Optimization of small molecule probes

描述（由申请人提供）：N-甲基-D-天冬氨酸（NMDA）受体是突触后配体门控离子通道，介导CNS中的兴奋性突触传递。NMDA受体是由2个甘氨酸结合的NR 1亚基和2个谷氨酸结合的NR 2亚基组成的异多聚体，其中有4种类型（NR 2A、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NMDA受体增效剂存在，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的（响应PAR-09-251“Optimization of small molecule probes for the nervous system”，RFA-NS-09-003的重新发布）是鉴定NMDA受体的有效亚基选择性增效剂，其可用于测试关于NMDA受体在神经系统疾病中功能的特定假设。为了实现这一点，我们开发了一种用于NMDA受体的非竞争性变构调节剂的测定法，并筛选了约100，000种化合物。我们鉴定了6种不同但相关的分子，它们在相似的位点起作用以增强含NR 2C/D的NMDA受体功能;一种支架似乎有利于NR 2C增强。我们的工作假设是，这些多个支架可以作为一个起点，为有效的亚基选择性NMDA受体增效剂的发展。我们建议使用药物化学来开发一种化合物，其EC 50值低于300 nM，最大增强> 2.5倍，对其他谷氨酸受体的选择性> 200倍，溶解度> 10倍EC 50，缺乏神经毒性，对72种其他CNS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NR 2C/D选择性NMDA受体增强剂的效力和功效？我们将使用平行的药物化学方法，每年合成和测试200种类似物，这一数字得到了我们初步数据的支持。将使用14个自动化双电极电压钳记录系统测定每种化合物在重组NR 1/NR 2A、NR 1/NR 2B、NR 1/NR 2C、NR 1/NR 2D、GluR 1受体上的EC 50值，该系统可记录/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.药物的脱靶倾向和药代动力学特性是什么？ NR 2C/D增效剂？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，并在研究完成时再次使用实验室和NIMH资助的脱靶筛选中心的自动多孔测定组合对抗72种受体，通道和转运蛋白。这些信息将有助于确定最佳支架（第1年）以及表征本研究中出现的两种最佳化合物（第2年）。我们将在体外评价血浆半衰期、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.