Toward drug treatment of spinal muscular atrophy: Mechanism of action

脊髓性肌萎缩症的药物治疗：作用机制

• 批准号: 8823350
• 负责人: ELLIOT J. ANDROPHY
• 金额: $ 26.07万
• 依托单位: INDIANA UNIV-PURDUE UNIV AT INDIANAPOLIS
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-08-15 至 2016-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8823350
• 关键词: 5q13; Adverse effects; Affinity; Alternative Splicing; Amino Acids; Applications Grants; Binding; Binding Proteins; Biochemical; Biological Assay; Brain; Breeding; Cell Culture Techniques; Cells; Chemicals; Chromosomes; Code; Complex; Cyclic AMP-Dependent Protein Kinases; Data; Databases; Defect; Development; Disease; Drug Interactions; Embryo; Exhibits; Exons; FDA approved; Genetic; Genetic Transcription; Glycogen Synthase Kinase 3; Grant; Half-Life; Human; In Vitro; Inherited; Investigation; Lead; Length; Ligands; Longevity; Maps; Mediating; Medicine; Messenger RNA; Modeling; Motor; Motor Neurons; Mus; Muscle; Muscle Weakness; Mutate; Neuromuscular Diseases; PI3K/AKT; PTEN gene; Pathway interactions; Pharmaceutical Chemistry; Pharmaceutical Preparations; Phosphorylation; Process; Production; Proteins; Proteomics; Publishing; RNA Interference; Regulation; Reporter; Reporting; Role; SMN protein (spinal muscular atrophy); SMN1 gene; SMN2 gene; Safety; Series; Severity of illness; Signal Pathway; Signal Transduction; Small Nuclear Ribonucleoproteins; Solid; Spinal Muscular Atrophy; Synthesis Chemistry; System; Therapeutic; Time; Toxic effect; Transcript; Transgenic Animals; Ubiquitin; Ubiquitination; base; beta catenin; clinically significant; combinatorial; design; exon skipping; functional loss; improved; infancy; inhibitor/antagonist; insight; motor neuron degeneration; mouse model; multicatalytic endopeptidase complex; novel; novel therapeutics; pre-clinical; programs; protein degradation; public health relevance; research study; scaffold; screening; success; survival motor neuron gene; validation studies

 DESCRIPTION (provided by applicant): Low levels of the SMN protein produced from the SMN2 gene are the cause of spinal muscular atrophy (SMA), an inherited disease of motor neuron degeneration. We successfully implemented a high throughput chemical diversity screen for compounds that increase cellular levels of this protein. Following a synthetic chemistry program, we identified two unique chemical scaffolds called LDN-75 and LDN-76 that increase SMN expression in cell culture. Critical validation studies in a severe murine model of SMA demonstrated these compounds increase SMN protein in brain, mean survival, and gross motor function. This grant proposal seeks to determine the mechanism of action of these active pre-clinical lead candidates. Our initial characterization reveals that the two series of compounds act through distinct mechanisms. Quantitative real-time PCR demonstrates that compounds from the LDN-76 series activate transcription with a proportional increase in inclusion of SMN2 exon 7. While the LDN-75 series had no detectable effect on SMN2 mRNA levels or processing, these compounds stabilize the SMN protein. Consistent with these results, we find that compounds from the two series show synergistic increases in SMN protein, further evidence that these act through distinct mechanisms. Aim 1 will determine whether LDN-75 compounds alter the ubiquitination or proteasome mediated destruction of SMN protein. It has been reported that protein kinase A (PKA) activation stimulated SMN incorporation into a stable complex. We will explore the possibility that LDN-75 regulates phosphorylation of SMN and increases its stability. In Aim 2, we will investigate the role of the PI3K/AKT/Wnt signaling pathway as a target for LDN-76. We hypothesize that transcription of SMN is modified by the beta- catenin pathway and that this is the downstream target for the LDN-76 based compounds. Aim 3 describes an affinity based approach to capture protein targets of our lead compounds. Mass sequencing will be performed for target identification. Genetic and biochemical experiments will be used to validate the candidate factors for effects on SMN expression and interaction with the lead compounds. There is no FDA approved treatment for SMA. While several new therapeutic designs are in development, there are no published data that any are clinically effective or will be absent of prohibitive side effects. The results generated in this R21 will supply invaluable insights into potential safety and toxicity concerns for our active lead compounds and potentially provide insights into chemical refinement of our leads.

 描述（由适用提供）：由SMN2基因产生的低水平的SMN蛋白是脊柱肌肉萎缩（SMA）的原因，这是一种运动神经元变性的遗传疾病。我们成功实施了高吞吐性化学多样性筛选，以增加该蛋白质的细胞水平。遵循合成化学计划，我们确定了两个称为LDN-75和LDN-76的独特化学支架，它们在细胞培养中增加了SMN表达。严重的SMA鼠模型中的批判性验证研究表明，这些化合物会增加大脑中的SMN蛋白，平均存活和总体运动功能。该赠款提案旨在确定这些活跃的临床前铅候选人的作用机理。我们的最初表征表明，这两个系列化合物通过不同的机制起作用。定量实时PCR表明，从LDN-76系列中的化合物激活转录，并成比例地增加了SMN2外显子7。尽管LDN-75系列对SMN2 mRNA水平或处理没有可检测到的效果，这些化合物使SMN蛋白稳定。与这些结果一致，我们发现两个系列的化合物表明SMN蛋白的协同增加，进一步证明了这些蛋白通过不同的机制作用。 AIM 1将确定LDN-75化合物是否改变了SMN蛋白的泛素化或蛋白酶体介导的破坏。据报道，蛋白激酶A（PKA）的激活刺激了SMN工业成稳定的复合物。我们将探索LDN-75调节SMN磷酸化并提高其稳定性的可能性。在AIM 2中，我们将研究PI3K/AKT/WNT信号通路作为目标的作用 对于LDN-76。我们假设SMN的转录是通过β-蛋白蛋白途径修饰的，这是基于LDN-76化合物的下游靶标。 AIM 3描述了一种基于亲和力的方法来捕获我们铅化合物的蛋白质靶标。将进行质量测序以进行目标识别。遗传和生化实验将用于验证候选因素对SMN表达和与铅化合物相互作用的影响。没有FDA批准的SMA治疗方法。尽管正在开发几种新的治疗设计，但尚无公开的数据，即任何临床上有效或将不存在禁止的副作用。 R21中产生的结果将为我们的活跃铅化合物提供潜在的安全性和毒性问题提供宝贵的见解，并有可能提供对铅化学细化的见解。