Optimization of small molecule probes

描述（由申请人提供）：n -甲基- d-天冬氨酸（NMDA）受体是介导中枢神经系统兴奋性突触传递的突触后配体门控离子通道。NMDA受体是由2个甘氨酸结合NR1亚基和2个谷氨酸结合NR2亚基组成的异聚体，有NR2A、B、C、D四种类型。NMDA受体参与正常的大脑功能，如发育、学习和记忆。此外，NMDA受体功能低下可能导致神经精神疾病，如精神分裂症，这导致了NMDA受体功能增强可能在治疗上有用的假设。然而，没有小分子的NMDA受体增强剂存在，以获得概念验证数据。鉴于缺乏亚基选择性工具化合物，我们在本提案中的目标（响应PAR-09-251“神经系统小分子探针的优化”，RFA-NS-09-003的再版）是鉴定有效的，亚基选择性的NMDA受体增强剂，可用于测试关于NMDA受体在神经系统疾病中的功能的特定假设。为了实现这一目标，我们开发了一种NMDA受体非竞争性变构调节剂的检测方法，并筛选了约10万种化合物。我们确定了6个不同但相关的分子，它们在相似的位点起作用，增强含有NR2C/ d的NMDA受体的功能；一个支架似乎有利于NR2C增强。我们的工作假设是，这些多种支架可以作为开发有效的亚基选择性NMDA受体增强剂的起点。我们建议利用药物化学开发一种EC50值低于300 nM， >最大增强2.5倍，对其他谷氨酸受体>选择性200倍，>溶解度10倍EC50，对72种其他中枢神经系统受体，通道和泵没有神经毒性和选择性在30-1000倍之间的化合物。实验将解决两个问题：1。哪些结构特征控制NR2C/ d选择性NMDA受体增强剂的效力？我们将使用平行药物化学方法，每年合成和测试200个类似物，我们的初步数据支持这个数字。每个化合物的EC50值将在重组NR1/NR2A， NR1/NR2B, NR1/NR2C, NR1/NR2D， GluR1受体上测定，使用14个自动化双电极电压钳记录系统，可记录/分析浓度-效应曲线，每年可记录/分析> 500个化合物。初步的研究已经确定了几个区域的支架控制效力和亚基选择性与可用的化学空间。2. 什么是脱靶负荷和药代动力学性质

DESCRIPTION (provided by applicant): N-methyl-D-aspartate (NMDA) receptors are postsynaptic ligand-gated ion channels that mediate excitatory synaptic transmission in the CNS. NMDA receptors are heteromultimers comprised of 2 glycine-binding NR1 subunits and 2 glutamate-binding NR2 subunits, of which there are four types (NR2A,B,C,D). NMDA receptors are involved in normal brain functions such as development, learning, and memory. In addition, NMDA receptor hypo-function may contribute to neuropsychiatric disorders such as schizophrenia, which has lead to the hypothesis that potentiation of NMDA receptor function could be therapeutically useful. However, no small molecule NMDA receptor potentiators exist with which to obtain proof-of-concept data. Given the lack of subunit- selective tool compounds, our objective in this proposal (in response to PAR-09-251 "Optimization of small molecule probes for the nervous system", a reissue of RFA-NS-09-003) is to identify potent, subunit-selective potentiators of NMDA receptors that can be used to test specific hypotheses about NMDA receptor function in neurological diseases. To accomplish this, we developed an assay for non-competitive allosteric modulators of NMDA receptors and screened ~100,000 compounds. We identified 6 distinct but related molecules that act at a similar site to potentiate NR2C/D-containing NMDA receptor function; one scaffold appears to favor NR2C potentiation. Our working hypothesis is that these multiple scaffolds can serve as a starting point for the development of potent subunit- selective NMDA receptor potentiators. We propose to use medicinal chemistry to develop a compound with an EC50 value below 300 nM, maximal potentiation of > 2.5-fold, selectivity against other glutamate receptors > 200-fold, solubility > 10-fold EC50, lack of neurotoxicity and selectivity against 72 other CNS receptors, channels, and pumps of between 30-1000 fold. Experiments will address two questions: 1. What structural features control potency and efficacy of NR2C/D-selective NMDA receptor potentiators ? We will use parallel medicinal chemistry approaches to synthesize and test 200 analogues per year, a number supported by our preliminary data. The EC50 value for each compound will be determined at recombinant NR1/NR2A, NR1/NR2B, NR1/NR2C, NR1/NR2D, GluR1 receptors using 14 automated two-electrode voltage-clamp recording systems that can record/analyze concentration-effect curves for > 500 compounds/yr. Preliminary studies have identified several regions of the scaffold that control potency and subunit-selectivity with available chemical space. 2. What are the off-target liabilities and pharmacokinetic properties of NR2C/D potentiators? We will determine solubility and metabolic stability for all active compounds, and screen the best compounds at key decision points and again at the completion of the study against 72 receptors, channels, and transporters using a combination of automated multi-well assays both in the lab and at an NIMH-funded off-target screening center. This information will help to identify the best scaffold to pursue (year-1) as well as characterize the two best compounds to emerge from this study (year-2). We will evaluate plasma half-life, brain:plasma ratio, and neurotoxicity in vitro.