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Development of Pyruvate-Ferredoxin Oxidoreductase Inhibitors for the Treatment of Clostridium Difficile Infection

Marshall, Alexandra
Thesis/Dissertation; Online
Marshall, Alexandra
Macdonald, Timothy
Clostridium difficile is an anaerobic, Gram-positive bacillus that can be found in normal human intestinal flora. Toxigenic strains of C. difficile can lead to C. difficile infection (CDI), which has recently overtaken Methicillin-resistant Staphylococcus aureus (MRSA) as the most frequently diagnosed hospital-acquired infection (HAI). The expense of drug development and looming threat of microbial resistance has stifled the introduction of novel antibiotics and current modes of treatment have little success in reducing recurrence of infection. Nitazoxanide (NTZ) is an FDA-approved antibiotic that is effective at treating and preventing the recurrence of CDI. NTZ inhibits bacterial growth by targeting pyruvate:ferredoxin oxidoreductase (PFOR), a metabolic enzyme in anaerobic bacteria that is responsible for the oxidative decarboxylation of pyruvate to form acetyl coenzyme A. It has been proposed that thiamine pyrophosphate (TPP), a coenzyme of PFOR, is inhibited by NTZ, presenting a novel therapeutic mechanism that could circumvent common modes of bacterial resistance. While effective against CDI, NTZ is not selective and lends itself to preventing complete recovery from infection. The development of NTZ-based PFOR inhibitors is presented here, including the initial elucidation of the mechanism of action of NTZ in PFOR and the optimization of the nitrothiazolide scaffold to the lead inhibitor amixicile (AMX) that inspired subsequent scaffold modifications. A variety of structure activity relationship (SAR) studies of nitrothiazolides were performed and used in the design of AMX which was more soluble, potent and non-toxic than NTZ. A series of second-generation inhibitors was proposed using the favorable properties of AMX and docking studies using a homology model based on the solved crystal structure of PFOR from Desulfovibrio africanus and the proposed mechanism of action of NTZ. The homology model was trained with PFOR inhibitors to predict the activity of additional NTZ derivatives. The library of PFOR inhibitors presented here represents a promising breakthrough in an otherwise dismal field of drug discovery. Although nitrothiazolide antibiotics are more active at the PFOR enzyme, they still do not perform as well as NTZ in whole cells, indicating that further structural modifications must be made to the scaffold of AMX in order to achieve an optimal lead compound for in vivo testing.
University of Virginia, Department of Chemistry, MS, 2013
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