Development of a protein palmitoylation assay to monitor treatment of CLN1 Batten Disease

摘要 在NIH SBIR第一阶段项目的以下提案中概述的具体目标中，我们将使用 多方面方法确定用于靶向分析开发的关键底物以监测治疗 CLN1巴顿病(CLN1)。CLN1是一种极其罕见的神经退行性疾病，影响儿童 棕榈酰蛋白硫酯酶1(PPT1)基因常染色体隐性突变。PPT1裂解 16碳脂链棕榈酸酯和许多在大脑中表达的蛋白质之间的硫酯酶键。 Evivent制药公司正在开发一种小分子硫代酯酶模拟物CIRC825，用于治疗 由于目前还没有针对CLN1患者的治疗方法。 我们提出了一种与同量异构体耦合的多路酰基树脂辅助捕获(Acyl-RAC)发现方法 利用串联质量标签(TMT)的多肽标记策略来描述一种新的神经细胞和血液中的棕榈酰基团 使用LC-MS/MS的Cln1/-小鼠模型的TMT分析将进行靶向平行反应 监测(PRM)质谱学，以定量验证目标。我们将为它们的亲缘关系选择蛋白质 在脑和血液中的表达，以实现与药物相关的常规血液采集标准 实习，临床检查。我们将调整我们的检测方法来评估CLN1患者的血液和培养。 用Acyl-RAC-PRM-MS发现PPT1底物的细胞以确定靶向分析的候选细胞 发展。培养的细胞将被CIRC825处理，并与未处理的对照组相比，通过 靶向酰基-RAC-PRM-MS为了允许基于再现性、稳定性、 在方法开发中，我们将确定PPT1的5-10个候选棕榈酰化蛋白底物 它们在患者来源的LCL或同样匹配的患者血斑样本中差异表达 我们在小鼠大脑和血液中的发现。 通过未来的NIH SBIR第二阶段项目，最佳衬底将用于开发一种 可扩展用于多种显示异常蛋白的疾病的商业临床检测方法 棕榈酰化。最后，我们计划在CLN1 I/II期临床期间为我们的最终化验进行概念验证 在提供我们的化验许可之前进行试验。

Summary In the Specific Aims outlined by the following proposal for an NIH SBIR Phase 1 project, we will use a multi-faceted approach to identify key substrates for targeted assay development to monitor therapy in for CLN1 Batten Disease (CLN1). CLN1 is an ultra-rare neurodegenerative disease that affects children with autosomal recessive mutations in the gene for palmitoyl-protein thioesterase 1 (PPT1). PPT1 cleaves the thioesterase bond between a 16-carbon lipid chain, palmitate, and many proteins expressed in the brain. Circumvent Pharmaceuticals is developing a small molecule thioesterase mimetic, CIRC825, for the treatment of CLN1 as there is currently no available therapeutic for CLN1 patients. We propose a multiplexed Acyl-Resin Assisted Capture (Acyl-RAC) discovery assay coupled to an isobaric peptide-labeling strategy using tandem mass tags (TMT) to profile the neuronal and blood palmitoylomes of a Cln1-/- mouse model using LC-MS/MS. TMT assays will be followed up with targeted, parallel reaction monitoring (PRM) mass spectrometry to validate targets quantitatively. We will select proteins for their relative expression in brain and blood to enable pharmaceutically-relevant routine blood collection for standard of practice, clinical examination. We will adapt our assay to the assessment of CLN1 patient blood and cultured cells for PPT1 substrates by discovery Acyl-RAC-PRM-MS to identify candidates for targeted assay development. Cultured cells will be treated with CIRC825 and assessed, compared to untreated controls, by targeted Acyl-RAC-PRM-MS. To allow for further optimization and selection based on reproducibility, stability, and behavior in method development, we will identify 5-10 candidate palmitoylated protein substrates of PPT1 which are differentially expressed in patient-derived LCLs or patient blood spot samples which are also match our findings in mouse brain and blood. Through a future NIH SBIR Phase II project, the optimal substrates will be used in the development of a commercial clinical assay which may be expanded to use in numerous diseases which display aberrant protein palmitoylation. Finally, we plan to perform proof-of-concept for our final assay during CLN1 Phase I/II clinical trials before offering our assay for licensing.