Discovery and Synthesis of Novobiocin Derivatives That Stimulate the Lipopolysaccharide Transporter and Synergize With Polymyxins

Abstract

Gram-negative bacterial infections are a worldwide problem and antibiotics capable of clearing such infections are an unmet clinical need. The polymyxins are highly potent drugs against Gram-negative bacteria but suffer from dose-limiting toxicity. Although the exact mechanism for polymyxin activity is unclear, it is known that polymyxin binds lipopolysaccharide. Lipopolysaccharide is a complex glycolipid that is synthesized inside the cell and transported through three cellular compartments to the cell surface by the seven essential lipopolysaccharide transport (Lpt) proteins. We discovered that novobiocin, a gyrase inhibitor, also binds the inner membrane Lpt complex LptB2FGC and stimulates lipopolysaccharide transport. The central finding of this dissertation is that stimulation of lipopolysaccharide transport by novobiocin derivatives sensitize bacteria to the polymyxins. The thesis presents preliminary evidence that polymyxin therapy can be safer when used in combination with a novobiocin derivative. We demonstrated that novobiocin derivatives that bind LptB2FGC and stimulate lipopolysaccharide transport can synergize with polymyxin B independent of gyrase inhibition. In Chapter 2, a semi-synthetic route was used to modify the novobiocin scaffold at the sugar and benzamide positions. An NMR binding assay was combined with a previously published cellular assay for measuring lipopolysaccharide transport to assess the ability of novobiocin derivatives to maintain their target engagement on LptB2FGC. In Chapter 3, we showed that novobiocin derivatives that maintained their ability to stimulate lipopolysaccharide transport synergized with polymyxin. By adding these two drugs together in a thigh infection model, we demonstrated that combination therapy is a therapeutically viable strategy for making polymyxin efficacious at a lower dose and therefore safer. Previously published work suggests that active lipopolysaccharide transport is important in the mechanism of action of the polymyxin antibiotics and ongoing work suggests that polymyxin acts at the inner membrane. In Chapter 4, we tethered novobiocin and polymyxin to leverage the co-localization of these molecules and their targets in the inner membrane, and we describe an efficient strategy to synthesize chimeric molecules in a modular fashion. This dissertation validates a novel therapeutic modality and describes the development of new chemical matter to treat Gram-negative infections by targeting active lipopolysaccharide transport.