PROJECT SUMMARY Molds cause devastating infections in diverse patient populations worldwide. The most life-threatening of these diseases are invasive mold infections (IMIs) which primarily affect immune compromised individuals including those undergoing chemotherapy, long term steroid treatment, and hemopoietic or solid organ transplant patients. IMIs can be caused by a handful of environmental molds but the most common species is Aspergillus fumigatus. Currently, there are three classes of antifungals used to treat IMIs; however, despite available treatments, mortality rates remain unacceptably high, typically ~50% but reaching 80-100% for some mold species and patient cohorts. One contributing factor to the high mortality rates is intrinsic resistance of some mold species to some or all clinical antifungals. In addition, acquired antifungal resistance, particularly among A. fumigatus isolates, is becoming a clinical problem worldwide. In the drug development space, antifungal drug screens often focus on yeast species. This is due in part to the technical ease of handling these organisms in high-throughput settings. Yeast growth is tightly correlated to optical density, facilitating large-format growth-based screens. In contrast, molds grow as filaments which create a heterogenous culture of tangled cells whose optical density is not well correlated to biomass. Additionally, the inability to handle these cultures with automated liquid handling devices creates additional challenges in high throughput settings. However, the high mortality rates and limited activity of clinical antifungal against molds warrants more focus on these organisms in antifungal drug screening. To address the need for mold-active antifungals, we developed and validated a high-throughput compatible screening assay to screen directly with A. fumigatus. This luciferase-based assay measures the release of the cytosolic enzyme, adenylate kinase (AK), as a readout for fungal cell lysis. Uniquely, when used with A. fumigatus this assay can also detect compounds that inhibit germination through the suppression of background AK release that occurs during normal germination and vegetative growth. Here, we propose a high- throughput screen of a large library of synthetic, drug-like molecules for novel scaffolds with anti-mold activity using our AK screening platform. Pilot large-format screens have already identified candidate molecules for further development; thus, based on the scale and diversity of our proposed screen, we expect to identify additional mold-active antifungals with novel mechanisms of action for preclinical development.