Summary Mechanosensitive Piezo ion channels enable eukaryotic cells to sense mechanical forces. In vertebrates, two Piezo homologs, Piezo1 and Piezo2, play central roles in all major physiological systems and are associated with numerous diseases including hypertension, xerocytosis, lymphedema, arthrogryposis, inflammation, pain, and cancer progression. Hence, pharmacological modulation of specific Piezo homologs could help treat many human ailments. Yet, this effort is currently limited by the paucity of homolog-selective pharmacological agents and the lack of a clear molecular understanding by which Piezo channels open and close their pore in response to physical and chemical stimuli. To bridge this gap, this proposal will leverage a recently identified pharmacological binding site in mammalian Piezo1 channels. Specifically, we will use reverse genetics, high-resolution electrophysiology, and calcium imaging to dissect structural pathways by which the binding of the small molecule Yoda1 energetically promotes an open state (Aim 1). In parallel, we will deploy an innovative, multi-pronged approach combining binding free energy calculations, structure-activity relationships, machine learning-based virtual screening of ultra- large billion-compounds library, and rapid experimental screening assays to identify novel molecules that preferentially bind conducting or non-conducting channel conformations, thus potentially acting as Piezo1 activators and inhibitors, respectively (Aim 2). Successful completion of this project will identify discrete mechanotrasnduction pathways associated with pharmacological activation of Piezo1 channels and identify novel pharmacological activators and inhibitors of Piezo1 channels with potential research and clinical value.