Neuroprotective properties of c-Rafi inhibitors

描述（由申请人提供）：神经系统疾病破坏了患者的生活质量，给家庭护理人员带来了巨大的负担，每年给社会造成数十亿美元的损失。神经系统疾病的一个共同特征是通过细胞凋亡引起的神经元变性。因此，抑制神经元凋亡的药物可能是神经退行性疾病治疗干预的候选药物。此外，确定这些神经保护药物的分子靶点并了解其作用中所利用的信号转导途径将导致开发更有效的治疗策略。通过使用大鼠小脑颗粒神经元的神经元凋亡的细胞培养范例，我们已经鉴定出完全抑制神经元凋亡的药物GW 5074 {5-碘-3-[（3，5-二溴-4-羟基苯基）亚甲基]-2-吲哚酮}。GW 5074在体外测试时是c-Raf的特异性和有效的抑制剂。然而，奇怪的是，用GW 5074处理培养的神经元导致c-Raf上活化修饰的积累。此外，GW 5074处理刺激B-Raf活性。在用GW 5074处理的神经元中，c-Raf下游受影响的分子是抗凋亡分子NF-κ B。GW 5074还抑制促凋亡转录因子c-jun，虽然GW 5074是最有效的，但其他两种c-Raf的化学抑制剂也观察到神经保护作用。c-Raf的小分子抑制剂作为神经保护剂的效用先前尚未描述。本提案的总体目标是使用GW 5074来了解c-Raf抑制剂发挥其抗凋亡作用的分子机制，并更彻底地研究GW 5074作为神经治疗剂的潜力。我们的具体目标是：（1）更好地了解GW 5074对c-Raf的影响，B-Raf了解GW 5074对c-Raf的影响，（2）分析GW 5074发挥神经保护作用的下游机制，（3）检查是否也在神经退行性疾病动物模型中观察到GW 5074对培养的小脑颗粒神经元的分子效应。我们希望GW 5074（或其他类似的c-Raf抑制剂）将成为一种高效和通用的治疗剂，可以在不久的将来进行临床试验，用于治疗神经系统疾病。

DESCRIPTION (provided by applicant): Neurological diseases disrupt the quality of the lives of patients, puts a tremendous burden on family caregivers, and cost society billions of dollars annually. A common feature of neurological diseases is the degeneration of neurons by apoptosis. Drugs that inhibit neuronal apoptosis could thus be candidates for therapeutic intervention in neurodegenerative disorders. Moreover, identifying the molecular targets of such neuroprotective drugs and understanding the signal transduction pathways that are utilized in their action would lead to the development of more effective therapeutic strategies. Working with a cell culture paradigm of neuronal apoptosis that uses rat cerebellar granule neurons we have identified a drug, GW5074 {5-Iodo-3-[(3,5-dibromo-4-hydroxyphenyl)methylene]-2-indolinone} that completely inhibits neuronal apoptosis. GW5074 is a specific and potent inhibitor of c-Raf when tested in vitro. Paradoxically, however, treatment of cultured neurons with GW5074 leads to the accumulation of activating modifications on c-Raf. Moreover, GW5074 treatment stimulates B-Raf activity. Among the molecules affected downstream of c-Raf in neurons treated with GW5074 are the antiapoptotic molecule NF-kappa b. GW5074 also inhibits the proapoptotic transcription factor, c-jun. Although GW5074 is the most effective, neuroprotection is also observed by two other chemical inhibitors of c-Raf. The utility of small molecule inhibitors of c-Raf as neuroprotective agents has not been described previously. The overall goal of this proposal is to use GW5074 to understand the molecular mechanism by which inhibitors of c-Raf exert their antiapoptotic effect and to more thoroughly investigate the potential of GW5074 as a neurotherapeutic agent. Our specific goals are to: (1) better understand the effect of GW5074 on c-Raf and B-Raf understand the effect of GW5074 on c-Raf, (2) analyze the downstream mechanisms by which GW5074 exerts its neuroprotective effect, and (3) examine whether the molecular effects of GW5074 in cultured cerebellar granule neurons are also observed in an animal model of neurodegeneration. It is our hope that GW5074 (or other c-Raf inhibitors like it) will emerge as a highly effective and versatile therapeutic agent that could proceed towards clinical trials for the treatment of neurological diseases in the near future.