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

开发蛋白质棕榈酰化测定来监测 CLN1 Batten 病的治疗

• 批准号: 10259433
• 负责人: Matthew Wolf Foster
• 金额: $ 30.48万
• 依托单位: CIRCUMVENT PHARMACEUTICALS, INC.
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-15 至 2024-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10259433
• 关键词: Adult; Affect; Affinity; Age; Alzheimer' s Disease; Archives; Behavior; Biological Assay; Biology; Blindness; Blood; Blood Cells; Brain; Brain Diseases; CLN1 gene; CLN2 gene; CLN4 gene; Carbon; Cell Line; Cells; Cessation of life; Child; Childhood; Cleaved cell; Clinical; Clinical Trials; Collection; Coupled; Cultured Cells; Development; Differentiated Gene; Disease; Enzymes; Excision; Exhibits; Fatty Acids; Fostering; Future; Genes; Human; Huntington Disease; Hydroxylamine; Impaired cognition; Inheritance Patterns; Inherited; Intervention; Knockout Mice; Label; Licensing; Lipids; Maps; Mass Spectrum Analysis; Measurable; Measures; Membrane; Methods; Modification; Monitor; Motor; Mus; Mutation; Neurodegenerative Disorders; Neurons; Palmitates; Pathology; Patient Monitoring; Patients; Pediatric Hospitals; Peptides; Peripheral Blood Mononuclear Cell; Pharmacologic Substance; Pharmacology; Phase; Phase I/II Clinical Trial; Pilot Projects; Plant Resins; Play; Post-Translational Protein Processing; Pre-Clinical Model; Protein Deficiency; Proteins; Reaction; Recombinants; Reproducibility; Role; Sampling; Schizophrenia; Seizures; Signal Transduction; Site; Small Business Innovation Research Grant; Specificity; Spielmeyer-Vogt Disease; Spottings; Symptoms; Technology; Testing; Therapeutic; United States National Institutes of Health; Universities; Validation; Vegetative States; Whole Blood; assay development; base; clinical examination; clinically relevant; design; differential expression; experimental study; follow-up; genomic locus; infancy; interest; lymphoblast; method development; mimetics; mouse model; palmitoylation; patient response; phase 2 study; protein function; protein transport; response; small molecule; thioesterase PPT1 gene product; treatment response

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.

概括 在以下NIH SBIR第1阶段项目的以下建议概述的具体目的中，我们将使用一个 多方面的方法来识别针对目标开发的关键基材，以监测治疗 CLN1板条疾病（CLN1）。 CLN1是一种超稀有神经退行性疾病，影响儿童 棕榈酰蛋白硫酯酶1（PPT1）的基因中的常染色体隐性突变。 PPT1切割 在大脑中表达的16个碳脂质链，棕榈酸酯和许多蛋白质之间的硫酯酶键。 Burnvent Pharmaceuticals正在开发一个小分子硫酯酶模拟物，Circ825，用于处理 CLN1的含量为CLN1患者目前没有可用的治疗性。 我们提出了一个多路复用的酰基乙烯辅助捕获（ACYL-RAC）的发现测定法与同骨相连 使用串联质量标签（TMT）的肽标记策略介绍了A 使用LC-MS/MS的CLN1 - / - 鼠标模型。 TMT分析将随访针对性的平行反应 监测（PRM）质谱法以定量验证目标。我们将为其亲戚选择蛋白质 在大脑和血液中的表达，以使药物相关的常规血液收集以标准 练习，临床检查。我们将根据CLN1患者血液的评估调整测定法和培养 通过发现Acyl-RAC-PRM-MS的PPT1底物的细胞，以鉴定有针对性测定的候选者 发展。培养细胞将通过Circ825处理，并与未经处理的对照进行评估， 靶向酰基-RAC-PRM-MS。为了基于可重复性，稳定性，允许进一步优化和选择 和方法开发中的行为，我们将确定PPT1的5-10个候选棕榈酰化蛋白底物 在患者衍生的LCL或患者血点样本中差异表达的，这也匹配 我们在小鼠大脑和血液中的发现。 通过未来的NIH SBIR II期项目，最佳基材将用于开发 商业临床测定法可能会扩展到许多显示异常蛋白质的疾病 棕榈酰化。最后，我们计划在CLN1 I/II临床期间为我们的最终测定法执行概念验证 在提供许可测定法之前进行试验。