Project Summary/Abstract Our long-term goals are to understand intracellular active Ca transport in cardiac muscle and to develop drugs that target Ca dysregulation in heart failure. We focus on the cardiac sarcoplasmic reticulum (SR) Ca- ATPase (SERCA2a), the large membrane enzyme that pumps Ca into the SR to relax myocytes after contraction, and on SERCA’s regulatory proteins (regulins, RLN): phospholamban (PLB), sarcolipin (SLN) and dwarf open reading frame peptide (DWORF). Ample experimental and clinical data indicates that activation of muscle Ca transport is a powerful approach to numerous severe and widely spread disorders, especially heart disease. Over decades, we have developed and used spectroscopic techniques to resolve the structural mechanism of SERCA regulation by RLN. The challenge is great due to the complexity of the Ca transport mechanism, which involves dynamic protein-protein interactions, and RLN mechanisms of action are unclear or controversial. We have recently demonstrated fluorescence lifetime (FLT) methods with exquisite precision and resolution of protein structural changes, with high-throughput acquisition enabled by rapid scanning in a microplate reader. In screens of chemical libraries of ≤50,000 small-molecules, we have identified multiple types of SERCA activators. We are poised to implement early-stage drug discovery campaigns targeting the SERCA-RLN interaction, to identify compounds with therapeutic potential to treat Ca dysregulation in heart failure. Our central hypotheses: (1) compounds that shift SERCA-RLN structural state or binding have tissue-specific effects (2) compounds that enhance Ca-transport enhance myocyte contraction, and (3) have antiarrhythmic properties. Aim 1 will accelerate discovery of compounds that target SERCA-RLN interaction. Aim 2 will test effects on SERCA function in cardiac SR, HEK cells expressing human SERCA2a, and in cardiomyocytes. These novel chemical probes will open new avenues to elucidate structure-function mechanisms characteristic of SERCA-RLN. Aim 3 will enable lead discovery, using medicinal chemistry to develop and test analogues of promising Hits from Aim 2. Outcomes of this program will be new lead-like compounds, and a demonstrated systematic process targeting cardiac Ca-transport for drug discovery and development. This process will be ready for implementation in large-scale discovery and development campaigns to be pursued in future academic-industrial partnerships. For impact, we bring together an innovative combination of techniques, technologies, and experts focused on cardiac Ca transport regulation. This project is designed to enable future translation, while also developing tools for mechanistic understanding. SERCA has emerged as a high-value therapeutic target for some of the greatest Public Health challenges, not only in the heart (heart failure, arrhythmia) but also in skeletal muscle (muscular dystrophy, sarcopenia) and non-muscle cells (Alzheimer’s, d...