Abstract The appearance of SARS-CoV-2 in early 2020 has spurred efforts to limit the disease spread and develop effective treatments. The most promising long-term approach is a vaccine. While some vaccines are entering accelerated clinical trials, it may take 12 or more months before an effective vaccine is available. Even if successful, it may not be possible to treat everyone with a vaccine or the effectiveness of the vaccine may be limited. Given the severity of the disease among a number of those patients, alternative approaches to limit infection should be developed. The goal of this proposal is to use human cardiac, vascular, and lung alveolar microphysiological systems (MPS) to identify possible compounds that block SARS-CoV-2 entry into cells and tissues. While cell binding assays can be used to screen drug candidates, human MPS offer the advantage of testing promising drug candidates under conditions encountered in the body. We propose a tiered approach in which primary cells and cells overexpressing angiotensin converting enzyme (ACE2) are used to identify promising candidates that block SARS-CoV-2 virus entry into cells, and vascular, cardiac, and lung alveolar MPS are used to provide a robust evaluation of drugs that block SARS-CoV-2 binding. The first tier with individual cell types enables a rapid screen and the screen with the microphysiological systems enables testing of the most promising candidates with the tissues most likely to be infected. We will develop the screening assays using a pseudovirus with the SARS-CoV-2 spike protein. In Aim 1, we will develop an assay for pseudovirus binding to ACE2 expressing cells by verifying binding and fusion to cells that express ACE2. We will whether the binding specifically involves the spike protein and determine the levels of binding sites on the cell types used in subsequent aims. In Aim 2, we will screen individual cells types for molecules that block entry into the cell of pseudovirus expressing the spike proteins. Potential drug candidates include those that potentially block spike protein binding (e.g. spike proteins, Captopril, Lisinopril, human recombinant soluble ACE2, and antibodies to the spike protein or ACE2) and those inhibiting Transmembrane Serine Protease 2 (TMPRSS2), activity (e.g. camostat mesylate, nafamostat mesylate). In Aim 3, we will test most promising compounds in vascular, cardiac and lung microphysiological systems and compare against results from 2D studies. We will also examine the relationship between drug blocking and factors that affect ACE2 expression.