Classical and rational approaches to antifungal drug design Jessica Louise Chitty BSc (Hons) A thesis submitted for the degree of Doctor of Philosophy at The University of Queensland in 2017 School of Chemistry and Molecular Biosciences Institute of Molecular Biosciences Abstract The emergence of human immunodeficiency virus (HIV) in the 1980s has led to an increase in infections from previously rare pathogens. Many of these now cause widespread infection among individuals with compromised immune systems, not just limited to AIDS patients but also to those placed on immunosuppressive medication. The encapsulated yeast Cryptococcus neoformans causes widespread disease in the immunocompromised population, particularly in sub-Saharan Africa where it is a major cause of AIDS-related mortality due in part to limited resources and variable drug availability. Current treatment options are restricted to three out-dated antifungals amphotericin B, flucytosine and fluconazole; where possible they are used in combination as nephrotoxicity and resistance are contributing factors in the unacceptably high mortality rates. Alternative therapeutic agents are urgently required to improve survival rates and combat antifungal drug resistance. Two main routes of compound development can be taken: classical drug screening or rational drug design. Classical design requires groups of compounds to be screened against pathogens and those identified with high efficacy and low cytotoxicity are pursued. Rational drug design requires a detailed characterization of the proposed target; exploitable differences between the pathogen and human host are sought out as potential druggable targets. In this thesis both classical and rational methods have been investigated. A classical approach was taken to investigate a class of octapeptin compounds, produced as secondary metabolites by the soil dwelling bacterium, Bacillus circulans. Related compounds, such as the polymxyins, have become last resort drugs against gram-negative bacteria. The physiological target of poylmxyins against gram-negative bacteria is thought to be its charged cell wall surface. Like gram-negative bacteria C. neoformans also has a charged surface; comprised of a polysaccharide capsule and melanin. These features have been investigated as a potential target of octapeptin C4. The fungal pathogen C. neoformans is commonly associated with bird guano, a particularly purine rich environment. Investigation into novel antifungals targeting enzymes of the purine biosynthetic pathway has been limited. Gertrude Elion’s pivotal work in developing inhibitors of the de novo purine biosynthesis pathway in a rational manner successfully led to the development of anticancer agents still used today. To enable development of such compounds against fungal pathogens, enzymes from the de novo purine biosynthesis pathway have been characterized in C. neoformans. ii In this thesis GMP synthase and ADS lyase were be presented. Gene deletion and subsequent complementation of the genes encoding GMP synthase and ADS lyase was performed. The deletion mutants in both showed defects in the production of virulence traits as well as being avirulent in a murine inhalation model of infection. Recombinantly expressed and purified protein was obtained for enzyme kinetics assays and structural studies. These have enabled the first in-depth analysis of these enzymes in fungi for comparison to previously characterized human enzymes. Collectively, these data highlight the potential of enzymes of the purine biosynthesis pathway to be exploited in the development of new therapeutic agents for the treatment of disseminated, life threatening fungal infections. iii Declaration by author This thesis is composed of my original work, and contains no material previously published or written by another person except where due reference has been made in the text. I have clearly stated the contribution by others to jointly-authored works that I have included in my thesis. I have clearly stated the contribution of others to my thesis as a whole, including statistical assistance, survey design, data analysis, significant technical procedures, professional editorial advice, and any other original research work used or reported in my thesis. The content of my thesis is the result of work I have carried out since the commencement of my research higher degree candidature and does not include a substantial part of work that has been submitted to qualify for the award of any other degree or diploma in any university or other tertiary institution. I have clearly stated which parts of my thesis, if any, have been submitted to qualify for another award. I acknowledge that an electronic copy of my thesis must be lodged with the University