Subtype specific NMDA receptor antagonists

描述(由申请人提供)： NMDA受体拮抗剂在实验性卒中模型中显示出良好的神经保护作用；然而，NMDA受体拮抗剂在卒中和创伤性脑损伤中的临床试验尚未成功。目前可用的药物在临床试验中失败的主要原因是它们的严重副作用，这需要使用远远低于有效剂量的药物。关键问题是开发降低NMDA活性而不产生这些剂量限制副作用的药物。这可以通过亚型(NR1或NR2)特异性拮抗剂来实现，这些拮抗剂已经明确地被证实具有更好的副作用。然而，目前可用的亚型特异性化合物由于许多问题而没有效果，例如在其他受体上的活性，不可溶，和/或在穿越血脑屏障方面效率低下。在拟议的研究中，我们将使用一种有前景的新药理学方法，基于使用RNA配体作为NMDA受体的亚型特异性拮抗剂，这将解决现有药物存在的问题。使用RNA配体的优势在于，它们可以从具有随机序列(1015个配体)的大RNA配体池中进化而来，以高度特异性地结合任何靶蛋白。此外，这些化合物是水溶性的，并已被证明可以穿越血脑屏障。选择方案将包括一个迭代的过程，基于对给定的NMDA受体亚单位的RNA配体的亲和力进行正选择，然后使用RT-PCR进行扩增。通过负选择步骤进一步选择如此获得的RNA配体的特异性，其中选择那些与NMDA受体的其他亚单位没有亲和力的配体。需要对通过上述步骤选择的RNA配体进行表征，以鉴定具有拮抗性质的RNA配体。我们在实验室开发的基于荧光的分析方法将极大地促进这一过程。这种分析提供了配体，即激动剂或拮抗剂的功能的高通量读数。通过该检测确定的候选RNA拮抗剂将在Co-Pi的实验室使用体外和体内模型进行抗兴奋毒性作用测试，从而为未来的临床前和临床试验提供基础。

DESCRIPTION (provided by applicant): NMDA receptor antagonists exhibit excellent neuroprotective efficacy in experimental models of stroke; however, clinical trials in stroke and traumatic brain injury with NMDA antagonists have not been successful. The failure of the currently available drugs in clinical trials can be primarily attributed to their severe side effects, which necessitate the use of significantly lower than effective doses. The key issue is to develop drugs that reduce NMDA activity without these dose-limiting side effects. This can be achieved by subtype (NR1or NR2) specific antagonists that have been clearly established to possess better side effect profiles. The currently available subtype specific compounds, however, have not been effective due to a number of problems such as activity at other receptors, insolubility, and/or inefficiency in crossing in blood brain barrier. In the proposed research we will use a promising new pharmacological approach, based on the use of RNA ligands as subtype specific antagonists for the NMDA receptors, which will address the problems with the currently available drugs. The advantage of using RNA ligands is that they can be evolved from a large pool of RNA ligands with random sequences (1015 ligands) to bind with high specificity for any target protein. Additionally, these are water soluble and have been shown to cross the blood brain barrier. The selection protocol will involve an iterative process of positive selection based on the affinity of the RNA ligand for a given subunit of the NMDA receptor followed by amplification using RT-PCR. The RNA ligands thus obtained are further selected for specificity by a negative selection step wherein those with no affinity for the other subunits of NMDA receptors are selected. The RNA ligands selected by the above steps need to be characterized to identify those with antagonistic property. This process will be greatly facilitated by the fluorescence based assay that we have developed in my laboratory. This assay provides high throughput readout of the functionality of the ligand, namely agonists or antagonist. The candidate RNA antagonists identified by the assay will be tested for antiexcitotoxic effects using in vitro and in vivo models in the Co-PI's laboratory, thus providing the basis for future preclinical and clinical trials.