Abstract Systemic fungal infections in immunocompromised patients have exceedingly high mortality rates. Current antifungal drugs are not sufficient to protect patients from increasing antifungal resistance and a need for new antifungals is now clearer than ever. However, similarities between targets in these eukaryotic pathogens and their human hosts have made the development of new antifungal drugs challenging. The natural product FK506 inhibits the serine-threonine specific protein phosphatase calcineurin in both fungi and humans by binding to the immunophilin FKBP12 and subsequently binding to calcineurin. In the pathogenic fungi Cryptococcus neoformans, Candida albicans, and Aspergillus fumigatus, calcineurin is a key virulence factor required for growth at human body temperature, growth in serum, and the yeast-hyphal dimorphic transition, respectively. In humans, calcineurin is required for T-cell activation and IL-2 production. In fact, FK506 is used clinically as a potent immunosuppressant. Although fungal and mammalian calcineurin and FKBP12 are highly conserved, we have recently identified key amino acid differences in the 80s loop of FKBP12 that are located at the FKBP12- FK506-calcineurin interface. A recently developed FK506 analog, APX879, is modified at a single moiety of FK506 (C22 keto oxygen) that approaches the FKBP12 80s loop. This analog exhibits significantly reduced immunosuppressive activity yet retains antifungal activity in vitro and in an animal model of cryptococcosis. Our central hypothesis is that with structure-guided rational design, FK506 analog calcineurin inhibitors can be generated with increased fungal specificity by introducing differential interactions in the 80s loop of FKBP12. In Aim 1, a defined library of FK506 analogs will be designed and synthesized based on the predicted interactions with known crystal structures for the calcineurin ternary complexes from fungi and mammals. The structure- activity relationship will then be determined by testing the spectrum of antifungal activity and the immunosuppressive activity of this library. In Aim 2 lead compounds will be tested for in vivo efficacy in murine models of cryptococcosis and in vivo immunosuppression. By defining the small molecule interactions between calcineurin, FKBP12, and FK506, compounds will be developed that shift the efficacy of calcineurin inhibition in vivo into the therapeutic window of higher antifungal activity and reduced immunosuppressive activity.