PROJECT SUMMARY/ABSTRACT Lyme disease is the most prevalent tick-borne infection in many parts of the world, including the U.S., where over 450,000 new cases occur annually. The disease is inflicted by a group of bacterial pathogens, Borrelia burgdorferi sensu lato, that thrive in nature through a complex enzootic infection cycle involving Ixodes scapularis ticks and a variety of vertebrate hosts. Despite much effort, the infection remains difficult to control, largely due to the absence of preventive strategies including vaccines, difficulties in the diagnosis of early infection, and failure to achieve complete cures using current antibiotics. Specifically, several months after standard-care antibiotic therapy, a subset of patients can experience a series of persistent or relapsing symptoms, which are termed as chronic Lyme disease or post-treatment Lyme disease syndrome (PTLDS). While the persistence of the bacterial infection may or may not contribute to the etiology or pathogenesis of PTLDS, its treatment options remain unknown. Therefore, the development of vaccines and new drugs is highly warranted. We have identified a critical virulence determinant called BbHtrA that plays an indispensable role in host infectivity and persistence of spirochetes. Deletion of the protein renders the pathogen non- infectious in mammalian hosts. Based on this information, we propose a therapeutics strategy that relies on disruption of the protein function by small molecule drugs. To this end, we will identify small molecules that disrupt BbHtrA protease activity by employing an assay designed for high-throughput screening (HTS) of large compound libraries based on nanoDSF and fluorescence protease inhibition technology. Hits of this screen will be validated by a secondary cellular assay, revealing potent compounds that are permeable across the B. burgdorferi membrane. Compound screening will be performed in collaboration with National Center for Advancing Translational Sciences (NCATS) investigators, who provide expertise, infrastructure, and modern compound libraries, and have extensive experience with HTS and drug discovery. We will also attempt to develop structural studies on BbHtrA, which will be important for the development of new therapeutic agents targeting the protein. The validation of selected hit molecules in a whole animal model of Lyme disease will be examined in future larger grant applications. The project employs cutting-edge technology that targets interacting virulence determinants to combat Lyme disease, a novel strategy that may avoid PTLDS. Moreover, the same approach may serve as a paradigm for combating other tick-borne infections, including those caused by the recently-discovered, more virulent strains of Lyme disease pathogens.