This project aims to develop synthetic multifunctional compounds as therapeutics for polycystic kidney disease (PKD). In collaboration with the Somlo group at Yale University, we have already shown that two parent compounds, 11-dichloro and 11-dipropyl, are effective at preventing and delaying cystic growth in two different mouse models of PKD. One arm of the project focuses on the synthesis of new molecules from the 11 family, which will inform, through a structure-activity study, the key molecular features required for activity and provide additional hints about the mechanism of action. A second arm of the project focuses on the development of a cell culture model that can be used to screen the new molecules for improved efficacy and selectivity; such molecules will be then validated in the established PKD mouse models and pave the way towards their preclinical and clinical development. During the last funding period, we continued the work on validating cell culture models that recapitulate the biological consequences of 11 compounds. We focused on the mouse kidney line IMCD3, which showed good promise for toxicity assays. However, to capture the selectivity of 11 compound for PKD1 null cells, a ratiometric assay was necessary (MultiTox-Glo), which measures both the fraction of live and dead cells. We also investigated aspects of the mechanism of toxicity in the IMCD3 cell culture model and showed that 11 toxicity can be attenuated by the antioxidant vitamin E. Together with our collaborators at Yale University, we also measured the transcriptional responses to 11 of a panel of oxidative stress inducible genes. The Yale team also continued their work probing the mechanism of toxicity of 11 compounds in animals (biomarkers of oxidative stress) and completed the testing of 11-dipropyl compound in the adult mouse model of PKD.