Optimization of small molecule probes

描述(申请人提供)：N-甲基-D-天冬氨酸(NMDA)受体是突触后配体门控离子通道，在中枢神经系统中介导兴奋性突触传递。NMDA受体是由2个甘氨酸结合的NR1亚基和2个谷氨酸结合的NR2亚基组成的异源多聚体，其中有四种类型(NR2A、B、C、D)。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障碍，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说。然而，没有小分子NMDA受体增强剂可以用来获得概念验证数据。鉴于缺乏亚基选择性工具化合物，我们在这项提案中的目标(针对PAR-09-251“神经系统小分子探针的优化”，RFA-NS-09-003的再版)是为了识别有效的NMDA受体亚基选择性增强子，这些增强子可用于测试有关NMDA受体在神经系统疾病中功能的特定假说。为了实现这一点，我们开发了一种NMDA受体的非竞争性变构调节剂的分析方法，并筛选了大约100,000种化合物。我们确定了6个不同但相关的分子，它们在相似的位置作用于增强含有NR2C/D的NMDA受体功能；一个支架似乎支持NR2C增强。我们的工作假设是，这些多重支架可以作为开发有效的亚单位选择性NMDA受体增强剂的起点。我们建议利用药物化学开发一种化合物，其EC50值低于300 NM，最大增强倍数为2.5倍，对其他谷氨酸受体的选择性为200倍，溶解度为EC50的10倍，对72个其他中枢神经系统受体、通道和泵的选择性在30-1000倍之间。实验将解决两个问题：1.什么结构特征控制NR2C/D选择性NMDA受体增强剂的效力和疗效？我们将使用平行的药物化学方法，每年合成和测试200个类似物，这一数字得到了我们初步数据的支持。每种化合物的EC50值将在重组的NR1/NR2A、NR1/NR2B、NR1/NR2C、NR1/NR2D、GluR1受体上使用14个自动双电极电压钳记录系统来确定，这些系统可以记录/分析500种化合物/年的浓度-效应曲线。初步研究已经确定了支架的几个区域，这些区域通过可用的化学空间来控制效力和亚单位选择性。2.什么是偏离目标的负债和药代动力学特性 NR2C/D增强器？我们将确定所有活性化合物的溶解度和代谢稳定性，并在关键决策点筛选最佳化合物，并在完成针对72个受体、通道和转运体的研究时，使用实验室和NIMH资助的非靶标筛选中心的自动化多孔分析组合再次筛选最佳化合物。这些信息将有助于确定要追求的最佳支架(第一年)，以及表征从这项研究中出现的两种最佳化合物(第二年)。我们将在体外评估血浆半衰期、脑/血浆比和神经毒性。

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.