抗癌药物舒尼替尼的抗帕金森神经保护作用及其分子靶标研究

Parkinson's disease (PD) is the second most common neurodegenerative disorder and become the third leading cause of death among the elderly worldwide. Although the molecular mechanisms of PD are not fully elucidated, the progressive loss of dopaminergic neurons caused by multiple genetic/environmental factors, including the mutant of leucine rich repeat kinase-2 (LRRK-2), has been proposed to be the fundamental pathogenesis of PD. Moreover, the over-activation of nitric oxide synthase (NOS) is also implicated in the pathogenesis of this disease. Currently used medications for PD could only offer moderate symptomatic benefits without obvious disease-modifying potential, partially because their primary target is dopamine deficiency, but not dopaminergic neuronal loss. Therefore, compounds with neuroprotection may offer therapeutic benefits with disease-modifying potential for PD. Compared with de novo development from new compound; the investigation of known drugs for novel diseases possesses many advantages, such as the better safety and the lower cost. Sunitinib is a novel multiple receptor tyrosine kinases inhibitor approved by FDA for treating both renal cell carcinoma and gastrointestinal stromal tumor. In our preliminary study, we have unexpectedly found that sunitinib substantially prevented apoptosis in MPP+ and 6-OHDA-pretreated primary cultured neurons. Moreover, sunitinib analogs were found to protect against MPTP/ MPP+-induced dopaminergic neuronal loss via directly inhibiting neuronal NOS (nNOS), possibly via the interaction between their 3-substitute-2-indolin structure and heme group inside nNOS. Additionally, sunitinib was also reported to inhibit leucine rich repeat kinase-2 (LRRK-2), a genetic factor implicated in the early onset of PD. Based on these novel findings, we hypothesized that sunitinib could be a promising drug for the neuroprotective therapy of PD via concurrently acting on nNOS and LRRK-2.. To test this hypothesis, we will first determine the affinity and the inhibition pattern of nNOS by sunitinib using in vitro assay of NOS activity. We will further investigate the molecular mechanisms, particularly those associated with nNOS and LRRK-2 inhibition, underlying the neuroprotective effects of sunitinib in various PD models in vitro. Finally, we will confirm therapeutic effects of sunitinib on C57BL6/J mice insulted with MPTP. Our study will provide not only new knowledge regarding sunitinib for the treatment of PD, but also insights into the mechanisms of and potential therapeutic strategies for this disorder. Ultimately, a novel and promising anti-PD drug might be expected from our study.

帕金森病是常见神经退行性疾病，是威胁老年人健康第三大杀手。现有治疗仅能缓解症状（即"对症"）， 治疗该病的首要任务是寻找具神经保护可改善病程"对因"药物。我们在寻找这类药物过程中，首次发现治疗肾细胞癌和胃肠间质瘤的临床抗癌药物舒尼替尼有显著的抗帕金森神经保护功能（已申请专利），提示该药可成为抗帕金森神经保护药物。一药多用有安全性好、研发周期短、成本低等优势。我们的前期创新性工作发现舒尼替尼类似物Semaxanib、SU4312可抑制帕金森病关键致病因子nNOS和LRRK-2，提示舒尼替尼也可能通过调控这些靶标发挥神经保护作用。本课题拟首先确证舒尼替尼与nNOS、LRRK-2的相互作用；进一步研究该相互作用介导舒尼替尼产生神经保护的机制；最后在帕金森病小鼠模型评价该药疗效。研究结果将阐明舒尼替尼产生神经保护的靶标和具体机制，为舒尼替尼发展成抗帕金森药物奠定基础，为抗帕金森药物研发提供新方向。

项目组圆满完成各项任务及指标。其中，课题研究期间在英国药理学杂志等国际期刊共发表本基金资助SCI论文9篇，获得中国专利授权1项。. 我们研究发现：舒尼替尼（sunitinib）通过直接抑制神经元型一氧化氮合酶（nNOS）酶活、降低nNOS表达、抑制乙酰胆碱酯酶（AChE）、抑制富亮氨酸重复激酶2（LRRK-2）等方式，产生神经保护；这一结果验证了我们提出的假说，即舒尼替尼可通过作用于nNOS、LRRK-2等，协同产生抗帕金森氏症（PD）的神经保护作用。我们还发现舒尼替尼可通过抑制成纤维细胞生长因子受体1（FGFR1）在细胞、动物模型中产生心脏毒性，可能影响其作为神经保护药物的应用。因此，在进一步的研究中，我们着重关注SU4312、SU5416、靛玉红-3-肟（I3O）等舒尼替尼类似物，希望从中找到有神经保护活性、无心脏毒性等副作用的抗PD先导化合物。. 我们的进一步研究发现：1）SU4312、SU5416通过作用nNOS、LRRK-2等方式产生神经保护活性，对抗1-甲基-4-苯基吡啶离子（MPP+）/1-甲基-4-苯基吡啶（MPTP）在细胞模型、斑马鱼模型产生的神经毒性；2）SU4312更可通过抑制单胺氧化酶-B（MAO-B），逆转MPTP诱导的大鼠运动障碍；3）I3O可通过增强肌细胞增强因子2（MEF2）转录活性方式对抗6羟基多巴胺（6OHDA）在PC12细胞产生的细胞毒性。这些结果有力地证明了舒尼替尼及其类似物可能通过多靶点作用产生抗PD神经保护作用，对其进一步研究有望获得一类结构全新、靶点明确、机制清晰的抗PD先导化合物。