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（由申请方提供）：SMN 2基因产生的低水平SMN蛋白是脊髓性肌萎缩症（SMA）的原因，SMA是一种运动神经元变性的遗传性疾病。我们成功地实现了高通量的化学多样性筛选的化合物，增加这种蛋白质的细胞水平。在一个合成化学项目之后，我们鉴定了两种独特的化学支架，称为LDN-75和LDN-76，它们可以增加细胞培养物中SMN的表达。在重度SMA小鼠模型中进行的关键验证研究表明，这些化合物可增加脑中的SMN蛋白、平均生存期和粗大运动功能。这项拨款提案旨在确定这些积极的临床前主要候选人的作用机制。 我们的初步表征表明，这两个系列的化合物通过不同的机制。定量实时PCR表明，LDN-76系列化合物激活转录，其中包含SMN 2外显子7的比例增加。虽然LDN-75系列对SMN 2 mRNA水平或加工没有可检测到的影响，但这些化合物可稳定SMN蛋白。与这些结果一致，我们发现这两个系列的化合物显示SMN蛋白的协同增加，进一步证明这些化合物通过不同的机制发挥作用。目的1将确定LDN-75化合物是否改变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）激活刺激SMN纳入一个稳定的复合物。我们将探索LDN-75调节SMN磷酸化并增加其稳定性的可能性。在目标2中，我们将研究PI 3 K/AKT/Wnt信号通路作为靶点的作用。 对于LDN-76。我们假设SMN的转录被β-连环蛋白途径修饰，并且这是基于LDN-76的化合物的下游靶标。目的3描述了一种基于亲和力的方法来捕获我们的先导化合物的蛋白质靶标。将进行质量测序以识别靶标。遗传和生化实验将用于验证影响SMN表达和与先导化合物相互作用的候选因素。 目前尚无FDA批准的SMA治疗方法。虽然有几种新的治疗设计正在开发中，但没有发表的数据表明任何一种治疗设计都是临床有效的，或者没有令人望而却步的副作用。R21中产生的结果将为我们的活性先导化合物的潜在安全性和毒性问题提供宝贵的见解，并可能为我们的先导化合物的化学精制提供见解。