Selective inhibition of nitric oxide synthase for multiple indications

项目摘要 本提案是R01 GM049725《选择性抑制一氧化氮》的延续 多个适应症的合成酶“。一氧化氮合酶(NOS)是一个值得注意的目标，就像我们 发现神经元型一氧化氮合酶(NNOS)抑制剂适用于潜在的治疗 神经退行性疾病(如帕金森氏症、阿尔茨海默氏症、脑瘫)、细菌感染和 黑色素瘤。NNOS的抑制剂可以阻止过量的NO，从而导致神经元退化。他们 还可以抑制导致黑色素瘤的黑素细胞产生的一氧化氮。细菌一氧化氮合酶抑制物(非常 类似于nNOS)可用于保护抗生素免受细菌降解(即抗药性)和 可以协同作用，抑制细菌生长的氧化应激分子，如氢 过氧化氢。我们在所有三个迹象上都取得了显著进展；其中两个不可能 在没有托马斯·普洛斯的结晶学专业知识的情况下取得了进展。在过去的四年里，我们有 能够缩短我们的抑制剂的合成，使抑制剂具有膜穿透性， 代谢稳定，血脑屏障(BBB)可穿透良好的口腔 生物利用度，同时保持良好的效力和nNOS的选择性。然而，我们还没有 得到了具有所有想要的性质的化合物。因此，这笔赠款将使我们能够 确定具有所有所需药效学和药代动力学特性的化合物。 我们计划优化我们的nNOS选择性化合物的药效学和 帕金森病和阿尔茨海默病小鼠模型及兔体内药代动力学研究 脑性瘫痪的模型。 我们已经确定了几种nNOS选择性化合物，它们可以抑制细菌的生长， 包括耐甲氧西林金黄色葡萄球菌，尤其是与抗生素或过氧化氢联合使用。我们计划制作 细菌一氧化氮合酶(BNOS)选择性化合物，以避免抑制 哺乳动物的一氧化氮合酶。此外，我们的化合物可能还有其他靶标，而不是bnos。 因此，我们将用光亲和标记的bnos进行蛋白质下拉实验。 并用蛋白质组学鉴定其与之结合的蛋白质。这应该会澄清更多的 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抑制剂进行测试 在抑制黑色素瘤生长和抑制黑色素瘤生长的异种移植黑色素瘤模型中 转移。杨博士已经确定了一种多肽载体，可以靶向黑色素瘤分子；我们将 将活性化合物偶联到该多肽上以用于药物输送。我们还将制作一个光亲和探测器 一种活性抑制物来识别靶标。

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.