Transition from classical methods to new strategies: Mechanistic evaluation of inhibitors against Mycobacterium tuberculosis shikimate kinase by Ngolui René Fuanta A dissertation submitted to the Graduate Faculty of Auburn University in partial fulfillment of the requirements for the Degree of Doctor of Philosophy Auburn, Alabama August 4, 2018 Keywords: Kinetics, spectrometry, tuberculosis, spectrofluorimetry, dissociation constants Copyright 2018 by Ngolui René Fuanta. Approved by Douglas Goodwin, Chair, Associate Professor, Chemistry and Biochemistry Holly Ellis, Professor, Chemistry and Biochemistry Chris Easley, Professor, Chemistry and Biochemistry Steve Mansoorabadi, Assistant Professor, Chemistry and Biochemistry Angela Calderón, Associate Professor, Drug Discovery and Development Abstract Tuberculosis (TB) still stands as one of the leading causes of mortality resulting from an infectious agent. In part, this stems from the emergence of multidrug and extensive drug-resistant strains of tubercle bacilli, demonstrating the pressing need for the development of novel anti tubercular agents. Interestingly, the shikimate pathway, found in plants, bacteria, fungi and algae, is essential for the survival of Mycobacterium tuberculosis. This pathway produces precursors to aromatic amino acids and other aromatic cellular metabolites. Fortunately, it has no mammalian analogue. This makes the enzymes of this pathway suitable targets for the development of novel anti-microbial agents. The research presented in this dissertation focuses on Mycobacterium tuberculosis shikimate kinase (MtSK). Our objectives were to develop LC- MS-based screening and characterization of potential anti tubercular agents – natural, synthetic or semi synthetic, develop fluorescence methods for rapid mechanistic evaluation of MtSK inhibitors and strategies for screening out promiscuous inhibitors. Using mass spectrometry, we characterized a group of marine derived compounds, manzamine alkaloids. These inhibitors showed mixed-noncompetitive mode of inhibition, with time-dependent and slow-binding components. Of this group, one, 6-cyclohexamidomanzamine A, a derivative of the parent manzamine A scaffold, showed slow, tight-binding kinetics with an inhibition constant in the nanomolar range. Another marine-derived inhibitor we characterized was ilimaquinone, which showed irreversible inhibition evident from formation of a covalent adducts with primarily serines and threonines, and to a lesser extent lysines and tyrosines. It is worth noting that due to ii the nonspecific nature of this inhibitor, as it has shown to modify other proteins (not only MtSK), further exploitation of this molecule as a potential antibacterial, antiviral, or anticancer agent should be approached with great care. With respect to developing rapid screening tools, we have generated a panel of MtSK variants with intrinsic fluorescent properties. Substitutions were made on the lid domain, V116W, the adenine binding domain, N151W, and the shikimate binding domain, E54W. These variants show differential responses to substrates and inhibitors. Interestingly, we noted that the binding of one of the substrates, shikimate produced changes in fluorescence in only one of the variants, supporting previously reported data that substrate binding induces conformational changes. Using these, we evaluated the inhibition kinetics of previously characterized inhibitor, so-called Compound 1. Our data supported a mixed-type mode of inhibition, with a slow, reversible component. Competition experiments indicated Compound 1 binds at or near the shikimate binding site, evident from spectral shifts in E54W in the presence of the inhibitor. Together, we have shown that the 6-cyclohexamido derivative of manzamine A is a potent inhibitor of MtSK, and this may be an indication of potential success in later drug discovery stages. The use of ilimaquinone for further drug screening and discovery purposes is at the discretion of the investigator. Our combinatorial approach to weeding out nonspecific inhibitors together with our panel of variants would increase turnover in the early drug screening stages and prevent wastage of long hours on fruitless drug investigations. Lastly, our panel of variants shows great potential for further development into high throughput screen platforms and possibly as sensors. iii Acknowledgments I dedicate this piece to my amazing parents Mr. and Mrs. Ngolui Stephen. Your encouragements and prayers have seen me through life, I stand tall because I stand on your shoulders. You are the very best a son could ever ask for. I love you. I want to give special thanks to my advisor Dr. Goodwin Douglas aka Good Doc for his tremendous support and insight into my graduate school