Subtype specific NMDA receptor antagonists

描述（由申请人提供）： NMDA受体拮抗剂在中风的实验模型中表现出优异的神经保护功效;然而，使用NMDA拮抗剂在中风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的失败可主要归因于其严重的副作用，这需要使用显著低于有效剂量的剂量。关键问题是开发降低NMDA活性而没有这些剂量限制性副作用的药物。这可以通过亚型（NR 1或NR 2）特异性拮抗剂来实现，这些拮抗剂已明确确立具有更好的副作用特征。然而，目前可用的亚型特异性化合物由于许多问题而不是有效的，例如对其他受体的活性、不溶性和/或在血脑屏障中穿越的效率低。在拟议的研究中，我们将使用一种有前途的新药理学方法，基于使用RNA配体作为NMDA受体的亚型特异性拮抗剂，这将解决目前可用药物的问题。使用RNA配体的优点是它们可以从具有随机序列（1015个配体）的大量RNA配体中进化而来，以与任何靶蛋白高度特异性结合。此外，它们是水溶性的，并且已被证明可以穿过血脑屏障。选择方案将涉及基于RNA配体对NMDA受体的给定亚基的亲和力的阳性选择的迭代过程，随后使用RT-PCR扩增。通过阴性选择步骤进一步选择由此获得的RNA配体的特异性，其中选择对NMDA受体的其它亚基没有亲和力的那些。通过上述步骤选择的RNA配体需要被表征以鉴定具有拮抗性质的那些。我们在实验室开发的基于荧光的测定法将极大地促进这一过程。该测定提供配体（即激动剂或拮抗剂）的功能性的高通量读出。将在Co-PI的实验室中使用体外和体内模型测试通过测定法鉴定的候选RNA拮抗剂的抗兴奋性毒性作用，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.