Type II Topoisomerases: Repurposing Old Enzymes for New Drugs

Abstract

Beyond their essential functions in many cellular processes, type II topoisomerases are the primary targets of some of the most widely prescribed anticancer and antibacterial therapies in the world. These drugs stabilize enzyme-generated DNA breaks and/or inhibit the catalytic activities of the type II enzymes. Unfortunately, the clinical utility of many chemotherapies and antibacterials targeted by type II topoisomerases has been curbed by off-target toxicities and rapidly growing resistance. The first part of this dissertation examines the interactions between human topoisomerase IIα and IIβ and naphthoquinones, quinone-based metabolites of naphthalene. 1,2-naphthoquinone, an environmental pollutant found in diesel exhaust particles, enhanced double-stranded DNA cleavage mediated by human topoisomerase IIα and IIβ and did so better than a series of naphthoquinone derivatives. Furthermore, 1,2-naphquinone increased enzyme-mediated DNA cleavage by closing the N-terminal gate of the enzyme. These findings suggest that some of the toxicities associated with 1,2-naphthoquinone may be attributed to its ability to enhance DNA scission mediated by human type II topoisomerases. The second part of this dissertation investigates the mechanism of target-mediated fluoroquinolone resistance in Neisseria gonorrhoeae and characterizes new drug classes that can overcome this resistance. Fluoroquinolones were found to mediate their interactions with gyrase and topoisomerase IV through a water-metal ion bridge in N. gonorrhoeae, and mutation of bridge-anchoring residues led to resistance. Moreover, fluoroquinlone-induced cytotoxicity was associated with the accumulation of gyrase-mediated DNA strand breaks. Two clinically advanced classes of gyrase/topoisomerase IV-targeted antibacterials, novel bacterial topoisomerase inhibitors (NBTIs) and spiropyrimidinetriones (SPTs), have been developed. Both classes interact with amino acid residues unique from fluoroquinolones. Examination of the NBTI OSUAB-185 revealed that members of this class can induce double-stranded (in addition to single-stranded) DNA breaks, which suggests that some NBTIs can adopt alternative binding configurations in the cleavage complex. Finally, the SPTs zoliflodacin and H3D-005722 were shown to enhance gyrase/topoisomerase IV-mediated DNA cleavage and inhibit the catalytic activites of these enzymes in N. gonorrhoeae, including fluoroquinolone resistant gyrases. Importantly, this work supports cellular findings that gyrase is the primary target of SPTs and reveals that the actions of SPTs varies across species and enzymes.