Development and characterization of peptidomimetic small molecule activators of peptidase neurolysin for stroke therapy.

总结 目前的中风研究更侧重于了解大脑的自我保护和修复机制。详细 这些机制的阐明是至关重要的，因为这些知识可以导致治疗干预的发展， 模拟或参与大脑的自我保护/修复机制，并可能导致成功的中风治疗。与拟议 我们寻求开发有效的和选择性的肽酶神经溶解素（Nln）的“药物样”小分子激活剂， 将被用作研究工具和领导化学实体，以推动药物发现进程， 新型药物。最近发表的和我们实验室的初步研究已经确定Nln是大脑的自我调节之一， 保护机制，对保护和恢复中风后的大脑功能。功能意义 NLN在中风后大脑中的作用是基于它能够抑制几种神经毒素并产生三种脑- 保护性/再生性神经肽，其从许多实验和临床研究中已知， 影响中风的预后基于这些证据，我们认为Nln是一种参与脑恢复的中心肽酶， 中风后的机制在这个合作申请中，我们将利用我们在药物多个方面的专业知识 发现过程，并将开发有效的'药物样'小分子，可以选择性地提高催化效率 可作为脑卒中后脑保护和恢复的实验性治疗药物。这一建议 基于我们令人信服的实验数据，表明Nln的催化活性可以通过两个 结构相关的二肽和不同的非肽化学型。我们的研究结果显示建议的研究是可行的。 鉴定的Nln激活剂的初始结构-活性关系研究和通过在两个不同的细胞中的体内实验， 小鼠中风模型表明中风后内源性Nln的抑制加重了中风损伤，而 Nln在脑中的过表达或其向中风后脑的递送实质上改善了中风结果。的目标 这一建议将在三个很好的综合目标完成：（1）设计和表征一个多样化和专有的 基于三个活性命中和指导生物测定的化合物库，以识别关键功能残基相互作用 在Nln结合位点内，并开发具有“药物样”的Nln的高效、脑渗透性、选择性激活剂。 性质;（2）进行生化和结构研究，以表征所鉴定的Nln的活化机制 激活剂开发;（3）确定Nln激活剂在卒中后脑保护和恢复中的治疗潜力， 一个中风的老鼠模型。这项工作是高度创新的，因为在科学文献中没有描述Nln的激活剂。 这些化合物从未被认为具有治疗潜力。合作调查小组， 包括药物化学和药物发现、晶体学和结构生物学、酶生物化学 和药理学，血脑屏障生理学和中风药理学，是非常合格的进行拟议的 问题研究我们的长期目标是将主要的Nln激活剂从实验室转移到床边，并开发有效的治疗方法， 这将改变目前大量中风患者的治疗方式。

SUMMARY Current stroke research focuses more on understanding the brain’s self-protective and repair mechanisms. Detailed elucidation of these mechanisms is crucial as such knowledge could lead to development of therapeutic interventions which mimic or engage the brain’s self-protective/repair mechanisms and can lead to successful stroke therapy. With the proposed research we seek to develop potent and selective ‘drug-like’ small molecule activators of peptidase neurolysin (Nln) which will be used as research tools and lead chemical entities to move the drug discovery process forward for development of a novel class of drugs. Recently published and pilot studies from our laboratory have identified Nln as one of the brain’s self- protective mechanisms, functioning towards preservation and recovery of the brain after stroke. Functional significance of Nln in the post-stroke brain is based on its ability to inactivate several neurotoxic and generate three cerebro- protective/regenerative neuropeptides, which are known from numerous experimental and clinical studies to critically contribute to the outcome of stroke. Based on this evidence we view Nln as a central peptidase involved in brain restorative mechanisms following stroke. In this collaborative application we will leverage our expertise in multiple aspects of the drug discovery process and will develop potent ‘drug-like’ small molecules which can selectively enhance the catalytic efficiency of Nln and can be used as experimental therapeutic agents for post-stroke brain protection and recovery. This proposal has been formulated based on our compelling experimental data indicating that catalytic activity of Nln can be enhanced by two structurally related dipeptides and a distinct non-peptide chemotype. Feasibility of the proposed studies is shown by our initial structure-activity relationship studies of the identified Nln activators and by in vivo experiments in two different mouse stroke models indicating that inhibition of endogenous Nln after stroke aggravates stroke injury, whereas overexpression of Nln in the brain or its delivery to the post-stroke brain substantially improves stroke outcome. The goals of this proposal will be accomplished in three well-integrated aims: (1) design and characterize a diverse and proprietary library of compounds based on three active hits and guided bioassays to identify critical functional residue interactions within the Nln binding site, and to develop high-potency, brain-permeable, selective activators of Nln with ‘drug-like’ properties; (2) conduct biochemical and structural studies to characterize the activation mechanism that the identified Nln activators exploit; (3) determine the therapeutic potential of Nln activators in post-stroke brain protection and recovery using a mouse model of stroke. This work is highly innovative because there are no activators of Nln described in the scientific literature and such compounds were never considered to have therapeutic potential. The collaborative investigative team, comprising experts in medicinal chemistry and drug discovery, crystallography and structural biology, enzyme biochemistry and pharmacology, blood-brain barrier physiology and stroke pharmacology, is highly qualified to conduct the proposed studies. Our long-term goal is to translate the lead Nln activators from bench to bedside and develop an effective therapy, which would transform the current treatment modalities for a vast number of stroke patients.