career. You pushed and supported me when all experiments seem to give up on me and still you pointed me to the light at the end of the tunnel. It is a rare privilege and an inexplicable experience to have been under your supervision. You truly stand out. My sincere thanks to my committee members; Holly Ellis for her helpful and comforting words through the trials of graduate school, Chris Easley for impacting your total knowledge of fluorimetry and analytical thinking unto me, and Steven Mansoorabadi aka Dr. Man, you made me feel like I was an expert in enzyme mechanisms and arrow pushing. Thank you for all the random visits you let me pay to your offices. I salute you. Many thanks to Dr. Angela Calderón for her continuous support and encouragements even when nothing seemed to work. Your positive attitude and outlook for better results and ground breaking findings didn‟t go without recognition. Thank you for the experience. I also want to thank former and current members of our group; Drs. Elizabeth Ndontsa (aka Prof), Haijun Duan (aka Diesel), Olive Njuma (aka Miss Olive/Lab machine/proximal iv tryptophan). I won‟t and can‟t leave out Dr. Johayra aka PI/PIC (Partner In Crime) for walking me through the intricacies of this research project. Hui Xu (aka Cong Cong), Jessica Krewall, Tarfi Azis, Rejaul Islam, Callie Jackson. Undergrads; Ethan McCurdy, Gobind Gill, Lauren Barr, Teddy Childers, Olivia Snider, Theresa Simermeyer, Michael Skinner, Daniel Zieman, Patrick Sarhmann and Kirklin McWhorter. With deep gratitude, I extend sincere appreciations to Mr. and Mrs. Soweh. Thank you for opening your home for me and for supporting all the way. You are truly an invaluable part of my achievement and I cannot thank you enough. Uncle Jonathan Tingume, Raymond Tingume, Daniel Weafua, my siblings - Marcellen Nian (aka capitaine), Alain Ngolui, Cpt. Serge Nsanyui, Emile Ngolui and Edmond Ngolui. Your support and guide throughout childhood days and even now has been impeccable. I would choose you again if asked for a second time. To all who have graced my life over the years. I say special thank you. To the friends I made in graduate school; Diana Forbes, Shamim Iqbal, Thankhoe Rantso, Mansour Alturki, Ibtisam Ibtisam, Katie Stanford, Claire Graham. Thank you for being a part of my experience and for sharing your story with me. You are boldly inscribed in my heart. Special thanks to the ladies of the chemistry office. Carol Nixon (retired), Lynn Walker (retired), Lavvorn Howard. You all have been inspirational to me throughout my stay here. You ladies rock! To Mark and Susie, you showed me great love and I am greatly indebted. Many a thank you to Auburn University, department of Chemistry and Biochemistry for accepting and believing in me. That a „crude‟ microbiologist could survive the storms of chemistry. v All in all, from the beginning to the end, THANKS go unto He who sits far above the heavens. For the Lord is good and His mercies endure forever. Gloria in excelsis! vi Table of Contents Chapter 1 ......................................................................................................................................... 1 Literature review ............................................................................................................................. 1 1.1 Overview of tuberculosis (TB) pathology ................................................................................ 1 1.2 Kinases: Phosphorylation of small molecules to proteins ........................................................ 2 1.2.1 Protein kinases.................................................................................................................... 4 1.2.2 Kinase groups and folds ..................................................................................................... 7 1.2.3 Nucleoside monophosphate (NMP) kinases .................................................................... 16 1.2.3.1. Shikimate kinase .......................................................................................................... 20 1.3 Shikimate pathway .................................................................................................................. 23 1.4 Natural products and drug discovery ...................................................................................... 26 1.4.1 By total synthesis ............................................................................................................. 26 1.4.2 By modification of existing scaffolds .............................................................................. 27 1.4.3 By immunoconjugation .................................................................................................... 28 1.4.4 Total synthesis of unmodified