The sarco/endoplasmic reticulum calcium ATPase (SERCA) is the calcium pump responsible for maintaining cellular calcium homeostasis. Diminished SERCA function has been directly linked to numerous degenerative disease states, such as heart failure. The pathological progression of heart failure is associated with an elevated level of cytosolic calcium, and impairs the function of the muscle contraction-cycle. The overarching goal of this research is to discover novel small-molecule effectors, capable of enhancing SERCA’s ability to pump and store calcium within the sarcoplasmic reticulum (SR). Drugs that increase the calcium pumping efficiency of SERCA will restore calcium homeostasis by reducing the calcium content in the cytosol, and enhance impaired cardiac function. The process of drug discovery is a high-risk effort, and involves screening millions of small-molecules to fortuitously discover a lead compound with high-therapeutic potential. The precise placement of two fluorescent proteins at specific locations along SERCA’s cytosolic headpiece, allows for the detection of fluorescence resonance energy transfer (FRET) between donor and acceptor fluorescent proteins. Human cell lines that overexpress this fluorescent fusion protein were generated, creating a live-cell biosensor. The rate of energy transfer (FRET) is dependent on the distance between the fluorescent probes and linked to the enzymatic activity of SERCA. FRET tracks SERCA’s structural status, while it pumps calcium into the sarcoplasmic reticulum. These biosensors are grown in vast quantities, harvested, and utilized for high-throughput drug screening. The cells are dispensed into high-density microplates, where each well contains a different compound. FRET is detected using proprietary fluorescence technology, capable of recording the nanosecond fluorescence decay rate (lifetime) and the full emission spectrum. Both lifetime and spectral modes offer incredibly fast speeds, with high resolution and precision. High-throughput screening by lifetime mode offers the advantage of resolving the structural status of the FRET biosensor because the mole fraction of each structural state is assessed, and candidate compounds found during the screening process can be characterized by their structural effect on the biosensor. High-throughput screening by spectral mode increases assays precision by taking into account the shape of the fluorescence emission spectrum. The shapes of these spectra are decomposed into the contribution of known components by a novel spectral unmixing method, and further used to accurately evaluate FRET. When coupled with lifetime mode, spectral-based drug screening increases assay precision and removes artifacts from cellular autofluorescence and fluorescent compounds. The complementary advantages of coupling spectral and lifetime fluorescence measurements significantly reduces the rate of false-positives from high-throughput drug screens. The development of the technology and FRET biosensor assay, drastically increases the probability of identifying a novel drug with great therapeutic potential.
University of Minnesota Ph.D. dissertation. February 2017. Major: Biochemistry, Molecular Bio, and Biophysics. Advisor: David Thomas. 1 computer file (PDF); ix, 131 pages.
Novel Fluorescence Tools for the Discovery of Cardiac Calcium Pump Therapeutics.
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