Library and, subject to the policy and procedures of The University of Queensland, the thesis be made available for research and study in accordance with the Copyright Act 1968 unless a period of embargo has been approved by the Dean of the Graduate School. I acknowledge that copyright of all material contained in my thesis resides with the copyright holder(s) of that material. Where appropriate I have obtained copyright permission from the copyright holder to reproduce material in this thesis. iv Publications during candidature Peer-reviewed journal articles J.L Chitty and J.A Fraser (2015) "Rethinking the targets for antifungal development." Microbiology Australia, 36(2), 88-89 S.D.M Arras, J.L Chitty, K.L Blake, B. Schulz, J.A Fraser (2015) “A genomic safe haven for mutant complementation in Cryptococcus neoformans” PLoS One, 10(4): e0122916. K.D Beattie, N. Ellwood, R. Kumar, X. Yang, P.C Healy, V. Choomuenwai, R.J Quinn, A.G Elliott, J.X Huang, J.L Chitty, J.A Fraser, M.A Cooper, R.A Davis (2016) “Antibacterial and antifungal screening of natural products sourced from Australian fungi and characterisation of pestalactams D-F” Phytochemistry, Apr; 124:79-85 R.D Blundell, S.J Williams, S.D.M Arras, J.L Chitty, K.L Blake, D.J Ericsson, N. Tibrewal, J. Rohr, Y.Q.A.E Koh, U. Kappler, A.A.B. Robertson, M.S Butler, M.A Cooper, B. Kobe, J.A Fraser (2016) “Disruption of de novo ATP biosynthesis abolishes virulence in Cryptococcus neoformans” ACS Infectious Diseases, Sep 9;2(9):651-663 J.L Chitty, T.L Tatzenko, S.J Williams, Y.Q.A.E Koh, E.C Corfield, M.S Butler, A.A.B Robertson, M.A Cooper, U. Kappler, B. Kobe and J.A Fraser (2017) “GMP synthase is required for virulence factor production and infection by Cryptococcus neoformans” Journal of Biological Chemistry, 292(7): 3049-59 S.D.M Arras, J.L Chitty, B. Schulz, M. Tanurdzic, J.A Fraser (2017) Sirtuins in the phylum Basidiomycota: A role in virulence in Cryptococcus neoformans” Scientific reports, 7, 46567 J.L Chitty, K.L Blake, Y.Q.A.E Koh, R.D Blundell, M. Thompson, M.S Butler, A.A.B Robertson, M.A Cooper, U. Kappler, S.J Williams, B. Kobe and J.A Fraser (2017) “Cryptococcus neoformans ADS lyase in an enzyme essential for virulence whose crystal structure reveals features exploitable in antifungal drug design” Journal of Biological Chemistry, 292(28): 11829-39 J.L. Chitty and J.A. Fraser (2017) “Purine acquisition and synthesis by human fungal pathogens” Microorganisms; 5(2) v Publications included in this thesis Jessica L. Chitty and James A. Fraser (Accepted 2 June 2017) “Purine acquisition and synthesis by human fungal pathogens” Microorganisms; 5(2) – incorporated as Chapter 1 Contributor Statement of contribution Jessica Chitty (Candidate) Wrote the paper (100%), edited paper (90%) James Fraser Edited the paper (10%) J.L Chitty, T.L Tatzenko, S.J Williams, Y.Q.A.E Koh, E.C Corfield, M.S Butler, A.A.B Robertson, M.A Cooper, U. Kappler, B. Kobe and J.A Fraser (2017) “GMP synthase is required for virulence factor production and infection by Cryptococcus neoformans” Journal of Biological Chemistry, 292(7): 3049-59 – incorporated as Chapter 3. Contributor Statement of contribution Jessica Chitty (Candidate) Concept and design of experiments (70%), Experimental work (75%), analysis and interpretation of the data (80%), wrote the paper (90%), edited paper (60%) Tayla Tatzenko Concept and design of experiments (10%), experimental work (15%) Simon Williams Concept and design of experiments (10%), analysis and interpretation of the data (5%), edited paper (5%) Y.Q. Andre Koh Experimental work (5%) Elizabeth Corfield Experimental work (5%) Mark Butler Edited paper (5%) Avril Robertson Edited paper (5%) Matthew Cooper Edited paper (5%) Ulrike Kappler Concept and design of experiments (10%), analysis and interpretation of the data (5%), edited paper (5%) Bostjan Kobe Edited paper (5%) James Fraser Concept and design of experiments (10%), analysis and interpretation of the data (10%), wrote and edited paper (10%) vi J.L Chitty, K.L Blake, Y.Q.A.E Koh, R.D Blundell, M. Thompson, M.S Butler, A.A.B Robertson, M.A Cooper, U. Kappler, S.J Williams, B. Kobe and J.A Fraser (2017) “Cryptococcus neoformans ADS lyase in an enzyme essential for virulence whose crystal structure reveals features exploitable in antifungal drug design” Journal of Biological Chemistry, 292(28): 11829-39 – incorporated as Chapter 4. Contributor Statement of contribution Jessica Chitty (Candidate) Concept and design of experiments (60%), Experimental work (75%), analysis and interpretation of the data (70%), wrote the paper (90%), edited paper (60%) Kirsten Blake Concept and design of experiments (10%), experimental work (10%) Y.Q. Andre Koh Experimental work (5%) Ross Blundell Experimental work (5%) Merinda