内化、下调以及受体介导的非G蛋白信号成分的支架。不幸的是，这些特性的生理相关性并没有得到充分的认识。因此，识别化合物的功能选择性不仅有助于揭示不同的生物过程，而且有助于揭示特定的功能结果。目前，功能选择性与精神病学的相关性尚不清楚。这对抗精神病药物尤其重要，其中多巴胺(DA)D2受体(D2R)拮抗基本上是所有这些药物的先决条件；然而，它们的其他内在活性尚不清楚。这项拟议研究的总体目标是检查抗精神病化合物的行为反应，并阐明在精神分裂症样行为的临床前遗传和药理学小鼠模型中抗精神病疗效所必需的信号转导机制。对于行为，抗精神病化合物对DA转运体(DAT)基因敲除(KO)、N-甲基-D-天冬氨酸(NMDA)受体NR1亚单位敲除(KD)以及安非他明(AMPH)或苯环利定(PCP)处理的C57BL/6小鼠复制分裂样状态的运动活性、脉冲前抑制(PPI)、潜在抑制(LI)和社会行为的影响。将采用信号转导通路的分子指纹图谱(MFSTP)来分析抗精神病化合物对各种信号转导的影响 包括蛋白激酶A(PKA)和多巴胺(DA)以及cAMP调节的磷酸蛋白32(DARPP-32)、Akt/蛋白激酶B(PKB或Akt)和糖原合成酶激酶33(GSK3)、磷脂酶C(PLC)以及细胞外信号调节的丝裂原活化蛋白激酶(ERK)通路。目前的项目#3中的实验将通过提供临床前模型来补充项目#1和#2中的实验，以测试各种抗精神病药物化合物在改善精神分裂症样行为方面的体内选择性和有效性，并将这些反应与信号转导中的变化相关联。我们的项目#3也将补充惠氏的核心项目，在该项目中，将分析精神分裂症样行为和抗精神病药物治疗行为的大鼠模型中的抗精神病药物反应。了解抗精神病药物功能选择性的相关性可能为治疗精神分裂症提供副作用更少、治疗选择性更强和疗效更高的新靶点。

In classical pharmacology, an agonist activates a single linear signal transduction pathway, whereas an antagonist blocks the action of the agonist and possesses no intrinsic activity. Over the past few years, it has become clear that signal transduction pathways are not merely linear cascades for signaling. Instead, they are organized into complex signaling networks that require high levels of regulation to generate precise and unique cellular responses. Hence, a given receptor, through various ligand-induced functional conformations, can engage multiple modalities through interaction with different signaling partners. In this way, a given ligand can bind a receptor and act as an antagonist for one signaling pathway while serving as an agonist at another or the converse. This property is established for several G protein-coupled receptors (GPCRs) - the most important targets for therapeutic intervention. Importantly, none of the drugs in clinical use have been developed with these multiple signaling considerations in mind. Additionally, agonists and antagonists are rarely completely selective and, for a given receptor, may alter signaling by influencing various receptor-mediated processes such as interaction with G proteins, desensitization, internalization, down-regulation, and receptor-mediated scaffolding of non-G protein signaling components. Unfortunately, the physiological relevance of these properties is not fully appreciated. Thus, identifying the functional selectivity of compounds may help reveal not only distinct biological processes, but also specific functional outcomes. Currently, the relevance of functional selectivity to psychiatry is unknown. This is particularly important for antipsychotic drugs, where dopamine (DA) D2 receptor (D2R) antagonism is essentially a prerequisite for all these drugs; however, their other intrinsic activities are obscure. The overall goal of the proposed research is to examine behavioral responses to antipsychotic compounds and to elucidate signal transduction mechanisms that are essential for antipsychotic efficacy in preclinical genetic and pharmacological mouse models of schizophrenia-like behaviors. For behavior, effects of antipsychotic compounds on motor activity, prepulse inhibition (PPI), latent inhibition (LI), and social behavior in DA transporter (DAT) knockout (KO), N-methyl-D-aspartate (NMDA) receptor NR1-subunit knockdown (KD), and C57BL/6 mice treated with amphetamine (AMPH) or phencyclidine (PCP) to reproduce schizophrenia-like states. Molecular fingerprinting of signal transduction pathways (MFSTP) will be performed to analyze effects of antipsychotic compounds on various signal transduction modalities that include the protein kinase A (PKA) and DA and cAMP-regulated phosphoprotein 32 (DARPP-32), Akt/protein kinase B (PKB or Akt) and glycogen synthase kinase 33 (GSK3), phospholipase C (PLC), and extracellular signal-regulated mitogen activated protein kinase (ERK) pathways. The experiments in the present Project #3 will complement those in Projects #1 and #2 by providing preclinical models to test the in vivo selectivities and efficacies of various antipsychotic compounds on amelioration of schizophrenia-like behaviors and will correlate these responses to alterations in signal transduction. Our Project #3 will complement also the Core Project from Wyeth where antipsychotic responses will be analyzed in rat models of schizophrenia-like and antipsychotic-treated behaviors. Understanding the relevance of functional selectivity of antipsychotic drugs may provide novel targets with fewer side-effects, greater therapeutic selectivity, and enhanced efficacy for treating individuals with schizophrenia.