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

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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.