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First-in-class peptide therapeutics for mitochondrial disorders: molecular mechanism of action and optimization of design

线粒体疾病的一流肽疗法：分子作用机制和设计优化

基本信息

批准号：
10727483
负责人：
NATHAN N ALDER
金额：
$ 8.75万
依托单位：
UNIVERSITY OF CONNECTICUT STORRS
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-09-30 至 2025-04-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10727483
关键词：
Acceleration Administrative Supplement Aging Anisotropy Binding Biological Assay Biological Availability Cells Chronic Complex Connecticut Coupled Data Disease Electronics Equilibrium Equipment Evaluation Failure Financial Hardship Fluorescence Fluorescence Resonance Energy Transfer Fluorescence Spectroscopy Frequencies Grant Health Heritability Housing Laboratories Libraries Measurement Membrane Membrane Potentials Mitochondria Mitochondrial Diseases Modeling Molecular Molecular Mechanisms of Action Optics Organelles Pathology Peptides Pharmaceutical Preparations Phase Photons Physiological Power Sources Production Property Protein Import Quality Control Reactive Oxygen Species Reproducibility Research Research Activity Research Support Resolution Sampling Scanning Services Techniques Therapeutic Therapeutic Agents Therapeutic Effect Time Universities Validation Variant Work Xenon amyloid fibril formation cost design experience instrument membrane model mitochondrial dysfunction mitochondrial membrane next generation novel peptide drug physical property productivity loss programs rational design release of sequestered calcium ion into cytoplasm repaired small molecule

项目摘要

SUMMARY This administrative supplement application (PA-20-272) is for the purchase of a single piece of equipment to support the research activities of the NIA grant, “First-in-class peptide therapeutics for mitochondrial disorders: molecular mechanism of action and optimization of design” (R01AG065879). The instrument being sought is a new research grade spectrofluorometer (Fluorolog-QM-75-22-C, HORIBA Scientific). This will replace the existing instrument in my laboratory (Fluorolog 3-22), which experienced a catastrophic failure in September 2022 (in the third year of this R01 project period), rendering the instrument inoperable. This current instrument was purchased in 2008 when I was establishing my laboratory at the University of Connecticut. Although it has consistently yielded high-quality and reproducible data, in recent years the Fluorolog 3-22 has needed major repairs with increasing frequency, on a nearly once-yearly basis. Each major repair costs thousands of dollars and results in extended downtime (typically several weeks) while instrument components are being serviced at the company. Hence, the cost of maintaining this instrument, in terms of financial burden and productivity loss, has become too high to manage. Furthermore, the Fluorolog 3-22 model has now been discontinued by HORIBA and replacement parts (electronics, optics, etc.) are being phased out, meaning that servicing of key instrument components is becoming impossible. The recent instrument failure occurred this month when the xenon arc lamp housing power supply failed. Hence, the only option is to acquire a replacement instrument. Analytical fluorescence spectroscopy is an essential part of my research program and is central to all aims of the grant activities under R01AG065879. These fluorescence-based activities include: (i) analysis of the equilibrium binding properties of peptides and other small molecules with model membranes and mitochondria; (ii) validation, analysis and quality control of novel, rationally designed mitochondria-targeted compounds as therapeutic agents for mitochondrial aging; (iii) assays for the effects of therapeutic compounds on membrane physical properties; (iv) analysis of mitochondrial function and physiological integrity, including membrane potential, calcium flux and reactive oxygen species production; and (v) evaluation of the effects of mitochondrial drugs on protein import and amyloid fibril formation on mitochondrial membranes. These measurements require multiple independent fluorescence techniques (precise emission and excitation spectral scans, anisotropy readings, Forster resonance energy transfer, static and dynamic quenching) and they are done using complex samples that contain model membranes, isolated organelles, and cells. This work therefore requires a top quality spectrofluorometer that can render high-resolution data from samples with complex backgrounds. In this regard, the Fluorolog-QM-75-22-C will be an excellent replacement instrument for steady-state readings. Furthermore, the addition of a Time-Correlated Single Photon Counting (TCSPC) modular add-on, available in new Fluorolog models, will provide fluorescence lifetime measurement capability that will enhance and accelerate this research.

摘要本行政补充申请(PA-20-272)用于购买单一设备为支持NIA基金的研究活动，“一流的多肽疗法治疗线粒体疾病：分子作用机理及优化设计“(R01AG065879)。正在寻找的工具是一种新的研究级荧光分光光度计(Fluorolog-QM-75-22-C，HORIBA Science)。这将取代我实验室现有的仪器(Fluorolog 3-22)，它在9月经历了一次灾难性的故障 2022年(在本R01项目期的第三年)，使仪器无法操作。这台当前的仪器是2008年购买的，当时我正在康涅狄格大学建立我的实验室。尽管它已经始终如一地产生高质量和可重复性的数据，近年来，Fluorolog 3-22需要主要维修频率越来越高，几乎每年一次。每次大修都要花费数千美元。并导致在维修仪器组件时延长停机时间(通常为几周那家公司。因此，就财政负担和生产力损失而言，维护这一工具的成本，已经变得太高，无法管理。此外，Fuorolog 3-22型号现已由HORIBA停产和更换部件(电子、光学等)正在逐步淘汰，这意味着关键仪器的维修组件正在变得不可能。最近的仪器故障发生在本月，当时氙弧灯住房供电出现故障。因此，唯一的选择是购买一台替代仪器。分析荧光光谱学是我的研究计划的重要组成部分，对所有 R01AG065879项下赠款活动的目标。这些基于荧光的活动包括：(I)分析多肽和其他小分子与模型膜和线粒体的平衡结合特性； (2)新的、合理设计的线粒体靶向化合物的确认、分析和质量控制线粒体老化的治疗剂；(Iii)治疗化合物对膜的影响的测定物理特性；(4)分析线粒体功能和生理完整性，包括细胞膜潜力、钙通量和活性氧的产生；以及(V)评估线粒体的作用关于蛋白质输入和线粒体膜上淀粉样原纤维形成的药物。这些测量需要多种独立荧光技术(精确发射和激发光谱扫描、各向异性读数，福斯特共振能量转移，静态和动态猝灭)，它们是使用络合物完成的包含模型膜、分离细胞器和细胞的样本。因此，这项工作需要一流的质量可从具有复杂背景的样品中呈现高分辨率数据的荧光分光光度计。在这方面， FLORLOG-QM-75-22-C将是稳定状态读数的极佳替代仪器。此外，增加了时间相关单光子计数(TCSPC)模块附加模块，可在新的荧光记录仪中使用模型，将提供荧光寿命测量能力，将加强和加快这项研究。