First and foremost, I would like to thank God for the opportunity He has given me to pursue my master's degree. I believe that He is the source of all blessings and I would not be where I am today without Him. Also, to all of the people who made me who I am today, thank you! It is impossible to mention each one of you, but know that I am forever grateful to all of you. With deep gratitude, I would like to thank my advisor, Dr. Baraem (Pam) Ismail, who always gave me tremendous support, continual guidance, and encouragement both in my academic journey and career path. This journey would not be possible without you! I would also like to thank Dr. George Annor, for being a tremendous mentor and friend who took a significant amount of time from his schedule to help me. Many thanks to Dr. Tonya Schoenfuss, my co-advisor, and to Dr. James Anderson, the final member of my thesis committee, for their support of my work. With a thankful heart, I would like to thank Forever Green Initiative, Minnesota Department of Agriculture, and The Land Institute for funding this project. Also to all plant agronomists who helped in providing the samples used in this project and work alongside with us, Xiaofei Zhang, Lee DeHaan, and Dr. Don Wyse. I would also like to thank research groups under the supervision of Dr. Senay Simsek, Dr. Jae-Bom Ohm, and Kristin Whitney at North Dakota State University, for their help and assistance with my research. Further, I would like to thank my research family at the Ismail Research Laboratory for their continuous support and assistance. I especially thank Chathurada and Catrin for their willingness to help and lend me their expertise. To Amy, my sister from another mother and father, for being a good friend who always listen to my ups and downs.

A major cause of tuberculosis (TB) resistance to the aminoglycoside kanamycin (KAN) is the Mycobacterium tuberculosis (Mtb) acetyltransferase Eis. Upregulation of this enzyme is responsible for inactivation of KAN through acetylation of its amino groups. A 123 000-compound high-throughput screen (HTS) yielded several small-molecule Eis inhibitors that share an isothiazole S,S-dioxide heterocyclic core. These were investigated for their structure–activity relationships. Crystal structures of Eis in complex with two potent inhibitors show that these molecules are bound in the conformationally adaptable aminoglycoside binding site of the enzyme, thereby obstructing binding of KAN for acetylation. Importantly, we demonstrate that several Eis inhibitors, when used in combination with KAN against resistant Mtb, efficiently overcome KAN resistance. This approach paves the way toward development of novel combination therapies against aminoglycoside-resistant TB.

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Tuberculosis (TB) remains one of the leading causes of mortality worldwide. Hence, the identification of highly effective antitubercular drugs with novel modes of action is crucial. In this paper, we report the discovery and development of pyrrolo[1,5-a]pyrazine-based analogues as highly potent inhibitors of the Mycobacterium tuberculosis (Mtb) acetyltransferase enhanced intracellular survival (Eis), whose up-regulation causes clinically observed resistance to the aminoglycoside (AG) antibiotic kanamycin A (KAN). We performed a structure–activity relationship (SAR) study to optimize these compounds as potent Eis inhibitors both against purified enzyme and in mycobacterial cells. A crystal structure of Eis in complex with one of the most potent inhibitors reveals that the compound is bound to Eis in the AG binding pocket, serving as the structural basis for the SAR. These Eis inhibitors have no observed cytotoxicity to mammalian cells and are promising leads for the development of innovative AG adjuvant therapies against drug-resistant TB.

A two-drug combination therapy where one drug targets an offending cell and the other targets a resistance mechanism to the first drug is a time-tested, yet underexploited approach to combat or prevent drug resistance. By high-throughput screening, we identified a sulfonamide scaffold that served as a pharmacophore to generate inhibitors of Mycobacterium tuberculosis acetyltransferase Eis, whose upregulation causes resistance to the aminoglycoside (AG) antibiotic kanamycin A (KAN) in Mycobacterium tuberculosis. Rational systematic derivatization of this scaffold to maximize Eis inhibition and abolish the Eis-mediated KAN resistance of M. tuberculosis yielded several highly potent agents. A crystal structure of Eis in complex with one of the most potent inhibitors revealed that the inhibitor bound Eis in the AG-binding pocket held by a conformationally malleable region of Eis (residues 28–37) bearing key hydrophobic residues. These Eis inhibitors are promising leads for preclinical development of innovative AG combination therapies against resistant TB.