Abstract: Cryptococcosis caused by infections of Cryptococcus spp. fungi. C. neoformans and C. gattii remains a leading killer of people living with HIV and post-organ transplant status. Recent outbreaks of infections of C. gattii in immune competent people raise significant concerns about the overall threat of cryptococcal species to public health. Cryptococcosis is one of the most difficult infections to treat with a mortality rate over 50%. New therapies with novel mechanism of actions (MOAs) are urgently needed to treat the infected. High throughput screening of target-based pharmaceutical libraries represents a very promising strategy to discover antifungals with novel MOAs because these libraries harbor well-defined molecules/compounds with desirable physicochemical properties, often with existing preclinical data for other therapeutic indications. Therefore, once identified, they can be quickly developed for novel applications such as antifungal. Cryptococcal fungi contain protein self-splicing elements, called inteins, in the essential gene, Prp8. Because inteins do not exist in multi- cellular organisms, such as humans, and often disrupt the functions of critical microbial genes, they are attractive drug targets. We have developed high throughput screening (HTS) strategies to allow identification of the Prp8 intein splicing inhibitors. In our preliminary results, we identified a few small molecule inhibitors that can inhibit the Prp8 intein splicing in vitro and inhibit the cryptococcosis in vivo. In this application, we will use our well- developed HTS assays to screen the predefined and target-based pharmaceutical libraries at the ICCB- Longwood screening facilities at the Harvard Medical School to identify the Prp8 intein splicing inhibitors. The identified candidate inhibitors will be characterized and optimized both in vitro and in vivo with the final aim to develop them as novel therapeutics for cryptococcosis.