Apolipoprotein-B (ApoB) is both a biomarker and a causal mediator of many central hallmarks of metabolic disease, including insulin resistance, fatty liver disease, atherogenesis, endoplasmic reticulum stress, and chronic inflammation. ApoB therefore serves as a useful phenotypic readout for the identification of compounds that engender diverse metabolic benefits. The present proposal will perform a high-throughput screen (HTS) to identify novel ApoB-lowering compounds using an automated robotics platform that enables screening to take place in live larval zebrafish using a genetically encoded chemiluminescent reporter to sensitively detect ApoB levels in individual fish. To accomplish this effort, we have brought together a team of scientists all located at Johns Hopkins University and leaders in their respective fields. Farber has established the zebrafish as a model for studies of vertebrate lipid metabolism, Mumm has created a powerful HTS zebrafish screening platform, Ahima is a world leader in mammalian energy metabolism and Lectka is an established chemist bringing significant expertise in screen hit prioritization to the effort. The HTS will take place in two iterations, with the first iteration screening a ~3,000 compound library of clinically approved compounds so that hits can be rapidly repurposed to treat a host of disease associated with Apob perturbations. The second iteration screening is much larger effort to maximize compound diversity (30,000 compounds) and discover potentially entirely new avenues for treatment. Hits from the both screens will be subjected to a high-content secondary screen that uses an automated imaging platform to monitor effects on disease progression using a panel of transgenic zebrafish carrying fluorescent reporters of several important metabolic disease risk factors. This secondary screen will efficiently classify and prioritize hits from the primary screen and identify the subset of compounds with validated metabolic benefits in live vertebrate organisms that justify further investigation and therapeutic development. Promising compounds from primary and secondary screening will also be validated for activity in mammalian models, including mouse and human cultured cells. The results of these efforts will be the first ever whole animal HTS for ApoB modifiers coupled with a high-content secondary screen that together will enable the rapid identification of compounds to ameliorate many metabolic disease phenotypes, as well as a collection of hits for the development of novel therapies to combat the growing global burden of metabolic disease.