A Dissertation entitled Development of Selective Inhibitors against Enzymes Involved in the Aspartate Biosynthetic Pathway for Antifungal Drug Development By Gopal P. Dahal Submitted to the Graduate Faculty as partial fulfillment of the requirements for the Doctor of Philosophy Degree in Chemistry ________________________________________ Dr. Ronald E. Viola, Committee Chair ________________________________________ Dr. Donald Ronning, Committee Member ________________________________________ Dr. Jianglong Zhu, Committee Member ________________________________________ Dr. Viranga Tillekeratne, Committee Member ________________________________________ Dr. Amanda C. Bryant-Friedrich, Dean College of Graduate Studies The University of Toledo August 2018 © Copyright by Gopal P. Dahal, 2018 This document is copyrighted material. Under copyright law, no parts of this document may be reproduced without the expressed permission of the author. An Abstract of Development of Selective Inhibitors against Enzymes Involved in the Aspartate Biosynthetic Pathway for Antifungal Drug Development by Gopal P. Dahal Submitted to the Graduate Faculty as partial fulfillment of the requirements for the Doctor of Philosophy Degree in Chemistry The University of Toledo August 2018 Aspartate semialdehyde dehydrogenase (ASADH) functions at a critical junction in the aspartate biosynthetic pathway and represents a validated target for antimicrobial drug design. This enzyme catalyzes the NADPH-dependent reductive dephosphorylation of β-aspartyl phosphate to produce the key intermediate aspartate semialdehyde. Production of this intermediate represents the first committed step for the biosynthesis of several essential amino acids in fungi and in bacteria. The absence of this enzyme in humans and other mammals will allow the selective targeting of any ASADH inhibitors against pathogenic microorganisms. We have accumulated significant structural and mechanistic information about the bacterial ASADHs, but have only limited knowledge of their fungal counterparts. To bridge this gap we have determined the high-resolution structures of three pathogenic fungal forms of ASADH, from C. neoformans, A. fumigatus, and from B. dermatitidis in both apo- and ligand-bound forms. While the overall structures of the fungal ASADHs are similar to the bacterial orthologs, some critical differences both in biological assembly and iii in secondary structural features can potentially be exploited for the development of species- selective drugs with selective toxicity against infectious fungal organisms. As an initial study ~1,000 compounds from our customized fragment libraries were screened against ASADH, followed by determination of the inhibition constant (Ki) values of the most potent hits. Encouragingly, these results showed that most of the hits obtained already have Ki values in the lower micromolar range. Several of these most potent and structurally diverse initial hits were selected for structural-activity relationship (SAR) evaluation, leading to optimized inhibitors against fungal ASADH with inhibition constants in the low micromolar/high nanomolar range, while still retaining high ligand efficiency values. The efficacy of these hits was further validated using a sensitive cell-based assay against the growth of Candida cells. These ASADH inhibitors suppress fungal growth, with most potent hits in this cell-based assay have low micromolar IC50 values. Further screening of ~1000 drug-like compounds from an NIH clinical collection library and a Prestwick drug library revealed that many of the hits from these libraries have core scaffolds that are representative of those obtained from the fragment library hits. These results confirm that fragments can serve as hit predictors and as a guide for optimized inhibitor selection. Native PAGE studies and small angle x-ray scattering (SAXS) analysis showed that the fungal ASADHs exist as a functional tetramer, in contrast to the bacterial ASADHs that exist as dimers. Some of these newly identified inhibitors show a non-competitive mode of inhibition, functioning to bind and cause oligomer dissociation into inactive dimers. These compounds are being used as starting points for designing protein-protein interaction inhibitors with a unique mode of action against our fungal enzyme target. High throughput docking against a sub-set of the ZINC compound library (70,000 compounds) also yielded iv hits with similar sub-structures from fragment library screening, with measured Ki values as low as 4 μM. These docking results are being used to guide the design and development of more potent inhibitors. The second project focused on purification, characterization and crystallization of membrane bound aspartate N-acetyltransferase (ANAT) enzyme, and performing inhibitor screening against mouse and human forms of this enzyme for the treatment of Canavan Disease (CD). ANAT catalyzes the biosynthesis of N-acetyl aspartate (NAA) in neurons, a major source of acetyl groups for lipid synthesis during brain development. CD is a fatal autosomal-recessive neurodegenerative disease caused by mutations in the acy2 gene that leads to the deficiency of the enzyme aspartoacylase, the enzyme responsible for the deacetylation of NAA in the oligodendrocytes of the brain. An approach in which selective ANAT inhibitors are used to lower brain NAA levels back into the physiological range has the potential to treat the symptoms of CD without introducing higher risks. We have now screened several bisubstrate analog inhibitors and their truncated series against ANAT, and the most potent hits have been found to inhibit ANAT in the lower nanomolar range. These bisubstrate inhibitors are also used for co-crystallization of ANAT in order to determine the crystal structure of the enzyme. This study will allow us to understand the mechanism of the enzyme and develop selective inhibitors which, upon further modifications, will produce lead compounds for the treatment of Canavan Disease. v To my Mom and Dad and my wife Sierra, who arouse my curiosity in learning new things and has always supported me ever since. vi Acknowledgements I would like to thank my academic advisor, Dr. Ronald E. Viola, for his immense support, guidance and encouragement over the past 5 years. His motivation and commitment to science inspired me and enriched my passion for research. I was fortunate enough to have a great person like him as my research advisor and all the academic accomplishments I achieved, I have learned from him and his research lab. His organization skills, patience and discipline inspired me to finish my projects in strict timelines, and the professional skills that I acquired in his lab helped me to shape myself as an independent scientist. I would like to thank my committee members Dr. Donald R. Ronning, Dr. Jianglong Zhu, and Dr. Viranga Tillekaratne for their constant support and motivation throughout my graduate studies. I would like to thank the Department of Chemistry and Biochemistry, the University of Toledo for providing me the opportunity to pursue my graduate studies. I would also like to thank all my past and present lab members for all their support, advice and words of encouragement, and for all the insightful scientific discussions and my friend’s Dr. Hem Raj Khatri, Pratik Pote, Subash Sapkota, Prabhat Napit, Bibek Joshi, Dr. Sikesh Manandhar, and Yogesh Pathak for their unwavering support. To my brother Kumar and his wife Deepa, my sister Laxmi and her husband Dinesh, my niece Yojana and my nephew Rishab, I thank them for their constant love, support, encouragement, and motivation throughout this PhD journey. vii Table of Contents Abstract……...................................................................................................................... iii Acknowledgements…........................................................................................................vii Table of Contents……......................................................................................................viii List of Tables….................................................................................................................xvi List of Figures……. ........................................................................................................xvii List of Abbreviations…………........................................................................................ xxi List of Symbols…...........................................................................................................xxiii Chapter 1: Introduction 1.1 Background and significance…………………………………………….…….1 1.2 Antifungals and their mode of action………………………………….……….3 1.3 Antifungal drug resistance……………………………………………….…….5 1.4 Mechanism of antifungal drug resistance……………………………………...6 1.5 Biofilm formation…………………………………………………….…….….7 1.6 Synopsis and chapter organization………………………………….………….8 Chapter 2: Aspartate biosynthetic pathway 2.1 Background and significance……………………………………….…………9 2.2 Aspartate semialdehyde dehydrogenase (ASADH) …………………….……10 viii 2.3 Catalytic mechanism…………………………………………………………11 2.4 Structure of ASADH…………………………………………………………12 2.5 ASADHs from human fungal pathogens…………………………..….……..13 2.6 Biophysical characterization of aspartate semialdehyde dehydrogenase….….15 2.6.1 Cloning, expression and purification of ASADH from Cryptococcus neoformans……………………………………………………...15 2.6.1.1 Cloning……………………………………………...……15 2.6.1.2 Yeast expression system……………………………....….16 2.6.1.3 Gene