Mechanisms of Action and Resistance for Dithiolopyrrolone Antibiotics against Staphylococcus aureus

Natural products are the source and inspiration of potent antibiotics. Characterization of the mode of action and resistance mechanisms of these chemical scaffolds are important in the design of new therapeutic agents. Antibiotic conjugates (i.e. bifunctional and hybrid antibiotics) have particularly high potential due to their ability to target specific pathogens or engage in multitargeting (Chapter 1). The focus of this thesis is investigating the mechanism of action of dithiolopyrrolone (DTP) natural product antibiotics, with a particular focus on the hybrid antibiotic, thiomarinol A. Thiomarinol A is the conjugate of two antibiotics: marinolic acid, a close analogue of the drug mupirocin that is a clinical treatment for methicillin-resistant Staphylococcus aureus (MRSA), and a dithiolopyrrolone (DTP) moiety. DTPs are a family of broad-spectrum prodrugs that enact their mode of action through metal chelation, consequently disrupting metal homeostasis. In Chapter 2, we investigate FAD-dependent oxidoreductases as potential intracellular DTP reducing agents and show that the thioredoxin reductase enzymes from Escherichia coli and S. aureus can reduce the prototypical DTP holomycin in vitro. We propose this mechanism of reduction activates DTP antibiotic activity in both Gram-positive and Gram-negative bacterial cells and is a contributing factor to cellular stress. In Chapter 3, we report that thiomarinol A has a dual mode of action identical to its components—thiomarinol A can chelate metals in S. aureus and is a potent inhibitor of the essential enzyme isoleucyl-tRNA synthetase (IleRS). Thiomarinol A binds MRSA IleRS 1600-fold more tightly than mupirocin. We show that holomycin and mupirocin are only additive in combination against MRSA, hinting that the efficacy of these pharmacophores is drastically enhanced only when the DTP and mupirocin analogue are covalently attached. To further understand thiomarinol potency we aimed to identify additional mechanisms of thiomarinol action and explore thiomarinol resistance in MRSA (Chapter 4). We also explore the ways in which thiomarinol can overcome mupirocin resistance.