PROJECT SUMMARY/ABSTRACT The overarching goal of our proposed research is to determine the mechanisms underlying small-molecule activation of the cardiac calcium pump (sarcoplasmic reticulum Ca2+-ATPase, SERCA), and the discovery of new classes of small-molecule inhibitors of the cardiac sodium pump (Na+/K+-ATPase, NKA). SERCA and NKA play a key role in the excitation-contraction-relaxation cycle in normal and pathological cardiac muscle and are validated pharmacological targets for heart failure (HF) therapies. Therefore, drug discovery and development targeting these pumps unlock exciting new opportunities for developing HF therapies that are directed at the heart. However, challenges limit drug discovery efforts targeting these ion pumps: (1) Despite the tremendous advances in crystallography and cryo-EM, there are no structures of SERCA bound to small-molecule activators, so the structural mechanisms for small-molecule activation of SERCA are still unclear; (2) Existing sodium pump modulators are primarily cardiotonic steroids that have a narrow therapeutic window and pro-arrhythmia cardiotoxic effects at therapeutically relevant doses. To overcome these challenges, we will use experimental and computational approaches to determine the structural mechanisms for small-molecule activation of SERCA and to discover new non-cardiotoxic, lead-like inhibitors of NKA. The proposal is significant because it will produce new mechanistic insights and small-molecule effectors targeting two major clinically relevant targets in the heart. The research is translational because it will expedite the discovery of lead molecules targeting cardiac pumps. We will pursue the following independent aims: (1) Determine the mechanisms and interactions for small-molecule SERCA activators, and (2) Discover novel non-cardiotoxic NKA inhibitors for congestive heart failure. We have performed preliminary studies that show the feasibility of the proposed research, including fresh evidence showing that small-molecule SERCA activation involves both a structural and a kinetic mechanism, and the discovery of a novel potent NKA inhibitor with a safe pro-arrhythmia cardiac toxicity profile.