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

总结 本行政补充申请（PA-20-272）用于购买单件设备 支持NIA资助的研究活动，“线粒体疾病的一流肽治疗： 分子作用机制和设计优化”（R 01 AG 065879）。正在寻找的工具是一个 新的研究级分光荧光计（Fluorolog-QM-75-22-C，HORIBA Scientific）。这将取代 我实验室现有的仪器（Fluorolog 3-22）在9月份发生了灾难性的故障 2022年（R 01项目期的第三年），使仪器无法运行。目前的文书 是2008年我在康涅狄格大学建立实验室时买的。虽然 由于Fluorolog 3-22始终产生高质量和可重复的数据，近年来，Fluorolog 3-22需要主要的 维修频率越来越高，几乎每年一次。每一次大修都要花费数千美元 并导致在维修仪器部件时延长停机时间（通常为几周）， 该公司因此，就财政负担和生产力损失而言， 已经高到无法管理了此外，HORIBA现已停产Fluorolog 3-22型号 和替换零件（电子、光学等）正在逐步淘汰，这意味着关键仪器的维修 组件变得不可能。最近的仪器故障发生在这个月， 外壳电源故障。因此，唯一的选择是购买替代工具。 分析荧光光谱是我的研究计划的重要组成部分，是所有 R 01 AG 065879项下赠款活动的目标。这些基于荧光的活动包括：（i）分析荧光素酶的活性。 肽和其他小分子与模型膜和线粒体的平衡结合性质; (ii)验证，分析和质量控制的新的，合理设计的靶向化合物， 用于线粒体老化的治疗剂;（iii）用于治疗化合物对膜的作用的测定 （iv）分析线粒体功能和生理完整性，包括膜 电位、钙通量和活性氧产生;以及（v）评估线粒体的作用 药物对蛋白质输入和线粒体膜上淀粉样纤维形成的影响。这些测量需要 多个独立的荧光技术（精确的发射和激发光谱扫描，各向异性 读数，福斯特共振能量转移，静态和动态淬火），他们是使用复杂的 包含模型膜、分离的细胞器和细胞的样品。因此，这项工作需要一个顶级的质量 这是一种可以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，委员会注意到， Fluorolog-QM-75-22-C将是稳态读数的优秀替代仪器。此外，委员会认为， 新Fluorolog中增加了时间相关单光子计数（TCSPC）模块插件 模型，将提供荧光寿命测量能力，将加强和加速这项研究。

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.