Student Theses and Dissertations
Date of Award
2024
Document Type
Thesis
Degree Name
Doctor of Philosophy (PhD)
RU Laboratory
Brady Laboratory
Abstract
Antibacterial resistant infections are an ongoing global health emergency. To combat this,novel potent antibiotics with unique modes of action are required. The classic arsenal of antibiotics used in clinical settings are largely natural products discovered by bulk fermentation of bacterial species isolates that are then extracted and purified to yield a bioactive compound.Unfortunately, this discovery pipeline no longer produces novel molecules, thus new discovery methodologies are essential to continue to identify antibiotic clinical candidate molecules. In the modern era, coupling natural product discovery with sequencing technologies has proven to bean efficient and fruitful method of identifying secondary metabolite natural products with unique bioactivity that could not previously be accessed through fermentation-based methods. This novel approach presents a promising reinvigoration to the field of antibiotics discovery. In our lab, we have developed a discovery method by which sequenced bacterial genomes are analyzed using bioinformatic algorithms to identify biosynthetic gene clusters (BGCs) and to make a structural prediction of the molecular product of a given cluster. This molecular prediction can be built using synthetic chemistry and assayed for its biological activity. The name given to this method and the resulting products that are synthesized is synthetic bioinformatic natural products (synBNP). It is using this method that our lab previously discovered cilagicin, a lipopeptide natural product that shows robust Gram-positive antibiotic activity and evades antibiotic resistance even after prolonged pathogen exposure. This resistance evasion is attributed to a dual polyprenyl phosphate binding mechanism. In this thesis, I present discovery and optimization efforts to expand this promising novel class of antibiotic natural products as well as to develop a singular lead clinical drug candidate that displays optimal bioactivity and in vivo efficacy. In Chapter 2, I present an investigation of bioinformatically screened predicted non-ribosomal polypeptide synthetase encoded structures to identify previously uncharacterized antibiotics that may possess the same molecular targets and resistance evasion ability seen with cilagicin. This structure-based screen yields three BGCs predicted to produce natural analogs of cilagicin. synBNPs for the products of each of these three clusters are synthesized and the resulting molecules are assayed for their biological activity. These compounds, called paenilagicin, bacilagicin, and virgilagicin, are shown to be potent antibiotics against multidrug-resistant Gram-positive pathogens. Paenilagicin and virgilagicin are further shown to engage both of the same polyprenyl phosphate targets as cilagicin, and both also demonstrate the ability to evade antibiotic resistance. Bacilagicin is shown to only bind a single molecular target and is susceptible to antibiotic resistance development. This discovery project expands the members of this family of polyprenyl phosphate binding antibiotics, which allows us to identify a conserved peptide moiety that we suspect may play a role in target engagement. In Chapter 3, building upon the structural diversity identified among polyprenyl phosphate binding antibiotics, I discuss a structural optimization project in which we sought to design an improved version of the most potent antibiotic in this family, cilagicin. To achieve our goal of a molecule with strong antibiotic activity and low serum protein binding to preserve activity invivo, we conduct two regional analyses of the overall molecular structure. In the first, we investigate the effect of structurally diverse lipid tail substituents on bioactivity. In the second, we conduct a series of orthogonal scans on the peptide core to explore the impact of different peptide moieties with various chemical properties on bioactivity. Ultimately, we identify an optimized compound, called dodecacilagicin, that maintains high Gram-positive antibiotic activity, shows minimal serum protein binding, and also evades antibiotic resistance. Overall, the work presented in this thesis represents the application and expansion of the synBNP discovery method as an ever evolving and robust means to discover novel structurally diverse natural products with unique bioactivity that were previously inaccessible by culture-dependent methods.
Recommended Citation
Rosenzweig, Adam, "Discovery and Optimization of a Family of Naturally Encoded Antibiotic Compounds that Evade Antibiotic" (2024). Student Theses and Dissertations. 774.
https://digitalcommons.rockefeller.edu/student_theses_and_dissertations/774
Comments
A thesis presented to the faculty of The Rockefeller University in partial fulfillment of the requirements for the degree of Doctor of Philosophy