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

总结 在NIH SBIR第1阶段项目的以下提案所概述的具体目标中，我们将使用 多方面的方法来确定靶向测定开发的关键底物，以监测 CLN 1巴滕病（CLN 1）。CLN 1是一种非常罕见的神经退行性疾病， 棕榈酰蛋白硫酯酶1（PPT 1）基因中的常染色体隐性突变。PPT 1切割 硫酯酶是16碳脂链、棕榈酸酯和大脑中表达的许多蛋白质之间的键。 Circumvent制药公司正在开发一种小分子硫酯酶模拟物，CIRC 825，用于治疗 CLN 1，因为目前没有可用于CLN 1患者的治疗方法。 我们提出了一种与同量异位素偶联的多重酰基树脂辅助捕获（Acyl-RAC）发现测定法。 肽标记策略，使用串联质量标签（TMT）来分析神经元和血液棕榈酰组， 使用LC-MS/MS的Cln 1-/-小鼠模型。TMT试验将采用靶向平行反应进行随访 监测（PRM）质谱以定量地验证目标。我们将选择蛋白质， 在脑和血液中的表达，使得能够进行药物相关的常规血液收集，用于标准的 实习，临床检查我们将调整我们的测定以评估CLN 1患者的血液和培养物。 通过discovery Acyl-RAC-PRM-MS鉴定用于靶向测定的候选物 发展将培养的细胞用CIRC 825处理，并通过与未处理的对照相比， 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，我们将鉴定5-10种候选的PPT 1棕榈酰化蛋白底物 其在患者来源的LCL或患者血斑样品中差异表达， 我们在老鼠大脑和血液中的发现 通过未来的NIH SBIR第二阶段项目，最佳底物将用于开发 其可扩展用于显示异常蛋白质的多种疾病 棕榈酰化最后，我们计划在CLN 1 I/II期临床试验期间对我们的最终检测进行概念验证。 在提供我们的检测方法以获得许可之前，

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.