Evaluating HMG-CoA reductase as a therapeutic target in ALS

描述(申请人提供)：肌萎缩侧索硬化症(ALS)是一种一致致命的神经退行性疾病，目前仍没有有效的治疗方法。对此的一种解释是，很少有治疗靶点--疾病途径中抑制其带来益处的分子事件--被确定，从而阻碍了合理的翻译方法。一 发现新靶点的方法是高通量体外筛选化学库，以确定可以改善疾病相关表型的化合物。被这些化合物抑制的酶构成了候选的治疗靶点。然而，为了验证它们的进一步发展，有必要证明它们的抑制作用延缓了体内疾病的发生或进展。在ALS疾病过程的早期发生的细胞事件中，有一种是运动终板上的运动轴突物理上的萎缩，直接导致肌肉瘫痪，并逐渐蔓延到全身。在突变的SOD1啮齿动物模型中以及患有散发性或家族性疾病的患者中，可以观察到早期肌肉失神经。防止轴突退化，或刺激再生，预计将推迟疾病的发生或进展。然而，由于潜在的机制尚不清楚，还不可能直接测试这一治疗假说。我们最近筛选了一个约50,000个化合物的文库，以确定在体外抑制环境下促进运动轴突再生的药物。最具冲击力的是他汀类药物，它在浓度低于基准化合物100倍的情况下，将轴突生长促进高达5倍。他汀类药物对轴突生长的影响完全依赖于其已知的靶酶HMG-CoA还原酶(HMGCR；3-羟基-3-甲基-戊二酰辅酶A还原酶)的抑制，HMG-CoA还原酶是胆固醇合成和蛋白质预烯基化途径的限速步骤。我们的数据确定HMGCR是ALS的一个新的候选治疗靶点。在这个为期两年的项目中，我们建议使用两种有效的配体西立伐他汀和辛伐他汀作为探针，在体内验证HMGCR作为靶点。我们的实验将使用分区的运动神经元培养来确定HMGCR抑制是否需要发生在细胞体或轴突末端。然后我们将建立他汀类药物体内给药方案，导致显著抑制运动神经元的HMGCR。 脊髓。最后，我们将确定他汀类药物能否延缓肌萎缩侧索硬化症突变型SOD1小鼠模型的肌肉失神经。总体而言，我们的实验应该允许我们确定HMGCR及其下游通路是否为ALS的有效治疗靶点。他们还应该为促进运动轴突生长可以延缓肌肉功能性去神经的想法提供原则性证据。最后，我们的数据应该会刺激对他汀类药物在运动神经元中调节的特定过程的进一步研究，从而有助于为未来的药物开发识别更具选择性的靶点，如蛋白质预烯基化。

DESCRIPTION (provided by applicant): Amyotrophic lateral sclerosis (ALS) is a uniformly fatal neurodegenerative disease for which there is still no effective treatment. One explanation for this is that very few therapeutic targets - molecular events in the disease pathway whose inhibition confers benefit - have been identified, hindering rational translational approaches. One means of discovering novel targets is high-throughput in vitro screening of chemical libraries to identify compounds that can ameliorate disease-related phenotypes. Enzymes inhibited by such compounds constitute candidate therapeutic targets. However, to validate them for further development, it is necessary to demonstrate that their inhibition delays disease onset or progression in vivo. Among the cellular events that occur early in the ALS disease process is the physical die-back of motor axons from motor endplates, leading directly to muscle paralysis that spreads progressively throughout the body. Early muscle denervation is observed in mutant SOD1 rodent models as well as in patients with either sporadic or familial forms of the disease. Preventing axonal degeneration, or stimulating regrowth, would be predicted to delay disease onset or progression. However, since the underlying mechanisms remain unclear it has not been possible to test this therapeutic hypothesis directly. We recently screened a library of ~50,000 compounds to identify agents that enhance motor axon regeneration in an inhibitory context in vitro. The strongest hits were the statins, which enhanced axonal growth by up to 5- fold at concentrations 100-fold lower than with benchmarking compounds. Statin effects on axonal growth depend entirely on inhibition of their known target enzyme HMG-CoA reductase (HMGCR; 3-hydroxy-3-methyl- glutaryl-CoA reductase), which is the rate-limiting step for cholesterol synthesis and protein prenylation pathways. Our data identify HMGCR as a novel candidate therapeutic target in ALS. In this two-year project, we propose to validate HMGCR as a target in vivo using two potent ligands, cerivastatin and simvastatin, as probes. Our experiments will use compartmentalized motor neuron cultures to determine whether HMGCR inhibition needs to occur in the cell body or in the axon terminal. We will then establish protocol for statin administration in vivo that lead to significant HMGCR inhibition in motor neurons in the spinal cord. Finally, we will determine whether statin administration can delay muscle denervation in the mutant SOD1 mouse model of ALS. Overall, our experiments should allow us to determine whether HMGCR and the pathways downstream of it are valid therapeutic targets in ALS. They should also provide proof of principle for the idea that enhancing motor axon growth can delay functional denervation of muscle. Lastly, our data should stimulate further research into the specific processes modulated by statins in motor neurons, and thereby help identify more selective targets - such as protein prenylation - for future drug development.