Sepsis plagues 19 million per year globally, with a mortality rate of ~ 30%, resulting in ~ five million deaths every year. Sepsis is caused by a dysregulated host response to infection. Over reaction of the immune system upon detection of the invasion of microbes triggers inflammatory reaction, the excessive reaction of which eventually lead to multiple organ failure and patient death. Pyroptosis is a form of programmed cell death in the immune response signaling pathway during infection. The detection of microbe invasion triggers the activation of the inflammatory caspases, which cleaves the linker between the N-terminal (NT) and C-terminal (CT) domains of the effector protein gasdermin D (GSDMD). This cleavage lifts the auto-inhibition of the GSDMD CT domain on the NT domain, which subsequently binds to the inner leaflet of the cell membrane, oligomerizes, and forms pores that are large enough for inflammatpry cytokines such as IL-1β to be released and trigger downstream responses. Studies have shown that GSDMD is the sole and direct effector of pyroptosis, and deletion of the GSDMD gene significantly improved the survival rate in a murine sepsis model. Therefore, we hypothesize that small molecule inhibitors that disrupt GSDMD function can effectively alleviate negative effects of pyroptosis on septic patient, and thus improve the outcome of sepsis treatment. To search for GSDMD inhibitors, we will pursue three specific aims to develop assays and validate them through screening a small collection of compounds. Aim 1. To develop an assay to identify compounds that strengthen GSDMD auto-inhibition. Aim 2. To develop an assay to identify compounds that prevent GSDMD interaction and pore-forming in membrane. Aim 3. To screen a small collection of compounds to validate and optimize assays. In preliminary studies, we established that the GSDMD is a tractable target. In addition, we have engineered several GSDMD constructs and developed several novel methods and primitive assays, which established the feasibility of the proposed approach. Outcomes of the proposed study include the establishment of useful assays for HTS and identification of a collection of active compound structures. In the next stage at the conclusion of this exploratory/developmental R21 project, we will seek additional funding and collaboration to test a larger number of compounds and develop medicinal chemistry and biology components to further test and develop identified compounds. It is our expectation that novel classes of inhibitors binding to original target sites on GSDMD to prevent its pore-forming activity will be identified, which will lead to the development of new anti-infection therapeutics.