1 Biochemistry Interdisciplinary Graduate Program, Molecular and Cellular Biochemistry Department, Indiana University, Bloomington, IN, USA.
2 Program in Neuroscience, Indiana University, Bloomington, IN, USA.
Mol Pain. 2018 Jan-Dec;14:1744806918801224. doi: 10.1177/1744806918801224. Epub 2018 Aug 29.
Elevated N-methyl-D-aspartate receptor activity contributes to central sensitization. Our laboratories and others recently reported that disrupting protein-protein interactions downstream of N-methyl-D-aspartate receptors suppresses pain. Specifically, disrupting binding between the enzyme neuronal nitric oxide synthase and either its upstream (postsynaptic density 95 kDa, PSD95) or downstream (e.g. nitric oxide synthase 1 adaptor protein, NOS1AP) protein partners suppressed inflammatory and/or neuropathic pain. However, the lack of a small-molecule neuronal nitric oxide synthase-NOS1AP inhibitor has hindered efforts to validate the therapeutic utility of disrupting the neuronal nitric oxide synthase-NOS1AP interface as an analgesic strategy. We, therefore, evaluated the ability of a putative small-molecule neuronal nitric oxide synthase-NOS1AP inhibitor ZLc002 to disrupt binding between neuronal nitric oxide synthase and NOS1AP using ex vivo, in vitro, and purified recombinant systems and asked whether ZLc002 would suppress inflammatory and neuropathic pain in vivo. In vitro, ZLc002 reduced co-immunoprecipitation of full-length NOS1AP and neuronal nitric oxide synthase in cultured neurons and in HEK293T cells co-expressing full-length neuronal nitric oxide synthase and NOS1AP. However, using a cell-free biochemical binding assay, ZLc002 failed to disrupt the in vitro binding between His-neuronal nitric oxide synthase and glutathione S-transferase-NOS1AP, protein sequences containing the required binding domains for this protein-protein interaction, suggesting an indirect mode of action in intact cells. ZLc002 (4-10 mg/kg i.p.) suppressed formalin-evoked inflammatory pain in rats and reduced Fos protein-like immunoreactivity in the lumbar spinal dorsal horn. ZLc002 also suppressed mechanical and cold allodynia in a mouse model of paclitaxel-induced neuropathic pain. Anti-allodynic efficacy was sustained for at least four days of once daily repeated dosing. ZLc002 also synergized with paclitaxel when administered in combination to reduce breast (4T1) or ovarian (HeyA8) tumor cell line viability but did not alter tumor cell viability without paclitaxel. Our results verify that ZLc002 disrupts neuronal nitric oxide synthase-NOS1AP interaction in intact cells and demonstrate, for the first time, that systemic administration of a putative small-molecule inhibitor of neuronal nitric oxide synthase-NOS1AP suppresses inflammatory and neuropathic pain.
N-甲基-D-天冬氨酸受体活性升高导致中枢敏化。我们的实验室和其他实验室最近报道，破坏 N-甲基-D-天冬氨酸受体下游的蛋白质-蛋白质相互作用可抑制疼痛。具体而言，破坏酶神经元型一氧化氮合酶与其上游（突触后密度 95 kDa，PSD95）或下游（例如一氧化氮合酶 1 衔接蛋白，NOS1AP）蛋白伴侣之间的结合可抑制炎症性和/或神经性疼痛。然而，缺乏小分子神经元型一氧化氮合酶-NOS1AP 抑制剂阻碍了验证破坏神经元型一氧化氮合酶-NOS1AP 界面作为镇痛策略的治疗效用的努力。因此，我们使用离体、体外和纯化的重组系统评估了假定的小分子神经元型一氧化氮合酶-NOS1AP 抑制剂 ZLc002 破坏神经元型一氧化氮合酶与 NOS1AP 之间结合的能力，并询问 ZLc002 是否会在体内抑制炎症性和神经性疼痛。在体外，ZLc002 减少了培养神经元中和共表达全长神经元型一氧化氮合酶和 NOS1AP 的 HEK293T 细胞中全长 NOS1AP 和神经元型一氧化氮合酶的共免疫沉淀。然而，使用无细胞生化结合测定法，ZLc002 未能破坏体外 His-神经元型一氧化氮合酶与谷胱甘肽 S-转移酶-NOS1AP 之间的结合，该结合需要该蛋白质-蛋白质相互作用的结合域的蛋白质序列，这表明在完整细胞中存在间接作用模式。ZLc002（4-10mg/kg 腹腔内注射）抑制了大鼠福尔马林诱发的炎症性疼痛，并减少了腰椎脊髓背角中的 Fos 蛋白样免疫反应性。ZLc002 还抑制了紫杉醇诱导的神经性疼痛小鼠模型中的机械性和冷性痛觉过敏。每日一次重复给药至少四天，抗痛觉过敏效果持续存在。ZLc002 与紫杉醇联合使用时还具有协同作用，可降低乳腺癌（4T1）或卵巢癌（HeyA8）肿瘤细胞系的活力，但无紫杉醇时不会改变肿瘤细胞活力。我们的结果验证了 ZLc002 破坏了完整细胞中的神经元型一氧化氮合酶-NOS1AP 相互作用，并首次表明系统给予神经元型一氧化氮合酶-NOS1AP 的假定小分子抑制剂可抑制炎症性和神经性疼痛。
