Selective inhibition of nitric oxide synthase for multiple indications

项目摘要 本提案是R 01 GM 049725“选择性抑制一氧化氮”的延续 用于多种适应症的合成酶”。一氧化氮合酶（NOS）是一个显着的目标，因为我们有 发现神经元型一氧化氮合酶（nNOS）抑制剂适用于潜在的治疗 神经变性疾病（例如，帕金森氏症、阿尔茨海默氏症、脑瘫）、细菌感染，以及 黑素瘤nNOS的抑制剂阻断可引起神经元变性的过量NO。他们 也能抑制黑色素细胞中产生的NO，从而导致黑色素瘤。细菌NOS抑制剂（非常 类似于nNOS）可用于保护抗生素免受细菌降解（即，电阻）和 可协同抑制氧化应激分子如氢的细菌生长 过氧化物我们在所有三个指标上都取得了显著进展;其中两个指标本来是不可能的。 没有托马斯普洛斯的晶体学专业知识，在过去的四年里， 能够缩短我们抑制剂的合成，使抑制剂是膜渗透， 从代谢中稳定下来，并且是血脑屏障（BBB）可穿透的，具有良好的口服 生物利用度，同时保持优异的效力和nNOS选择性。然而，我们还没有 得到的化合物具有所有所需的特性。因此，这笔赠款将使我们能够 鉴定具有所有所需药效学和药代动力学性质的化合物。 我们计划优化我们的nNOS选择性化合物的药效学， 帕金森病和阿尔茨海默病小鼠模型和兔中的药代动力学和试验 脑性瘫痪的模型。 我们已经鉴定了几种抑制细菌生长的nNOS选择性化合物， 包括MRSA，特别是与抗生素或过氧化氢联合使用。我们计划 细菌NOS（bNOS）-选择性化合物，以避免抑制 哺乳动物NOS此外，除了bNOS之外，可能还有其他靶点，我们的化合物 因此，我们将用光亲和标记的bNOS进行蛋白质下拉实验 抑制剂并用蛋白质组学鉴定其所连接的蛋白质。这应该澄清一个更 bNOS抑制剂的完整作用机制。 我们最好的nNOS选择性抑制剂也将由孙杨博士作为多种的抑制剂进行测试 和在异种移植黑素瘤模型中用于抑制黑素瘤生长， 转移杨博士已经确定了一种肽载体，以靶向黑色素瘤分子;我们将 将活性化合物偶联到该肽用于药物递送。我们还将制作一个光亲和探针 活性抑制剂来识别目标

Project Summary This proposal is a continuation of R01 GM049725, “Selective Inhibition of Nitric Oxide Synthase for Multiple Indications”. Nitric oxide synthase (NOS) is a remarkable target, as we have found that neuronal nitric oxide synthase (nNOS) inhibitors are applicable to the potential treatment of neurodegenerative diseases (e.g., Parkinson's, Alzheimer's, cerebral palsy), bacterial infection, and melanoma. Inhibitors of nNOS block the excess NO that can cause degeneration of neurons. They also inhibit NO produced in melanocytes that leads to melanoma. Inhibitors of bacterial NOS (very similar to nNOS) can be used to protect antibiotics from bacterial degradation (i.e., resistance) and can be synergistic for bacterial growth inhibition by oxidant stress molecules, such as hydrogen peroxide. We have made outstanding progress on all three indications; two of which could not have progressed without the crystallography expertise of Thomas Poulos. In the last four years we have been able to shorten the syntheses of our inhibitors, make inhibitors that are membrane penetrable, are stabilized from metabolism, and are blood-brain barrier (BBB) penetrable with good oral bioavailability while maintaining excellent potency and nNOS selectivity. However, we have not yet gotten compounds that possess all of the desired properties. Consequently, this grant will allow us to identify compounds that have all of the desired pharmacodynamics and pharmacokinetic properties. We plan to optimize our nNOS-selective compounds for pharmacodynamics and pharmacokinetics and test in mouse models of Parkinson's and Alzheimer's diseases and in a rabbit model for cerebral palsy. We have identified several nNOS-selective compounds that inhibit the growth of bacteria, including MRSA, especially in combination with an antibiotic or hydrogen peroxide. We plan to make bacterial NOS (bNOS)-selective compounds to avoid potential side effects from inhibition of mammalian NOS. Also, there may be additional targets, other than bNOS with which our compounds interact; therefore, we will carry out protein pulldown experiments with a photoaffinity-labeled bNOS inhibitor and identify proteins to which it attaches with proteomics. This should clarify a more complete mechanism of action of bNOS inhibitors. Our best nNOS-selective inhibitors will also be tested by Dr. Sun Yang as inhibitors of a variety of melanoma cell lines and in a xenograft melanoma model for inhibition of melanoma growth and metastasis. Dr. Yang has identified a peptide carrier to target molecules to melanoma; we will conjugate active compounds to this peptide for drug delivery. We also will make a photoaffinity probe of an active inhibitor to identify targets.