Our goal is to develop small-molecule drugs for treatment of skeletal muscle disorders related to dysregulation of intracellular calcium, focusing on specific proteins in the sarcoplasmic reticulum (SR). Each Aim starts with the design of fluorescent biosensors (specific SR proteins labeled with fluorescent donor and acceptor), to be used in high-throughput screening (HTS) of small molecules. A key innovation is our recently developed HTS approach based on fluorescence lifetime (FLT) detection of protein structural changes by fluorescence resonance energy transfer (FRET). Our combination of FRET biosensor engineering with unique FLT detection has produced an unprecedented combination of sensitivity, specificity, speed, and precision in protein structure-based studies of mechanism for drug discovery. We previously validated this approach through applications to cardiac muscle. We now focus on skeletal muscle, targeting the two key SR proteins involved in Ca regulation, the Ca release channel (RyR1) and the calcium pump (SERCA1a). Aim 1: Targeting RyR1 leak reduction. Our biosensor is based on FRET between two regulatory proteins (FKBP12.0 and CaM) bound to RyR1. In pilot screens, we have shown that this FLT-based FRET assay can detect small molecules that restore aberrant RyR1 function, in which the Ca channel leaks Ca from the SR into the cytoplasm, inducing myopathies. We will carry out larger-scale screening, to identify new drug candidates, then use cellular and in vivo muscle assays to test the reversal of undesirable calcium leak in fibers and mice. Aim2: Targeting SERCA1a activation. We seek a complementary solution to combat Ca leak – enhancing SERCA1a activity to pump Ca back into the SR lumen. This approach also targets factors (e.g., mutation or oxidation) that impair SERCA1a activity. We will use two complementary approaches, building on our previous studies with SERCA2a (cardiac), with fluorescent biosensors expressed in live cells. (A) We will use an intramolecular FRET biosensor (donor and acceptor attached to different domains of SERCA1a), to screen a small-molecule library to detect compounds that bind to SERCA, alter enzyme structure, and activate Ca transport. (B) We will use an intermolecular biosensor, with donor on SERCA1a and acceptor on the SERCA1a regulator sarcolipin (SLN), to detect compounds that activate the enzyme by uncoupling the inhibitory effects of SLN. We will evaluate potency and efficacy of drug candidates, using assays on myofibers and muscles, both in vitro and in vivo in mouse models including pre-clinical longitudinal drug testing. We have assembled a multi-PI team with complementary expertise and decades of successful collaboration, led by David Thomas (SERCA1a, FLT-FRET), Razvan Cornea (RyR1, biosensor engineering), and Dawn Lowe (skeletal muscle functional analysis). We will also be joined by collaborators with complementary expertise in medicinal chemistry (Aldrich) and myofiber Ca assays (Launikonis)...