Aspergillus fumigatus is a prevalent fungal pathogen that primarily infects individuals through inhalation of conidia, which subsequently germinate and send invasive hyphal extensions throughout the lungs, resulting in clinical bronchopulmonary and invasive aspergillosis. Individuals who are immunocompromised, such as burn patients, transplant recipients, cancer patients undergoing chemotherapy, and individuals suffering from HIV/AIDS or other immunodeficiency syndromes, as well as those suffering from chronic lung disease exhibit a higher risker developing aspergillosis. In addition, asthmatics with fungal hypersensitivity are susceptible to developing allergic bronchopulmonary aspergillosis or severe asthma with fungal sensitization. Current antifungal therapies are generally unsuitable for the treatment of aspergillosis due to the toxicity and side effects associated with these drugs. These shortcomings underscore an urgent clinical need to develop new antifungal drugs that offer greater efficacy and a lower toxicity profile. One such target for drug development is homocitrate synthase (HCS), which catalyzes the first and committed step in fungal lysine biosynthesis. HCS is conserved in A. fumigatus, but is absent in humans, rendering it an attractive target for antifungal intervention. Further, recent studies have demonstrated that HCS is essential to the bronchopulmonary virulence of A. fumigatus, validating this enzyme as a novel target for antifungal drug design. The overall objective of this proposal is to identify and characterize HCS inhibitors that block the growth of A. fumigatus. Toward this goal, we have developed a sensitive, low volume fluorescent assay for HCS and employed this assay to conduct an in vitro high-throughput screen for small molecule inhibitors of A. fumigatus HCS (AfHCS) in the UM Center for Chemical Genomics. This screen has identified over 20 compounds that inhibit AfHCS with IC50 values less than or equal to ten micromolar, which have been prioritized for testing in fungal growth assays using A. fumigatus clinical isolates. We envision that these compounds will yield chemical probes for studying aspergillosis in cell- and animal-based models and will provide a basis for developing new therapeutics for treating a broad spectrum of bronchopulmonary diseases caused by A. fumigatus.