Developing Novel Antifungal Compounds for Use as Single-Agent and in Multi-Drug Combination Therapies for Treating Invasive Fungal Infections

Abstract

Invasive fungal infections are recognized as a global health threat with the World Health Organization listing 4 fungal species as critical pathogens for global study. With the rise of resistance and new pathogenic species continuing to evolve, there is an immediate need for antifungal drug development. Current antifungal therapies are antiquated, have numerous side effects, and consist of only four available classes. The development of novel drugs to treat fungal disease is hindered by the evolutionary conservation between fungi and mammals. With these considerations in mind, this thesis aims to investigate repurposing established therapeutics for use in treating invasive fungal infections.Chapter 1 focuses on the global impact of fungal disease and highlights Cryptococcus neoformans as a primary pathogen to study. It begins by outlining the current state of fungal pathogenesis and the antifungal therapeutic armament. The development of antifungal agents will additionally be addressed by giving an overview of investigational drug candidates including FKBP12 ligands, Gwt1 inhibitors, and prenylation inhibitors. In Chapter 2, prenylation inhibition is investigated as a drug development strategy. Novel synthesized compounds and the Food & Drug Administration (FDA) approved carcinoma product Tipifarnib are assessed for their in vitro antifungal activity through microdilution broth minimum inhibitory concentration (MIC) assays. The mechanism of action of Tipifarnib was visualized using fluorescence microscopy before resistance mechanisms were interrogated by isolating fungal colonies with reduced tipifarnib susceptibility and genetically assessed mutations responsible for resistance. In Chapter 3, the anticancer and immunosuppressive drug rapamycin is investigated for application as an antifungal drug. Treatment with rapamycin mimics starvation and induces cell death through autophagy. Using antifungal susceptibility MIC assays, rapamycin antifungal efficacy is shown to be significantly impacted by the nutritional environment. Additionally, synthesized analogs of rapamycin are investigated for their potential to specifically inhibit the fungal target of FK506 binding protein (FKBP12). Analog specificity is measured by comparing in vitro antifungal activity to immunosuppressive activity. A lead fungal specific analog is tested during an in vivo infection model demonstrating no adverse effects or therapeutic benefit at the concentrations tested. In Chapter 4, immunosuppressive therapeutic agent FK520 is structurally modified and assayed for fungal specificity. Structure-guided drug design is used to develop two libraries of FK520 analogs. Through in vitro assessment of their antifungal and immunosuppressive activities, the lead compound JH-FK-08 is identified and used as an antifungal therapy during an in vivo cryptococcosis model. JH-FK-08 is shown to significantly reduce fungal burden and extend survival in addition to demonstrating a similar pharmacokinetic profile to parent compound FK506. In Chapter 5, combination therapies are investigated for FK506 and JH-FK-08. Checkerboard assays are applied to test the interaction of two drugs when inhibiting fungal growth in vitro. JH-FK-08 is shown to have synergistic activity with Gwt1 inhibitors against C. neoformans and Candida albicans. FK506 is shown to have synergistic interactions with rapamycin which is unexpected due to the shared requirement of binding chaperone protein FKBP12. An FKBP12 mutant is shown to have no defect during virulence and competition in vivo experiments. This indicates that an FKBP12 mutation could confer resistance to a combination of FK506 and rapamycin.Chapter 6 summarizes the results presented throughout this dissertation. The conclusions, impact of findings, and future directions are further considered within the broader context of antifungal therapeutic approaches. In addition, the benefits of repurposing established medications for novel use are highlighted and discussed here. Targeting proteins found in both humans and fungi can be an effective approach especially when designing structurally specific fungal inhibitors. This thesis demonstrates the efficacy of this approach through the development of promising antifungal agent JH-FK-08 shown to significantly extend survival and reduce fungal burden in mice undergoing an invasive fungal infection.