Browsing by Author "Hoy, Michael J"

Invasive fungal infections are a major burden on the global healthcare system and represent a significant threat to human health for future generations. The majority of invasive fungal infections in the world are caused by Cryptococcus, Candida, and Aspergillus species. With the changing climate, growing antifungal drug resistance, and an increasing immunocompromised patient population, the need for novel and effective antifungal treatments is higher than ever. Despite this clear unmet need, the FDA has approved no new antifungal drugs for the treatment of life-threatening systemic fungal infection in over 20 years. Moreover, only 4 classes of antifungal drugs are available to treat these invasive fungal infections and all suffer from issues associated with either toxicity, resistance, or bioavailability. Due to the high degree of conservation between the eukaryotic cell targets in fungi and mammals, fungal selectivity and low patient toxicity is a significant issue. In this dissertation, I have taken protein crystal structure-guided design and genetic target elucidation approaches to develop novel, fungal-specific antifungal compounds. I focus primarily on two targets: the serine-threonine specific protein phosphatase, calcineurin, and the putative membrane flippase, Apt1. In Chapter 1, I will detail the global threats faced by opportunistic fungal pathogens and emerging mechanisms of drug resistance. I then summarize the currently available treatment options for invasive mycoses and their associated weaknesses. I conclude by reviewing some of the novel antifungal compounds in development today and explain some of the methods utilized by researchers in modern drug development. In Chapter 2, I summarize our efforts to develop a fungal-specific calcineurin inhibitor called APX879. Both fungal and mammalian calcineurin are inhibited naturally by the small molecule FK506 when bound to its molecular chaperone, FKBP12. Here, I describe how we utilize recently solved protein crystal structures of the fungal calcineurin inhibitory complex (Calcineurin-FK506-FKBP12) from the major fungal pathogens to design an FK506 analog that is increased for its fungal specificity. We identified key structural differences in the 80s loop of FKBP12 that could be exploited by making structural modifications to the C22 of FK506. APX879 is a C22-modified FK506 analog that is reduced for immunosuppressive activity and toxicity as a result of decreased mammalian calcineurin inhibition. Additionally, we show that APX879 still maintains broad-spectrum antifungal activity and is efficacious in an animal model of Cryptococcus infection. In Chapter 3, I describe the development of a second-generation calcineurin inhibitor, JH-FK-05, that is further increased for its fungal selectivity. We began by utilizing protein crystal structures of Aspergillus FKBP12 bound to a natural FK506 analog, FK520 to design additional compounds to test for fungal specificity. These FK520 analogs were screened for a shift in balance of antifungal and immunosuppressive activity, and we found that C22-modified JH-FK-05 maintained broad-spectrum activity and was non-immunosuppressive in vivo. Moreover, when treated with JH-FK-05, mice in two different models of murine cryptococcosis exhibited a significant improvement in survival and tissue fungal burden. We then applied molecular dynamic simulations to JH-FK-05 bound to fungal vs mammalian FKBP12 to identify FK520 residues in the future that could be targeted to increase fungal specificity. In Chapter 4, I briefly illustrate the progress in developing additional FK506 and FK520 analogs. We have synthesized an additional 15 FK506 or FK520 analogs and screened them for their in vitro antifungal activity against Cryptococcus and their in vitro immunosuppressive activity in primary murine T cells. Several of these compounds presented a promising degree of fungal selectivity and have been selected for further study in animal models of fungal infection. In Chapter 5, I provide detailed methods for the elucidation of unknown antifungal targets. In many cases of drug development, a compound is isolated with promising activity before its molecular target is known. An understanding of the mechanism of action for any compound is an essential step before it can be utilized clinically to treat patients. Here, I describe step by step procedures for generating spontaneously resistant mutant strains, testing for antifungal susceptibility, and identification of the causative mutation conferring resistance through genetic crosses and whole-genome sequencing. In Chapter 6, I describe the mechanism of action for Butyrolactol A (ButA), a natural polyketide compound with potent antifungal activity against Cryptococcus. ButA had antifungal activity alone but also could potentiate the activity of caspofungin, which is an antifungal drug that Cryptococcus is naturally resistant to. Although the antifungal activity of ButA had previously been described, the mechanism of action remained unknown. We isolated two resistant mutants to ButA and identified mutations in the gene encoding lipid flippase APT1 by utilizing whole-genome sequencing. ButA had low toxicity in mice at doses as high as 50 mg/kg. However, despite robust in vitro antifungal activity, we detected no therapeutic efficacy of ButA treatment at multiple doses and in combination with caspofungin. Additional pharmacokinetics and pharmacodynamics may shed some light on the discrepancy between the in vitro and in vivo activity of ButA. In Chapter 7, I will summarize the findings presented in this dissertation and provide future directions for each of the conclusions. Additionally, Appendices A and B contain supplementary methods for protein production associated with Chapters 2 and 3, respectively.