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Saturation Mutagenesis and Structure-Activity Relationship of a Natural Product Antibiotic


Antibiotics are some of the most important drugs ever developed to save human lives. These drugs are essentially cures for microbial infections. Despite how important these drugs are, the current antibiotic drug discovery and development pipeline has been met with many challenges. With the constant and rapid rise of resistance to our current antibiotics, new antibiotics are needed more than ever. This problem is exacerbated by the lack of new antibiotics approved for use in the past few decades. In the search for new antibiotics, natural products are a great place to look since most FDA approved antibiotics are natural products or derivatives thereof. This thesis project studies a particular antibiotic called thiocillin, produced by the soil bacterium Bacillus cereus. Thiocillin is a ribosomally synthesized and post-translationally modified peptide (RiPPs) natural product. Thiocillin exhibits potent antimicrobial activity against a broad spectrum of gram-positive bacteria including dangerous pathogens such as methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococci (VRE). This thesis project seeks to identify new and more potent analogs of thiocillin as well as to gain a better understanding of its properties which will enable better drug development in the future.

Chapter 1 describes the genetic exploitation of B. cereus biosynthetic machinery to generate a saturation mutagenesis library of thiocillin’s macrocycle in order to study the structure-activity relationship of this potent antimicrobial natural product. Since thiocillin is built from a ribosomally translated peptide scaffold, this enables the use of site-directed mutagenesis as a powerful tool to rapidly generate thiocillin analogs. This work discovered many new potent thiocillin analogs, the best showing an 8-fold increase in potency over wild-type. Combining our experimental work with BRIKARD computational modelling, we gain a deep appreciation for the role of thiazoles and dehydro post-translational modifications in further restricting entropy beyond simple macrocyclization. Our results exemplify nature’s chemical logic of using rigidifying elements in macrocycle design and we hope that this work may impact future macrocycle drug design.

Chapter 2 explores the use of a redox-reactive oxaziridine reagent to site-specifically label thiocillin analogs engineered with a methionine residue. Probes such as biotin, an alkyne, or an azide are installed onto the engineered methionine. These probes can be used to further study the biology of thiocillins or as a starting point for semi-synthetic medicinal chemistry. Here, we attempt to increase compound solubility by generating thiocillin prodrugs via click chemistry. We demonstrate the ease of site-specific labelling with the oxaziridine reagent, but at the expense of a loss of activity. This exemplifies some of the semi-synthetic challenges in the drug development of thiocillins.

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