Goal: To create a well-tolerated, orally available therapeutic that can be employed alone or in combination with other antifungal agents to improve treatment outcomes for patients suffering from cryptococcal meningitis (CM). The worldwide disease burden of infections caused by Cryptococcus is over one million cases annually. The disease is normally caused by inhalation of the fungus, which spreads from the lungs to the central nervous system in immunocompromised individuals, where it is classified as cryptococcal meningitis (CM). CM is always fatal if left untreated. First-line therapy is limited to amphotericin B/5- flucytosine, which is largely unavailable in the developing world. Our recent efforts to identify novel therapeutics to treat CM have focused on targeting the fungal protein Gwt1 acyl transferase, an enzyme that is required for the assembly of glycosylphosphatidylinositol (GPI) anchored proteins that are later attached to the fungal cell wall. Amplyx Pharmaceuticals has in-licensed a portfolio of potent Gwt1 inhibitors from Eisai, including APX001, the first-on-class molecule which has completed Phase 1 clinical trials. APX001A, the active moiety of APX001, has some activity against Cryptococcus, however, its potency in mouse animal models is best observed in combination with fluconazole. We have recently synthesized and evaluated additional Gwt1 inhibitors and have identified several molecules with significantly improved microbiological activity (> 30-fold) against C. neoformans and C. gattii. In this proposal, Linnaeus Bioscience Inc. will develop a microscopy based assay for C. neoformans and use it to confirm that the new molecules target Gwt1, evaluate synergy with other antifungal agents, and explore in vitro and in vivo phenotypic effects. Dr. John Perfect, Duke University School of Medicine, will evaluate compounds for efficacy in mouse models of CM. The ultimate outcome of this proposal will be the selection of a candidate for IND-enabling studies that demonstrates a significant improvement vs APX001 in both CM survival and fungal burden mouse models. We will begin by developing a rapid microscopy assay for determining the mechanism of action (MOA) of Gwt1 inhibitors in Cryptococcus and apply it to studies of APX001A analogs. We will then characterize 30 analogs of APX001A molecules that are significantly more potent (4 to >32 fold) than APX001A vs Cryptococcus. We plan to use our new microscopy based assay to ensure these more potent molecules are on target. We will also characterize these molecules for their potential cytotoxicity and metabolic stability. The top 10 compounds will be selected and further studied to characterize their microbiological activities, including determining their spectrum of activity against a panel of C. neoformans and C. gattii isolates. We will successfully scale-up the synthesis of 8-10 selected analogs in prodrug form and assess appropriate formulations for use in animal efficacy studies. We will characterize the PK, MTD and safety for 8-10 compounds, identifying molecules with over 40% oral bioavailability that have an MTD, potential for hERG inhibition, and potential for cytochrome P450 isoform interaction that is equivalent to or better than APX001 (APX001A). In the final step, we will demonstrate the efficacy of 5-10 compounds in Cryptococcus neoformans animal models of infection. We will test molecules for their ability to reduce fungal burden in lung and brain models and to improve survival in CM models. We will also determine if these compounds reduce fungal burden when used in combination with fluconazole (or other antifungals that are used for the treatment of CM). We will use our microscopy based assay to assess Gwt1 inhibitor effectiveness and mode of action in vivo in brain and lung infection models. When completed we will declare a candidate ready for IND enabling studies.